Principles of Bacterial Pathogenesis
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Principles of Bacterial Pathogenesis EDITED BY
Eduardo A. Groisman Howard Hughes Medical Institute Washington University School of Medicine Department of Molecular Microbiology St. Louis, Missouri
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Contents
Contributors Preface
xi XV
1. Evolution of Bacterial Pathogens HOWARD OCHMAN
I. II. III. IV. V. VI. VII. VIII. IX.
Introduction 2 The Genetic Basis of Virulence 2 Identification of Sequences Involved in Bacterial Pathogenesis 6 Recovery of Genes Contributing to Virulence 8 The Population Genetics of Pathogens 9 Studying Bacterial Population Genetics 10 The Organization of Genetic Diversity in Pathogenic Microorganisms Population Genetics of Representative Bacterial Pathogens 14 Conclusions 28 References 29
13
2. Germ Warfare: The Mechanisms of Virulence Factor Delivery JILL REISS HARPER AND THOMAS J. SILHAVY
I. II. III. IV. V VI. VII. VIII.
Introduction 43 The General Secretory Pathway 45 Autotransporters: Type V 47 Two-Step Secretion: Type II 49 ABC Transporters: Type I 52 Conjugal Transfer Systems: Type IV 55 Contact-Dependent Secretion: Type III 57 Concluding Remarks 61 References 61
vi
CONTENTS
3. Regulation of Virulence Gene Expression in Bacterial Pathogens CHARLES J. DORMAN AND STEPHEN G . J. SMITH
I. II. III. IV. V. VI. VII. VIII. IX. X. XL XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII.
Introduction 76 Transcription Initiation 77 Regulatory Protein Families 79 Covalent Modification of Transcription Factors 82 Regulatory Networks 86 The Oxidative Stress Response 87 The Modular Nature of Bacterial Regulatory Proteins 89 The Overlap between Genome Structure and Gene Regulation 92 Other Classes of Protein Regulators 94 DNA Structure and Gene Regulation 94 Stereotypical and Stochastic Events in the Control of Gene Expression The Switch Controlling Type 1 Fimbrial Expression in E. coli 101 Pap Pilus Gene Transcription 103 Contact-Dependent Gene Regulation 105 The Virulence Gene Regulatory Cascade of 5. y7exA2^n 106 A Thermometer Protein from the Salmonella Virulence Plasmid 109 Cell-Density-Dependent Regulation 110 Adaptive Mutation 114 Rare tRNAs and Translation Modulation 114 Protein Splicing 115 AntisenseRNA 115 Perspective 116 References 117
97
4. Strategies to Identify Bacterial Pathogenicity Factors ANDREW CAMILLI, D . SCOTT MERRELL, AND JOHN J. MEKALANOS
I. II. III. IV. V VI.
Introduction 134 Biochemical Strategies 135 Genetic Screens 149 Genetic Selections 159 Genomic Approaches 165 Concluding Remarks 169 References 170
5. Mechanisms of Bacterial Pathogenesis in Plants: Familiar Foes in a Foreign Kingdom JAMES R . ALFANO AND ALAN COLLMER
I. Introduction 180 II. An Overview of Bacterial Plant Pathogens and Plant Diseases 181 III. Tumorigenic Agrobacterium tumefaciens: Using the Type IV Secretion System to Transform the Host into a Factory for Bacterial Nutrients 186
CONTENTS
Vli
IV. Necrogenic, Stealth Pathogens: Parasites Strongly Dependent on the Hrp (Type III) Protein Secretion System 189 V. Necrogenic, Brute-Force Pathogens: Soft-Rotters Dependent on Type II Secretion of Pectic Enzymes 200 VI. Other Virulence Factors of Gram-Negative Plant Pathogens Compared with Those of Animal Pathogens 201 VII. Host Innate Immune Systems: Common Components in Pathogen Recognition and Defense Signaling 206 VIII. The R Gene Surveillance System: An Innate Immune System with Elaborate Recognition Specificity 207 IX. Pseudomonas aeruginosa: Dual-Kingdom Pathogenesis 209 X. Conclusions 210 References 211
6. Yersinia AoiFE P. BOYD AND GUY R . CORNELIS
I. II. III. IV. V. VI. VII.
Introduction 228 The Adhesive Factors 231 Iron Acquisition 236 Pathogenicity Islands 237 Yst Enterotoxin 238 The Yersinia Virulence Plasmid Conclusion 252 References 253
238
7. Molecular Pathogenesis of Salmonellae CHRISTINA A. SCHERER AND SAMUEL I. MILLER
I. II. III. IV. V. VI. VII. VIII. IX.
Introduction 266 History 266 Taxonomy 267 Epidemiology and Clinical Disease 268 Clinical Course and Basic Immunology 272 In Vitro Models of Salmonella Virulence 280 Virulence Factors 290 Antibiotic-Resistant Salmonellae 312 5a/mon^//<3-Based Vaccines 314 References 316
8. Shigellosis: From Disease Symptoms to Molecular and Cellular Pathogenesis PHILIPPE J. SANSONETTI, COUMARAN EGILE, AND CHRISTINE WENNERAS
I. Introduction 336 II. Bacteriology 337 III. The Somatic Antigen
338
viii
CONTENTS
IV. V. VI. VII. VIII. IX. X. XI. XII.
Epidemiology and Transmission 338 Disease Symptoms and Complications: Orientations for Future Research? Histopathology of Shigellosis: A Window on Pathogenesis 342 Animal Models: Strengths and Weaknesses 343 Cellular Models of Infection: The Contribution of Shigella to the Concept of Cellular Microbiology 344 Pathogenic Mechanisms: In Vitro Expression of the Invasive Phenotype Pathogenic Mechanisms: In Vivo Expression of the Invasive Phenotype Role of Chromosomally Encoded Genes in the Virulence of Shigella Conclusions 373 References 373
340
345 365 369
9. Pathogenic Escherichia coli JOSE L . PUENTE AND B . BRETT FINLAY
I. II. III. IV. V. VI. VII. VIII. IX. X.
Introduction 388 Enterotoxigenic E. coli (ETEC) 390 Enteroinvasive E. coli (EIEC) 396 Enteropathogenic E. coli (EPEC) 398 Enterohemorrhagic E. coli (EHEC) 408 Enteroaggregative £". CY?//(EAEC) 414 Diffusely Adhering E. coli (DAEC) 417 Uropathogenic £". C6>// 418 E. coli That Cause Sepsis and Meningitis 426 Conclusions 428 References 428
10. Molecular Basis of Vibrio cholerae Pathogenesis VICTOR J. DIRITA
I. II. III. IV. V. VI.
Introduction 457 Vibrio cholerae 458 Cholera 463 Molecular Mechanisms of Disease 465 Natural and Induced Immunity against Vibrio cholerae Infection Future Studies: The Past Is Prologue 493 References 495
\\. H. pylori Pathogenesis TIMOTHY L . COVER, DOUGLAS E . BERG, MARTIN J. BLASER, AND HARRY L . T. MOBLEY
I. II. III. IV. V.
Introduction 510 Epidemiology 510 Gastric Histology and Physiology 512 Clinical Diseases Associated with H. pylori Infection Microbiology 519
516
489
CONTENTS
VI. VII. VIII. IX. X. XI. XII.
ix
Genetic Diversity and Population Structure of H. pylori 521 Initial Gastric Colonization 524 Gastric Inflammation 529 Interactions of H. pylori with the Gastric Epithelium 531 Vacuolating Cytotoxin 532 Persistence of H. pylori Infection 536 Factors Influencing Development of Clinically Evident Disease References 542
12. Neisseria SCOTT D . GRAY-OWEN, CHRISTOPH DEHIO, THOMAS RUDEL, MICHAEL NAUMANN, AND THOMAS F. MEYER
I. II. III. IV. V. VI. VII. VIII. IX.
Introduction 559 Natural Competence for Transformation Surface Structures 567 Tissue Colonization 570 PorB31 586 IgAl Protease 590 Iron Acquisition in Vivo 592 Immune Response 594 Summary 599 References 600
566
13. Bordetella PEGGY A. COTTER AND JEFF F. MILLER
I. II. III. IV. V. VI. VII. VIII. IX.
Introduction 620 Respiratory Infections by B6>rj£'r6'//rt Species 621 Evolutionary Relationships among Bordetella Subspecies 624 Bordetella Virulence Factors 628 The Bordetella-Host Interaction 639 The BvgAS Sensory Transduction System 642 Phenotypic Modulation 646 Transcriptional Control of Bvg-Regulated Genes 640 The Role of Bvg-Mediated Signal Transduction in the Bordetella Life Cycle 654 References 658
14. Pathogenesis of Haemophilus influenzae Infections CHRISTOPH M . TANG, DEREK W . HOOD, AND E . RICHARD MOXON
I. II. III. IV. V.
Introduction 676 Population Biology 680 Molecular Determinants of Pathogenicity Pathogenesis 699 Conclusions 705 References 705
682
539
CONTENTS
15. Pathogenic Mechanisms in Streptococcal Diseases MICHAEL CAPARON
I. II. III. IV. V. VI. VII.
Introduction 717 Three Basic Mechanisms of Pathogenesis: Example of 5. pyogenes Steps Common to All Three Pathogenic Mechanisms 721 First Mechanism: Invasion and Multiplication in Tissue 728 Second Mechanism: Toxin-Mediated Disease 739 Third Mechanism: Immunopathological-Based Diseases 742 Concluding Remarks 743 References 743
719
16. Listeria monocytogenes HAFIDA FSIHI, PIERRE STEEPEN, AND PASCALE COSSART
I. General Overview of Listeria monocytogenes and Listeriosis II. Genetic Tools and Cell Biology Techniques to Study L. mono cytogenes Infection 755 III. Molecular Mechanisms for Entry and Spread of L. monocytogenes in Nonphagocytic Cells 758 IV. Regulation of L. monocytogenes Virulence Gene Expression V. Conclusion 787 References 787
Index
805
752
782
Contributors
Numbers in parentheses indicate the pages on which the authors' contributions begin.
(179), Department of Biological Sciences, University of Nevada, Las Vegas, Nevada 89154-4004
JAMES R. ALFANO
(509), Departments of Molecular Microbiology and Genetics, Washington University Medical School, St. Louis, Missouri 63110-1093
DOUGLAS E. BERG
J. BLASER (509), Department of Medicine, New York University School of Medicine, New York, New York 10016
MARTIN
AoiFE P. BOYD (227), Microbial Pathogenesis Unit, Universite Catholique de Louvain, ICP et Faculte de Medecine, Brussels 1200, Belgium (133), Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
ANDREW CAMILLI
(717), Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
MICHAEL CAPARON
(179), Department of Plant Pathology, Cornell University, Ithaca, New York 14853-4203
ALAN COLLMER
(227), Microbial Pathogenesis Unit, Universite Catholique de Louvain, ICP et Faculte de Medecine, Brussels 1200, Belgium
GUY R. CORNELIS
(751), Unite de Interactions Bacteries-Cellules, Institut Pasteur, Paris, Cedex 15, 75724, France
PASCALE COSSART
A. COTTER (619), Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, CA 90095-1747
PEGGY
(509), Department of Medicine and Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232
TIMOTHY L. COVER
(559), Abteilung Infektionsbiologie, Max-Planck-Institut-fur Biologic, Tubingen 72076, Germany
CHRISTOPH DEHIO
XI
xii
CONTRIBUTORS
J. DIRITA (457), Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 48109
VICTOR
J. DORMAN (75), Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland
CHARLES
COUMARAN EGILE (335), Unite de Pathogenic Microbienne Moleculaire & Unite INSERM 389, Institut Pasteur, 75724 Paris, Cedex 15, France B.
(387), Biotechnology Laboratory, and the Departments of Biochemistry & Molecular Biology and Microbiology & Immunology, University of British Columbia, Vancouver, BC V6TIZ3, Canada
BRETT FINLAY
(751), Unite des Interactions Bacteries-Cellules, Institut Pasteur, Paris, Cedex 15, 75724, France
HAFIDA FSIHI
(559), Department of Medical Genetics and Microbiology, University of Toronto, Toronto M5S 1A8, Canada
SCOTT D . GRAY-OWEN
(43), Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
JILL REISS HARPER
DEREK W. HOOD (675), Molecular Infectious Diseases Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, 0X3 9DU, United Kingdom J. MEKALANOS (133), Department of Microbiology and Molecular Genetics, Shipley Institute of Medicine, Harvard Medical School, Boston, Massachusetts 02115
JOHN
D.
(133), Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111
SCOTT MERRELL
(559), Max-Planck-Institut fur Infektionsbiologie, Department of Molecular Biology, 10117 Berlin, Germany
THOMAS F. MEYER
(619), Department of Microbiology, Immunology, and Molecular Genetics, UCLA School of Medicine, Los Angeles, CA 90095-1747
JEFF F. MILLER
I. MILLER (265), Departments of Medicine and Microbiology, University of Washington, Seattle, Washington 98195
SAMUEL
(509), Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 212011559
HARRY L.T. MOBLEY
E.
(675), Molecular Infectious Diseases Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, 0X3 9DU, United Kingdom
RICHARD MOXON
(559), Department of Molecular Biology, Max-Planck-Institut-fur Infektionsbiologie, Berlin 10117, Germany
MICHAEL NAUMANN
CONTRIBUTORS
Xili
(1), Department of Ecology and Evolutionary Biology, University of Arizona, Tucson, Arizona 85721
HOWARD OCHMAN
(387), Department of Molecular Microbiology, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Cuemavaca 62210, Mexico
JOSE L. PUENTE
(559), Department of Molecular Biology, Max-Planck-Institutfiir Infektionsbiologie, Berlin 10117, Germany
THOMAS RUDEL
J. SANSONETTI (335), Unite de Pathogenic Microbienne Moleculaire & Unite INSERM 389, Institut Pasteur, 75724 Paris, Cedex 15, France
PHILIPPE
A. SCHERER (265), Departments of Medicine and Microbiology, University of Washington, Seattle, Washington 98195
CHRISTINA
J. SILHAVY (43), Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544
THOMAS
(75), Department of Microbiology, Moyne Institute of Preventive Medicine, Trinity College, Dublin 2, Ireland
STEPHEN G J . SMITH
(751), Unite des Interactions Bacteries-Cellules, Institut Pasteur, Paris, Cedex 15, 75724, France
PIERRE STEFFEN
(675), Molecular Infectious Diseases Group, Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, 0X3 9DU, United Kingdom
CHRISTOPH M . TANG
(335), Department of Medical Microbiology and Immunology, University of Goteborg, S41345 Goteborg, Sweden
CHRISTINE WENNERAS
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Preface
Infectious diseases are the leading cause of morbidity and mortality worldwide. While some of these infections are caused by eukaryotic parasites, bacterial pathogens continue to present a threat to the well-being of humans and animals both in the developing and developed worlds. The use of vaccines and antibiotics, together with changes in sanitary practices, has contributed to an important increase in the life span of humans in the last century. However, these significant improvements are now challenged by the appearance of microbes resistant to multiple antibiotics, the emergence of new bacterial pathogens, and the use of health care treatments that, while prolonging life, render individuals susceptible to opportunistic pathogens. Then, what can be done to sustain the improvements in health care developed during the last 100 years? Novel strategies are currendy being tested to prevent and/or treat bacterial infections. An increasing number of these strategies are based on our understanding of the mechanisms by which pathogenic microorganisms cause disease. This is possible due to exciting developments in the field of bacterial pathogenesis, which started some 20 years ago with the use of molecular genetics to investigate the microorganisms responsible for causing disease, and are now complemented with cell biological and biochemical approaches aimed at unraveling the consequences that infection by such microorganisms has on their hosts. We now have a basic understanding not only of the varied nature of virulence determinants but also of their origin and acquisition by pathogenic microbes. We appreciate that expression of virulence determinants is most often regulated in response to host signals and that microbes use different devices to deliver toxic products to host cells. These studies have revealed a set of principles that govern bacterial pathogenesis and, as the tide indicates, constitutes the subject matter of this book. The motivation for Principles in Bacterial Pathogenesis was twofold: first, to provide in-depth coverage of the best-characterized bacterial pathogens, with the goal of uncovering the salient features that these microbes have in common which allow them to conquer new niches and to circumvent host defense mechanisms, and, second, to group contributions by the world experts in bacterial pathogenesis
XV
XVI
PREFACE
in which they present a general discussion of the subject beyond the work performed in their own laboratories. This book is divided in two parts that comprise a total of 16 chapters, each of which can be read independent of the rest. The first part consists of five chapters, three of which discuss aspects of bacterial pathogenesis that are common to all pathogens: evolution, secretion, and regulation of virulence determinants. The fourth chapter presents a thorough description of the strategies currently used to identify virulence determinants. The fifth chapter discusses bacterial pathogens of plants, highlighting the similar mechanisms that bacterial pathogens of animal and plants employ when interacting with their respective hosts. These first 5 chapters serve as a general introduction to the 11 pathogen-based chapters that comprise the second part of the book. Each of the latter chapters provides a broad discussion of the best-understood human pathogens. In sum, while novel aspects of pathogenic organisms will continue to be discovered, a basic understanding of the principles governing bacterial pathogenesis will not only allow us to appreciate the sophisticated mechanisms used by microbes in their pathogenic lifestyle, but will also be essential in beginning to understand the plethora of information emerging from genomics, and to develop new rational approaches to the treatment and prevention of infectious diseases. Eduardo A. Groisman Department of Molecular Microbiology Howard Hughes Medical Institute Washington University School of Medicine St. Louis, Missouri
CHAPTER 1
Evolution of Bacterial Pathogens HOWARD OCHMAN
I. II. HI. IV. V. VI.
Introduction The Genetic Basis of Virulence Identification of Sequences Involved in Bacterial Pathogenesis Recovery of Genes Contributing to Virulence The Population Genetics of Pathogens Studying Bacterial Population Genetics A. Multilocus Enzyme Electrophoresis B. DNA Sequencing C. Multilocus Sequence Typing vn. The Organization of Genetic Diversity in Pathogenic Microorganisms VIII. Population Genetics of Representative Bacterial Pathogens A. Bordetella B. Borrelia C. Escherichia coli and Shigella D. Haemophilus E. Helicobacter F. Listeria G. Mycobacterium H. Neisseria I. Salmonella J. Staphylococcus K. Streptococcus L. Vibrio IX. Conclusions References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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HOWARD OCHMAN
/. Introduction In what ways do pathogenic microorganisms differ from nonpathogenic forms? For an organism to be considered a pathogen, it must, during some phase of its life-cycle, advance disease and alter the health or behavior of another organism, that is, its host. Every organism can serve as a host for pathogens, and pathogens come into contact with a very large number of species; however, most pathogenic microorganisms are virulent to relatively few, and often only one, host species, and infection may cause disease in only a limited segment of the host population. So despite the wide range of mechanisms deployed by pathogens to disable their hosts (and to promote their own replication and transmission), there is one common theme: virulence depends upon the susceptibility of a host. Therefore, the identification of pathogens, the differences between pathogenic and nonpathogenic microorganisms, and the specific factors required for virulence must each be defined with regard to its relevance to the host. This chapter addresses two general issues about the evolution of microbial pathogenesis. First, we consider the differences between the genomes of pathogenic and nonpathogenic bacteria, the specific types of genes that contribute to the virulence phenotype, and the evolutionary history of these sequences in the genome of pathogens. Next, we discuss the molecular population genetics of microbial pathogens and the factors that govern the organization of genetic diversity within these populations. Because the origins and genetic bases of virulence influence the population structure of pathogens, these topics are interconnected and broadly relevant to the emergence, outbreak, control, and prevention of the diseases caused by microbial pathogens.
//. Thie Genetic Basis of Virulence One way to gain insight into the specific factors contributing to virulence has been to identify the genetic differences between pathogens and closely related nonpathogenic bacteria. In this regard, there are potentially four types of genetic events that might be responsible for the differences in pathogenic potential among related bacteria: (1) virulence as a result of genes specific to the pathogen; (2) virulence as a result of the absence of a suppressor locus in the pathogen; (3) virulence as a result of allelic differences between genes shared by the pathogen and nonpathogen; and (4) virulence as a result of the differential regulation of the same complement of genes in the pathogen and nonpathogen.
1.
VIRULENCE AS A RESULT OF SPECIES- OR STRAIN-SPECIFIC GENES
The most common approach to investigate virulence genes—on both analytical and technical grounds—has been to search for sequences that are restricted to
1.
THE EVOLUTION OF BACTERIAL PATHOGENS
3
pathogenic organisms. Virulence determinants are often acquired through horizontal transfer, which may explain why virulence traits are distributed sporadically among bacterial taxa (Fig. 1) or within some bacterial species (Fig. 2). In many bacterial species, there is an association between species-specific genes and virulence. Pathogenicity islands (or Pai)—i.e., segments of the chromosome that encode virulence genes and are absent from related nonpathogenic bacteria [1-3]—have been identified in pathogenic strains of E. coli [4-6], Shigella flexneri [7, 8, 24], Salmonella enterica [9], Yersinia pestis [10-12], Vibrio cholerae [13], Haemophilus influenzae [14, 15], Helicobacter pylori [16], and Staphylococcus aureus [17]. In each of these cases, a specific chromosomally encoded gene, or cluster of genes, has been implicated in the virulence of the microorganism, and the corresponding region was not present in avirulent strains or related species. Although it has long been known that species-specific regions confer traits that are unique to a bacterial species, the term "pathogenicity island" was first used to describe a DNA segment harbored by uropathogenic strains of E. coli [18]. Further characterization of pathogenicity islands revealed that many were situated at transfer RNA loci, which are commonly used as integration sites for foreign sequences [19]. For example, certain phages detected in E. coli, such as the retronphage (t)R73 [20] and the bacteriophage P4 [21] insert at, or near, tRNA genes, suggesting that pathogenicity islands are often transferred and acquired through phage-mediated events [1, 22]. The frequent insertion of foreign DNA sequences at tRNA genes is presumably due to the high degree of sequence conservation of tRNA genes across species. In fact, there is recurrent use of the
H:
Escherichia Shigella Salmonella Citrobacter Klebsiella Serratia Yersinia Proteus
Fig. 1 Phylogenetic relationships among enteric bacteria showing taxa that are normally capable of invading eukaryotic cells (denoted with darkened branches).
HOWARD OCHMAN
E. CO//K-12 ETEC0159:H4 EPEC0in:H12 ETEC078:Hn EIEC0n2:NM EPEC0m:H12 ECOR 5 EC0R6 ECOR 10 ECOR 14 (UTI)
-
ECOR27 ETEC0148:H28
EIEC0124:NM • Shigella flexneri Shigella boydii Shigella flexneri ETEC0159:H4 I ECOR 69 ' ECOR 30
LT ^
J ECOR 70 I ECOR 58 EIEC028:NM
ECOR 38
ECOR 61 ECOR 62 (UTI) ECOR 52 ECOR 64 (UTI) ECOR 59 ECOR 66 EPEC055:H6 Shigella sonnei I ECOR 50 (UTI) ^ 1 FPOR 4q ECOR 49
EHEC0157:H7 ECOR 37 Fig. 2 Relationships among commensal and pathogenic strains of Escliehchia coli and Shigella spp. based on nucleotide sequences of the gene encoding malate dehydrogenase (adapted with permission from Pupo et al. (1997) [56]). Abbreviations are as follows: UTI = urinary tract infection; EHEC = enterohemorrhagic E. coli; EIEC = enteroinvasive E. coli; EPEC = enteropathogenic E. coli; ETEC = enterotoxigenic E. coli. ECOR strains are from the E. coli reference collection [100], and the 0:H serotypes of pathogenic E. coli are noted.
tRNA^^^^ locus, which is targeted by (\>R\13 and as the integration site for several pathogenicity islands: Pai-1 of uropathogenic E. coli [4], the LEE island of enteropathogenic E. coli [23], the SHI-2 island of Shigella flexneri [8, 24], and the SPI-3 island of Salmonella enterica [25] each represent independent insertions of different virulence gene clusters into similar chromosomal locations. Flanking many pathogenicity islands there are signature sequences, such as short direct repeats, reminiscent of the integration of mobile elements (or even, in the case of Yersinia pestis, copies of the IS elements themselves) further attesting that these species- or strain-specific regions can be acquired laterally through a variety of transfer mechanisms. Although research on pathogenicity islands focuses on chromosomally encoded regions, genes involved in bacterial virulence are also carried on extrachromosomal elements that are maintained within the genome of pathogens. For example.
1.
THE EVOLUTION OF BACTERIAL PATHOGENS
5
many of the genes required for Shigella virulence reside on a 220-kb plasmid [26, 27], and, similarly, all virulent strains of Yersinia harbor a 70- to 75-kb plasmid that encodes proteins necessary for their antihost properties [28, 29]. In this regard, the acquisition of plasmid-borne antibiotic resistance genes will also allow previously sequestered pathogens to exploit new hosts. Other virulence determinants in these enteric species have been acquired by the organism in phage-mediated events. For example, the cytotoxins first characterized in Shigella are encoded on a bacteriophage that has subsequently been transferred to enterohemorrhagic strains of E. coli [30, 98]. In the case of Vibrio cholerae, two coordinately regulated factors contribute to virulence: cholera toxin, which is encoded by a filamentous bacteriophage (termed CTX(t)) related to the coliphage Ml3 [31], and the toxin-coregulated pili, which is encoded within a large pathogenicity island. This pathogenicity island of Vibrio cholera is in itself another filamentous bacteriophage [32], and this demonstrates a novel case where one horizontally acquired phage encodes the receptor for the products specified by a second phage, both of which are required for full virulence.
2.
VIRULENCE RESULTS FROM THE ABSENCE OF A SUPPRESSOR LOCUS
Similar to the mechanisms described above—whereby a microbe has acquired certain genes that render it virulent—it is also possible that the pathogen has either lost a gene encoding a product capable of diminishing its virulence potential, or that such a determinant was acquired by the related avirulent forms. An early example of a virulence suppressor in enteric bacteria is the surface protease OmpT, which is absent from the genomes of Shigella and enteroinvasive E. coli (EIEC). The presence of ompT results in attenuation of virulence because the encoded protease interferes with expression of the VirG protein, which is required for intercellular spread [33]. The ompT gtUQ is probably not ancestral to enteric bacteria: it is located within the 21-kb cryptic lambdoid phage, suggesting that avirulent strains of E. coli acquired ompT through horizontal gene transfer. In addition to lacking ompT, Shigellae are also devoid of genes whose products suppress virulence. Representatives of the four species of Shigella, as well as enteroinvasive strains of E. coli (Fig. 2), harbor deletions for the region containing cadA, which encodes lysine decarboxylase. When the cadA gene from an avirulent strain of E, coli was introduced into Shigella flexneri, the resulting strain was still able to invade cells in tissue culture, but did not exhibit the toxic effect that induces the fluid secretion normally associated with infection. In contrast to the situation where a microorganism gains genes that enhance its pathogenic potential (i.e., pathogenicity islands), these regions have been termed "black holes" to denote deletion of genes that reduce the pathogenic potential of an organism [34].
6
HOWARD OCHMAN
3.
VIRULENCE RESULTS FROM ALLELIC DIFFERENCES
BETWEEN HOMOLOGOUS GENES
Because pathogenic and nonpathogenic have often diverged in sequence, it is possible that the differences in pathogenic properties result from allelic variation in homologous genes due to nonsense or missense mutations. For example, point mutations in the fimH gene of E. coli can change the binding of fimbrial adhesins and confer increased virulence in the mouse urinary tract [35, 36].
4.
VIRULENCE RESULTS FROM THE DIFFERENTIAL REGULATION OF THE SAME COMPLEMENT OF GENES
In addition to genetic polymorphisms among bacterial strains and species, it is possible that the differences in pathogenic properties are caused by differential regulation of essentially the same set of genes. For example, the invasion gene complexes of S. enterica and S. flexneri are largely homologous, but controlled by very different environmental signals: invasion in S. enterica is regulated by oxygen tension [37], whereas the expression of virulence genes by Shigella is controlled by temperature [38]. The origin of virulence properties in many pathogenic species is likely to result from a combination of the factors presented above. For Shigella and enteroinvasive E. coli, it is clear that virulence is the result of the incorporation of a large virulence plasmid into a strain lacking the ompT gene and the deletion of cadA from their genomes. And while the virulence genes on the Shigella and EIEC plasmids are 99% identical, these species exhibit large differences in their median infective doses, which could be due to allelic variation or to differential regulation of homologous genes or to species-specific chromosomal genes. Also note that these types of genetic changes do not pertain to the analysis of most opportunistic or newly emerging pathogens. Because these microbes are displaced to nonstandard hosts, tissues or environments, the genes contributing to virulence are not preadapted to the host and are not likely to differ from the repertoire required for growth in their customary environments.
///. Identification of Sequences Invoived in Bacterial Pattiogenesis From the previous discussion, it is obvious that most of the differences in pathogenic potential among related bacteria are due to changes in gene content (mechanisms 1 and 2) rather than to changes in the ancestral genes themselves (mechanisms 3 and 4). Although the specific approach, as well as technical
1.
THE EVOLUTION OF BACTERIAL PATHOGENS
7
considerations, bias the identification of the particular genetic events and the recovery of genes involved in virulence (see below), the vast majority of traits that are unique to a species are encoded on segments of the genome that arose through horizontal transfer [39]. Stepwise mutational changes in existing genes only rarely confer novel functions, whereas traits encoded by acquired DNA will occasionally confer the ability to explore new hosts or environments and can have a large impact on bacterial evolution [40]. As a result, none of the phenotypic characteristics that distinguish E. coli from S. enterica are attributable to the divergence of homologous genes by mutation; instead, all of the species-specific traits derive from functions encoded by horizontally transferred genes (e.g., lactose utilization, citrate utilization, indole production) or from the loss of ancestral DNA (e.g., alkaline phosphatase) [39]. The broad association of species-specific traits with unique portions of the chromosome does not imply that all (or even the majority of) genes contributing to the virulence phenotype are restricted to pathogens and absent from the related nonpathogenic bacteria. The classification of genes as being involved in pathogenesis depends largely on the particular approach used to define and identify these factors [41]. The traditional approach to recognizing virulence determinants was purification of microbial products, which, upon introduction into a susceptible host, produced some of the symptoms advanced by the whole organism. This biochemical approach resulted in identification of a variety of factors, usually toxins, produced by several pathogens such as Vibrio cholerae and Clostridium botulinum. The classical bacterial genetics approach defines virulence genes as those that, on mutation, give rise to strains with median lethal doses (LD50) higher than that corresponding to the wild-type parent [42]. This interpretation of virulence includes all relevant loci—apart from those essential for growth under laboratory conditions—without assumptions about the precise role that particular virulence determinants play in the pathogenicity of the microorganism. The molecular genetic approach is used to isolate virulence genes based on their capacity to confer certain pathogenic properties on normally benign strains, such as E. coli. A prime example of this approach was the recovery of DNA segments contributing to the invasive character of Yersinia by selecting for clones that could render an E. coli laboratory strain capable of eliciting its uptake by epithelial cells [43]. Similarly, the introduction of a plasmid containing the LEE island into a laboratory isolate of E. coli creates strains that produce attachment and effacing lesions in host cells [23]. It is not surprising that these three strategies have led to the recovery of somewhat different subsets of genes involved in pathogenesis. However, if virulence is to be characterized in terms of the consequences of bacterial infection on the health of the host, the classical bacterial genetics approach—whose aim is to identify all genes that affect host fitness—provides the most comprehensive definition of virulence genes.
8
HOWARD OCHMAN
Given this perspective on bacterial pathogenicity, many of the "virulence" genes required for propagation of pathogens within a host would be identical to those required in commensal or benign interactions with hosts. In fact, the molecular genetic experiments, which attempt to convert nonpathogenic E. coli into pathogens, suggest that E. coli, as normal constituents of the human intestinal flora, already contain genes necessary for interaction with animal cells and are, thus, predisposed to become pathogens on acquisition of a particular virulence gene cluster. In addition, many of the genes implicated in Salmonella virulence are also present in nonpathogenic strains of E. coli [9]. These genes encode enzymes responsible for the biosynthesis of nutrients that are scarce within host tissues, transcriptional and posttranscriptional regulatory factors, proteins necessary for the repair of damaged DNA, and products necessary for defense against host microbicidal mechanisms. The presence of these genes in nonpathogenic species suggests that they promote survival within the nutritionally deprived and/or potentially lethal environments that microbes encounter inside and outside animal hosts.
IV. Recovery of Genes Contributing to Virulence Although the identification and isolation of virulence genes largely depends on how these genes are defined, many pathogens require genes that are absent from related nonpathogenic bacteria. Therefore, several researchers have applied molecular and genetic techniques to recover segments of the genome that are specific to particular bacterial lineages. These procedures yield anonymous DNA fragments and have been typically employed to obtain diagnostic probes for the identification of particular bacterial strains or species [44-49]. However, in a few cases, these techniques have been exploited to find new pathogen-specific genes having a potential role in virulence. A subtractive hybridization procedure [50] was used to recover DNA sequences present in an avian pathogenic strain of Escherichia coli but absent from a nonpathogenic laboratory strain of Escherichia coli K-12 [51]. The pathogen-specific sequences recovered by this method mapped to at least 12 positions in the chromosome. Subsequently, the phenotype of mutant strains harboring deletions for each of these unique fragments was tested, and two were found to contain genes required for virulence in avian hosts. Other studies have examined the unique DNA in the genome of pathogens, but have yet to directly assess the function of these sequences. For example, a subtractive hybridization procedure used to examine the differences in gene content among strains of Helicobacter pylori yielded 18 clones, several of which were presumed to have a role in the
1.
THE EVOLUTION OF BACTERIAL PATHOGENS
9
specific virulence characteristics of H. pylori strains [52]. Although genome subtraction and physical mapping techniques allow one to identify and clone the differences between two genomes, there is usually no rapid way to determine if these pathogen-specific sequences are indeed relevant to pathogenesis. A thorough review of the strategies used to identify virulence determinants is presented in the chapter by Camilli et al. in this book (see Chapter 4).
V. The Population Genetics of Pattiogens What is the genetic structure of pathogen populations, and how much genetic variation is present in these populations compared with that in related nonpathogenic microorganisms? Moreover, what is the apportionment of genetic diversity among pathogenic strains in relation to that in species at large? Although there are several genetic mechanisms by which microorganisms change their pathogenic potential, it has become evident that the bacteria responsible for disease outbreaks are distinct clones that are frequendy characterized by unique combinations of virulence genes or of alleles at virulence genes. The situation is not as clear for pathogenic species, principally because the classification of a pathogenic "species" is somewhat arbitrary, and historically reflects the ability of epidemiologists to classify strains. On one hand, pathogens have been overclassified—that is, they are typed into a multitude of genetically narrow groups or species—compared to nonpathogens. This is probably judicious, given the importance of assigning an identity of each isolate implicated in human disease. For example, based on serological characteristics, the Salmonellae were assorted into thousands of distinct species (now termed serovars), but a recent taxonomic revision based on DNA-DNA hybridization reclassified these strains into a single species. Salmonella enterica [53, 54]. Similarly, the Shigellae have traditionally been subdivided into four species—Shigella boydii, S.flexneri, S. dysenteriae, and S. sonnei—although the total amount of genetic variation within this genus is contained within E. coli [55-58]. The classification of Shigellae based on serologic and metabolic characteristics illustrates two additional points. First, the amount of diversity can vary widely among species: while Shigella sonnei consists a single genetically uniform clone, each of the other three species comprises a heterogeneous array of clones. Second, the classification of strains does not always reflect their true genetic similarities or relationships. As shown in Figure 2, certain strains of S. flexneri can be more closely related to S. boydii than to any other strains typed S. flexneri, and evidence from other studies have shown that Shigella boydii and S. dysenteriae have multiple origins from within E. coli [55-58].
10
HOWARD OCHMAN
In contrast to these enteric pathogens, the classification of strains into other pathogenic species is more liberal and diffuse, due, in part, to the inability to rapidly differentiate among isolates that display a diagnostic phenotype or produce a specific pathology in hosts. However, the subsequent analysis of such species using additional genetic and/or phenotypic markers often reveals the true nature of the diversity and the relationships among strains.
VL Studying Bacterial Popuiation Genetics Studies addressing the genetic structure of bacterial populations began relatively recently [59, 60] by evolutionary geneticists who had originally examined the amount and distribution of genetic variation among natural populations of eukaryotes. Bacteria, particularly E. coli and Salmonella, were an attractive group of organisms on account of their phenotypic diversity, haploid chromosomes, large populations sizes, short generation times, and ease of propagation and experimental manipulation. Hence, bacterial population genetic research was originated by population geneticists interested in bacteria, rather than by bacterial geneticists or medical microbiologists interested in population genetics. The information gathered by population geneticists has broad implications for medicine and epidemiology, and these studies typically go beyond the simple identification of strains and address questions pertaining to their genetic relationships and their levels of allelic diversity [61, 62]. Numerous molecular techniques—such as RAPDs, IS (and other repetitive element) fingerprinting, ribotyping, phage typing, macrorestriction mapping by pulsed-field gel electrophoresis, and plasmid profile analysis—have been applied to establish the identity of strains for the epidemiological purposes. But these methods typically do not supply the information necessary to establish the relationships among strains, infer the genetic structure of natural populations, or assess the relative roles of natural selection, random drift (i.e., the change in gene frequencies caused by the chance event of random sampling in small populations), new mutations, and horizontal gene transfer (including intragenic and intergenic recombination) on the organization of allelic diversity [63]. Once the evolutionary relationships of clones of a species is available, it is possible to examine the manner in which the total genetic diversity is apportioned with respect to host species, geographic populations, and the specific disease pathology. The following methods have been applied to establish the evolutionary relationships and genetic structure of bacterial populations.
A.
Muitilocus Enzyme Electrophoresis
Multilocus locus enzyme electrophoresis (or MLEE) has been the primary method used to assess genetic variation in bacterial populations [64]. The main advantage
1.
11
THE EVOLUTION OF BACTERIAL PATHOGENS
of this technique is that many genes can be readily examined in hundreds, if not thousands, of isolates. However, this method rehes on the discrimination of alleles of distinct electrophoretic mobilities—also called allozymes or electromorphs in this context—and, therefore, can detect only a portion of the sequence variation at a locus (Fig. 3). The key concept underlying the use of MLEE in population genetics is that the electromorphs can be direcdy equated with alleles of the corresponding structural gene and that electromorph profiles over the sample of different enzymes (frequently termed electrophoretic types or ETs) correspond to multilocus chromosomal genotypes [65]. The proteins assayed by this method are usually metabolic enzymes, such as those involved in glycolysis, which are expressed in all isolates of strains, and the allelic variation is unaffected by environmental conditions, including host, culture medium, or laboratory storage. Moreover, the allelic variation detected at these enzyme loci is selectively neutral, or nearly so, such that there is minimal convergence to the same allele through adaptive evolution [66-68]. Hence, this technique provides a rapid way to index the overall levels of genetic diversity at numerous loci throughout the chromo-
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12
HOWARD OCHMAN
some and to infer genetic relationships among strains. Because the number of alleles at a locus is fairly large in bacterial populations, we expect that recombination would, by chance, only rarely generate strains of identical multilocus chromosomal genotypes. Therefore, strains of the same ET are considered to be similar by descent and shared ancestry. Over the past two decades, MLEE has become established as the standard procedure for assessing genetic variation in bacterial populations and the one against which the discriminatory power of all other techniques is measured.
B.
DNA Sequencing
Although MLEE offers a rapid and inexpensive way to appraise the genetic variation in bacterial populations, many laboratories have turned to directly sequencing the genes specifying several of the enzymes originally indexed by MLEE or the genes encoding proteins involved in virulence. In contrast to MLEE, nucleotide sequencing offers a means of uncovering all of the allelic variation at a locus and detecting events of intragenic recombination. Moreover, nucleotide sequences are composed of discrete characters—i.e., the four bases—as opposed to MLEE, which can only provide the relative mobilities of electromorphs. This allows for the unambiguous identification of alleles, and for comparison and portability of data, from different studies and laboratories. Furthermore, the use of PCR to generate sequencing templates permits the analysis of noncultivable organisms. Although nucleotide sequencing provides the most complete information about the genetic variation and relationships among strains, it is still relatively cosdy and time consuming, especially for the analysis of variation at several loci in a large number of strains. And in many applications, the level of variation detected by MLEE has been sufficient to answer all but the subdest questions about the genetic diversity and structure of natural populadons.
C.
Multilocus Sequence l o p i n g
Maiden et al [69] have devised a method for the identification and typing of bacterial clones based on the determination of sequences of several gene fragments (Fig. 4). Multilocus sequence typing (MLST) exploits the advantages of nucleodde sequence data, but also constructs chromosomal genotypes, which can be used to detect intergenic recombination in the manner of MLEE, through an analysis of multiple chromosomally encoded loci [70]. In the initial application of MLST, the nucleodde sequences of PCR-amplified fragments from 11 housekeeping genes were obtained for more than 100 isolates oi Neisseria meningitis. For MLST, the gene fragments (i.e., alleles) are 400 to 500 nucleoddes in length—a convenient size for the direct automated sequencing of a DNA fragment
I.
13
THE EVOLUTION OF BACTERIAL PATHOGENS
Chromosomal DNA
Amplify -450-bp fragments of several (7-10) house-keeping genes
Sequence gene fragments on both strands
Compare sequences of each gene fragment with the known alleles at the locus
Assign alleles at the loci to give the allelic profile
Compare the allelic profile with those of isolates within a central database via the internet
Fig. 4 Multilocus sequence typing (MLST). The method for allocation of the allelic profile, or sequence type (ST), of a bacterial isolate is shown. As in MLEE, the relationships among isolates can be visualized as a dendrogram, constructed from a matrix of pairwise differences between the allelic profiles of the isolates. Reprinted with permission from Spratt (1999) [70].
with a single primer—and each unique combination of alleles over loci is referred to as a sequence type (ST). As the number of sequencing facilities increase, and the costs of DNA sequencing fall, MSLT is certain to become the method of choice for assessing variation in bacterial populations.
VIL The Orgonization of Genetic Diversity in Pattiogenic Microorganisms Early studies on microbial pathogens, particularly enteropathogenic E. coli [71, 72], suggested that very few clones, as identified by serotyping, were associated with disease outbreaks. Subsequent analysis using MLEE provided the first indication that the species E. coli as a whole was clonal, as evident from the repeated recovery of strains of the same chromosomal genotypes from different
14
HOWARD OCHMAN
times and geographic locations [60,73]. Three generalizations have emerged from the broad-scale application of MLEE to the study of common human pathogens [61, 63]. First, most species of bacteria are highly polymorphic for electrophoretically detectable alleles at each enzyme locus, such that a typical locus may have 10 to 20 electromorphs. Second, despite harboring large amounts of genetic variability, most bacterial species are clonal and consist of a relatively small number of genotypes. This implies that rates of recombination between genetically distinct clones, which would serve to generate new combinations of alleles over loci, must be very low. Finally, in most pathogenic species, only a very small proportion of clones promotes most of the disease worldwide (which indicates large differences in virulence among strains). Furthermore, the same clones that cause disease can be recovered over long periods of time.
VIIL Population Genetics of Representative Bacterial Pattiogens Having considered the connections among the molecular, genetic, and evolutionary perspectives on bacterial virulence, we can now turn our attention to the population genetic analysis of specific pathogens. Most of the information summarized below is based on data obtained through the application of MLEE; however, for several organisms, we can also integrate information on the relationships and diversity among strains, as achieved through the analysis of DNA sequences. The species are discussed in alphabetical order (with the exception of Shigella, which, based on its proper taxonomic position, is included within E. coli), and the key characteristics of their genetic variation and population structure are summarized in Table I.
A.
Bordetella
Bacteria belonging to the genus Bordetella are of primary importance in pediatric and veterinary medicine because of their ability to colonize the epithelium of the respiratory tract of a variety of vertebrate hosts, there causing bronchial and pulmonary pathology (see Chapter 13 herein, by Cotter and Miller). Based on phenotypic characteristics, the genus Bordetella nominally consists of four species: B. pertussis, an obligate human pathogen causing whooping cough, B. parapertussis, which has been isolated from humans and sheep, B. bronchiseptica, which is the etiologic agent of canine kennel cough, and B. avium, which causes respiratory disease in fowl. These four species have been further subdivided on the basis of serology and biotyping; however, evidence from MLEE indicates that
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from an evolutionary genetic standpoint B. pertussis and human isolates of B. parapertussis should be considered clones of B. bronchiseptica that adapted to the human host relatively recendy [74]. Furthermore, many electrophoredc types (ETs) within each species are strongly host adapted. For example, ETs 22 through 26 of B. parapertussis are confined to sheep, while B. parapertussis FT 28 and the four ETs of B. pertussis are only recovered from humans [75]. Although laboratory experiments have shown the Bordetellae to be naturally competent and capable of recombinadon, their populadon structure is predominandy clonal, with several of the same ETs recovered from different localises and at different dmes. While there is no evidence of gene exchange between distandy related strains, recombinadon occurs among some of the more closely related strains with broader host ranges [75].
B.
Borrelia
The spirochete Borrelia burgdorferi is the causal agent of Lyme disease and is transmitted to humans and animals by the bite of infective Ixodes dcks [76]. In humans, the clinical features of Lyme disease progress from a rash and flu-like symptoms to arthritis, cardids, and neurologic disorders; this pathology was first described in Europe several decades ago. Based on genetic criteria, strains from Europe, Asia, and the United States that were originally classified as Borrelia burgdorferi (sensu lato) were reassorted into a complex of at least four highly divergent species—B. burgdorferi (sensu stricto), B. garinii, B, afzelii, and B. japonica—and there are likely to be additional species in other geographic regions [77-79]. Among the ETs defined by MLEE, there were highly nonrandom associations of alleles over the chromosome (i.e., linkage disequilibrium), indicating a clonal population structure. All isolates from the United States (including the type strain from Shelter Island, New York isolated in 1982) and many from west-central Europe formed a monophyletic clade of lineages descended from a single common ancestor, which is presendy considered B. burgdorferi sensu stricto [77]. In addition, comparisons of the branching orders of phylogenetic trees based on the DNA sequences of two chromosomally encoded genes (fla and p93) and a plasmid-bome gene (ospA) provide no evidence of lateral gene transfer—again indicating that Borrelia burgdorferi (sensu lato) is strictly clonal—and that plasmid transfer between clones is very rare [80]. Recent analysis of the two outer surface lipoproteins encoded by ospA and ospC within Borrelia burgdorferi (sensu stricto) revealed a strong association between ospA and ospC alleles [81], despite the fact that these genes are encoded on separate plasmids. The ospC gene is highly variable, and even in local populations there are large numbers and high frequencies of divergent alleles, as expected if the diversity is maintained through some form of selection acting to maintain
1.
THE EVOLUTION OF BACTERIAL PATHOGENS
17
variation in the population. Furthermore, the geographic distribution of opaC alleles confirmed that Borrelia burgdorferi (sensu stricto) originated in the United States and only recendy spread to Europe [81],
C. Escherichia coli and Shigella Although typically a benign consdtuent of the mammalian intestinal flora, E. coli is occasionally an infecdous agent responsible for several human diseases. Unlike many of the pathogenic microbes discussed so far, most information on the evoludonary and population genetics of E. coli was obtained from studies of ''wild" strains from healthy hosts. And for the past 20 years, the genedc structure and level of variadon observed in these commensal populations of E, coli have served as the basis for interpreting results from pathogenic and nonpathogenic bacteria alike [82]. Given that the organization of genetic diversity is known for natural populations of E. coli isolated from a wide range of sources [83], we can begin to ask questions about the emergence of pathogenic strains and the extent of genetic variation in pathogenic strains of E. coli. Strains of E. coli have been tradidonally typed on the basis of polymorphism in their O (somatic), K (capsular) and H (flagellar) antigens [72, 84]. Despite more than 1000 combinadons of antigens of the O, K, and H groups, certain serotypes were repeatedly associated with certain diseases, providing the first indication that E. coli populadons were clonal [85, 86]. Enteropathogenic E. coli (EPEC) (see Chapter 9 herein, by Puente and Finlay) were first idendfied from outbreaks of infantile diarrhea in Great Britain nearly 50 years ago [87]. The EPEC strains constitute very few serotypes, many of which are represented by a single temporally stable, and geographically widespread, clone [88]. Based on MLEE, the EPEC strains form two clonal complexes, each possessing the plasmid-borne adherence phenotype and distandy related to other pathogenic E. coli [89]. In the early 1980s, there were several outbreaks of hemorrhagic colids associated to the rare E. coli serotype 0157:H7, the so-called Jack-in-the-Box strain [90, 91]. These isolates are serologically distinct from the EPEC strains, and over the past decade there have been numerous outbreaks of hemorrhagic colitis and hemolytic uremic syndrome in North America and Asia linked to a variety of foodstuffs [92-94]. The virulence characteristics of enterohemorrhagic E. coli 0157:H7 derive from several sources: strains produce one or more forms of Shiga cytotoxin, contain a 92-kb virulence plasmid (plus a 3.3-kb plasmid in Japanese strains) and harbor a 43-kb pathogenicity island conferring the ability to evoke attachment and effacing (A/E) lesions [95-98]. MLEE was used to determine the reladonships of enterohemorrhagic E. coli 0157:H7 isolates to one another and to other strains causing enteric infections. A majority of 0157:H7 strains belong to a clone complex that is only distantly
18
HOWARD OCHMAN
related to other Shiga-toxin-producing strains of E. coli or to other ETs of the 0 1 5 7 group causing enteric infections in animals [99]. The 0157:H7 clone is most closely related to strains 0 5 5 :H7, also capable of producing A/E lesions. The most hkely hypothesis for the origin of 0157:H7 is that it emerged from an 055:H7 progenitor after acquiring the phage-encoding Shiga toxins and plasmid-encoded adhesins through horizontal transfer [89]. An alternative approach to studying the evolutionary genetics of virulence in E. coli has been to examine the phylogenetic distribution of pathogenic strains in relation to the species as a whole. To index the degree of genetic diversity and relationships among strains from natural populations of E. coli, most researchers have relied on the ECOR collection, which includes 72 strains originally selected to encompass the range of genetic variation in the entire species [100]. Based on MLEE [101] and on the nucleotide sequences of several genes [102], the phylogeny of ECOR strains consists of four major subgroups (A, B l , B2, and D), and the characterization of the ECOR collection by most other techniques has yielded essentially the same groupings [83, 103]. From such studies, it is clear that pathogenic strains do not form a phylogenetically distinct group, nor do they have a single evolutionary origin within E. coli [56, 104, 105]. In addition, strains of E. coli are predisposed to become human pathogens on the disruption of chromosomal genes and/or the acquisition of additional plasmids, phages, or pathogenicity islands. Yet, some generalizations can be made: commensal strains of E. coli are principally within subgroups A and B1 (and these strains typically lack virulence determinants), whereas many of the genes associated with pathogenic isolates of E. coli are prevalent among ECOR strains in subgroups B2 and D [106, 107]. Despite the tendency for pathogens to be related to commensal strains in certain subgroups and the phylogenetic clustering of certain virulence-associated genes, there are pathogenic isolates in each of the E. coli subgroups. In a comprehensive analysis of the relationships between nonpathogenic (as represented by ECOR) and a variety of pathogenic strains of E. coli, analysis by MLEE and mdh (malate dehydrogenase) sequences shows that enteropathogenic (EPEC), enterotoxigenic (ETEC), enteroinvasive (EIEC), and enterohemorrhagic (EHEC) strains were distributed among the ECOR subgroups [56]. Shigella is the etiologic agent of bacillary dysentery and has long been known to be closely related to E. coli (see Chapter 8 herein, by Sansonetti et al). Although classified into a separate genus, the four subspecies of Shigella constitute lineages that actually fall within the range of variation detected in E. coli [55-58]. Strains of Shigella sonnei form a single clone that has remained virtually unchanged for at least the past 40 years [108], but each of the other Shigella species have multiple independent origins from within E. coli (Fig. 2). Based on genetic criteria, the Shigellae should not be considered species separate from E. coli, and, like other agents of enteric disease, they appear to be secondarily derived from commensal strains of E. coli.
1.
D.
THE EVOLUTION OF BACTERIAL PATHOGENS
19
Haemophilus
Isolates of Haemophilus influenzae have traditionally been serotyped on the basis of six chemically distinct polysaccharide capsule types, but many isolates are unencapsulated and, hence, untypable (see Chapter 14 herein, by Tang et al). These unencapsulated forms are generally noninvasive, but encapsulated strains are invasive; and, in particular, those expressing serotype b are the major agents of meningitis in children. There were originally few useful epidemiological markers for serotype b strains of H. influenzae because most isolates were of identical biotype. However, the application of MLEE to Haemophilus [109-111] provided the first genetic framework for studying the population structure and relationships among strains and species. Like several other pathogens, the population structure of//, influenzae is clonal, with certain ETs distributed worldwide and persisting over long periods of time [109, 112]. Among the encapsulated strains (serotypes a-f), there were two major phylogenetic divisions. Strains producing a and b capsules occur within each of these divisions, most probably as a result of transfer of the serotype-specific capsule genes between lineages [113, 114]. Although serotype a strains are rarely virulent, some have been implicated in cases of septicemia and meningitis. These isolates harbor a "virulence-enhancing" mutation usually detected in type b strains, providing additional evidence of horizontal transfer [115]. Although it was originally thought that unencapsulated forms of //. influenzae would simply represent a subset of the encapsulated strains that recendy lost the polysaccharide capsule, these nontypeable strains belonged to distinct clusters and are, as a group, much more genetically diverse than clones expressing capsuletype b [74, 116, 117]. In general, there is little relationship between ET and particular disease conditions; however, the nonencapsulated strains isolated from healthy carriers were distinct from those causing various diseases [118].
E.
Helicobacter
Nearly half the human population is colonized by Helicobacter pylori, but clinical symptoms, ranging in type from peptic ulcer to gastric carcinoma and lymphoma, manifest in only a very small proportion of those infected ([119]; see Chapter 11 herein, by Cover et al). Although host and environmental factors—such as blood type, cigarette smoking, and gender—affect the risk of gastric ailments [120, 121], strains of //. pylori causing disease contain genetic determinants not found in avirulent strains [16, 122]. As a result, strains have been classified into two groups (type I and type II) that differ in the presence of the cytotoxin-associated gene cagA, which is encoded on a pathogenicity island, and of the vacuolating cytotoxin tox, whose phenotype is controlled by expression of the vacA gene
20
HOWARD OCHMAN
[123]. Type I strains, which represent 60% of all isolates examined, are cagA'^/tox^ and are associated with duodenal and gastric ulceration, while type II strains are cagA~/iox~ and are largely asymptomatic [124-126]. The simple classification of H. pylori as type I or type II does not impart the true extent of genetic diversity within the species as a whole, or the degree of genetic differentiation among strains. Most typing methods can discriminate among isolates from different individuals [127], and minor genetic changes, arising from rearrangements occurring within a lineage, have been detected in samples from a single host [128]. The mean genetic diversity of H. pylori, as assessed by MLEE, exceeded that detected for any other bacterial species examined by this technique [129]. Moreover, the index of association (Z^) value between loci [130] did not differ significantly from zero, implying free recombination within natural populations of H. pylori. Such high levels of genetic exchange obscure the clonal descent and genealogical relationships among strains, and can also impede the use of certain therapies against this pathogen. As a step in the control and treatment of H. pylori infections, the sequence diversity at several virulence-associated loci has been investigated, often in an attempt to identify the association between sequence variants and disease pathology [131-133]. All loci examined display unusually high levels of genetic variability, and in some cases unique alleles have been formed through intragenic recombination [132, 134-137]. In an analysis of the partial nucleotide sequences from two flagellins, flaA and flaB, and from vacA, it was noted that the vast majority of H. pylori strains had unique sequences at all three loci and that approximately 20% of the nucleotide positions were polymorphic in different strains, due principally to changes at synonymous sites [138]. This pattern of substitutions, characterized by high levels of divergence at silent sites but very low levels at nonsynonymous sites, suggests that these genes are under strong selection. Moreover, analysis of these data confirms that recombination is so frequent within H. pylori that the alleles at different loci, as well as the polymorphisms within loci, are effectively at linkage equilibrium [138].
F.
Listeria
Among the species of this genus, only Listeria monocytogenes is commonly pathogenic for humans, causing several invasive diseases including septicemia, meningitis and meningoencephalitis (see Chapter 16 herein, by Fsihi et ai). L monocytogenes have been isolated from environmental sources and a wide variety of raw and processed foodstuffs, particularly dairy products, which has led to the application of numerous techniques for the detection, identification, and epidemiological tracing of strains [139-145]. Based of MLEE, strains of L monocytogenes belong to two major clusters [146-148], and the existence of two primary lineages has been confirmed by ribotyping [149], restriction mapping [150], and DNA sequencing [151]. One cluster contained strains serotyped as l/2a
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THE EVOLUTION OF BACTERIAL PATHOGENS
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and l/2c, and the other included strains of serotypes l/2b, 3b, and 4b; and, except in a very few cases, each ET contained strains typed to only one serotype. Strains recovered from animals and humans were distributed into each of these phylogenetic groups, indicating that there is no genetic differentiation among strains infecting a variety of mammalian hosts. Subsequendy, on the basis of the DNA sequences of several genes, a third evolutionary lineage of L. monocytogenes has been defined [152, 153], and this group comprises strains recovered from animals, but not from humans. Several lines of evidence are consistent with the hypothesis that recombination of chromosomal genes is an infrequent event in natural populations of L monocytogenes. First, there is no overlap in the serotypes of isolates assigned to the two phylogenetic clusters. Second is the nonrandom association of alleles among ETs for many pairs of loci (i.e., linkage disequilibrium). Third is the finding that ETs are very stable, such that genetically indisdnguishable isolates have been recovered in widely separated geographic locations. Despite the large number of clones detected within L monocytogenes, clones typed as ETl have been implicated in disease outbreaks in California and Switzerland, suggesdng that this ET is either very abundant in the environment (or in foodstuffs), or highly pathogenic to humans [147].
G.
Mycobacterium
Despite a steady decline in the incidence of tuberculosis in the United States beginning in the 1950s, there has been an alarming increase in the number of individuals infected by Mycobacterium tuberculosis, and perhaps one-third of the world's population harbor this pathogen ([154]). In spite of this very large population size, the species as a whole displays almost no sequence variation, suggesting that all strains of M. tuberculosis shared a common ancestor an estimated 15,000 years ago [155]. Due to the close reladonships among strains, the epidemiological tracing of M. tuberculosis is typically based on RFLP analysis of a mobile genedc element, IS6J10, which is variable in its copy number and genomic location among strains [156, 157]. Analysis of more than two megabases of sequence data from a total of 26 loci has provided the first comprehensive view of the origin, ancestry, and genetic populadon structure of M. tuberculosis [158]. Unlike most bacterial species that gain antibiodc resistance genes through the acquisidon of plasmids or other elements, the vast majority (>95%) of sequence variation within this species is attributable to nonsynonymous substitutions or other mutations in chromosomal loci that confer resistance to antibiotics. Despite very low levels of neutral sequence variation—in fact, the M. tuberculosis species has the lowest level of nucleotide diversity of any bacterial pathogen—strains could be assigned to three genotypic groups based on combinations of polymorphisms at two sites: codon
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HOWARD OCHMAN
463 in katG, and codon 95 in gyrA. (These two sites are not involved in antibiotic resistance and, hence, serve as useful genetic markers to examine the evolutionary history and relationships of strains.) All isolates of the predominandy nonhuman pathogens, M microti and M. bovis, and the human pathogen, M. africanum, have the same combination of polymorphisms characteristic of M. tuberculosis genotypic group 1, suggesdng that this genotypic group is ancestral to groups 2 and 3. Because the most ancient group is expected to contain the most variadon, this view of the relationships among the genotypic groups is further supported by the fact the genotypic group 1 contains the most variation in IS6100 copy numbers and in the nucleodde sequences at other loci [158]. Despite the low rate amount of nucleotide sequence diversity, the three genotypic groups of M. tuberculosis have diverged with respect to IS6110 copy numbers. \S6110 profiles can change reladvely rapidly, and in many cases isolates resampled after 90 days from the same patients displayed changes in IS6100 genotype, particularly among strains with greater numbers of these IS elements. And due to reladvely minor changes in the \S6110 fingerprint patterns of strains from single individuals, this variation could not have been produced by reinfecdon by a different strain [159].
H.
Neisseria
Due to the widespread and recurrent epidemics of meningococcal disease, there has been extensive work on the population biology of Neisseria meningitidis (see [160-163] for comprehensive reviews and Chapter 12 herein, by Meyer et al). In general, the variation among strains of Neisseria is generated through horizontal exchange, but epidemics are often caused by the spread of specific genedc variants, which results in clonal replacement. After their descent for a common ancestor, strains rapidly diversify through mutation and recombination [162]. A^. meningitidis are conventionally typed on the basis of capsular polysaccharides, and serogroups A, B, and C account for more than 90% of the cases of meningococcal disease worldwide ([164]). In an early study, it was demonstrated that the European epidemic of serogroup B disease that began in the 1970s was caused by a group of 22 very closely related clones, designated as the ET-5 complex, that have no close genedc relationship to other groups of clones [165]. Clones of the ET-5 complex were also the causative agents of later outbreaks in Africa, South America, Cuba, and the United States (where it was likely to have been introduced by Cuban immigrants). N. meningitidis is carried asymptomadcally in the upper respiratory tract by about 15% of the human population, and the clones isolated from carriers are only rarely represented among those causing disease, showing that certain complexes of clones have a low virulence potendal [166].
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Unlike most other serogroups of N. meningitidis, which are generally associated with endemic disease, isolates of serogroup A are unusual in that they may cause very large epidemics. Among the serogroup A organisms responsible for 23 epidemics between 1915 and 1983, there were 34 distinctive ETs constituting four clone complexes, each representing a group of related clones [167]. Most epidemics were caused by a single clone, and the same clone was responsible for concurrent epidemics in different countries. Clonal analysis has also demonstrated that serogroup A isolates are a restricted phylogenetic subpopulation of the species [168], which probably arose no more than a few hundred years ago [169]. Although recombination produces genetic variation within clone complexes, these results would indicate that only limited amounts of genetic exchange occur between phylogenetically unrelated strains of A^. meningitidis. Sequence analysis of meningococcal genes provides clear evidence that the evolution of N. meningitidis has been characterized by high levels of intra- and intergenic recombination, which is perhaps not surprising for a naturally transformable species. For example, allelic variants of the gene encoding adenylate kinase (adk), whose variation is regularly assayed by MLEE, have a mosaic structure produced by recombination between genes from different strains [170]. Moreover, within and among species of Neisseria, patterns of sequence divergence for adk, recA, aroE (shikimate dehydrogenase), and glnA (glutamine synthetase) are very different [171, 172], and the phylogenetic trees based on each of these genes are not congruent, as expected for species that undergo frequent recombination. Note that even in recombining species, such as A^. meningitidis, the epidemic increase of certain ETs can give the appearance of a clonal population structure despite high levels of gene exchange [130, 173].
I.
Salmonella
Under the original serotyping schemes of White [174] and Kauffman [175], the Salmonellae were assigned to nearly 3000 serotypes or serovars, with each considered a distinct species. However, based on the biotyping and molecular genetic evidence, all strains were subsequently typed as single species, S. enterica, which comprises eight subspecific groups (designated I, II, Ilia, Illb, IV, V, VI, and VII) [176, 177]. Hence, the nomenclature has changed such that Salmonella typhimurium would now be referred to as Salmonella enterica serovar Typhimurium or, simply, Typhimurium. Over 60% of the serotypes belong to subspecies I, including those strains causing >99% of the cases of human salmonellosis (see Chapter 7 herein, by Scherer and Miller). Due to its genetic relationships to the other subspecific groups, subspecies V has recently been reclassified as a separate species. Salmonella bongori [54], and MLEE as well as nucleotide sequence analysis of several genes have confirmed its divergent phylogenetic position [178, 179, 63, 180-182].
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MLEE was originally used to investigate the allelic variation in genes in large samples of Salmonella [183-189]. The total genetic diversity in Salmonella, as assessed by MLEE, is among the highest reported for any bacterial species (Table I)—nearly twice that observed in E. coli. Clonal aspects of the genetic structure of S. enterica are well illustrated by the serovars Typhi and Paratyphi A, B, and C, all of which are agents of enteric fever in humans. MLEE analysis has demonstrated that there are no close relationships among these serovars, implying independent evolutionary derivation [187, 188]. Typhi is an unusually distinctive and homogenous serovar, and over 80% of the worldwide isolates are of a single ET (with a second ET comprising 16% of strains, all from West Africa). Paratyphi B consists of a large and heterogenous group of lineages that are closely related to Typhimurium. However, the ability of Paratyphi B to cause human enteric fever arose in a single globally distributed clone, and only recendy, since it is only weakly differentiated. The genetic variation and relationships among strains have also been assessed by the nucleotide sequencing of several housekeeping genes, including proline permease (putP) [178], glyceraldehyde-3-phosphate dehydrogenase (gapA) [190], malate dehydrogenase (mdh) [180], 6-phosphogluconate dehydrogenase (gnd) [179], isocitrate dehydrogenase (icd) [191], and isocitrate dehydrogenase kinase/phosphatase (aceK) [192]. For these five housekeeping genes, on average, about 16% of nucleotides and 5% of amino acids are polymorphic. With the exception of gnd, the level of sequence diversity is greater in S. enterica than in E. coli, which is attributable to the unusually high divergence of subspecies V {S. bongori) from the other subspecies. Comparisons of the individual trees based on the nucleotide sequences have revealed several cases in which the branching orders of lineages are not congruent. These disparities are due both to intragenic recombination events, which can involve regions ranging from six basepairs to more than 1 kb, and to the exchange of entire genes [63]. Notwithstanding low levels of recombination at some loci, the relationships among strains based on nucleotide sequences matched those established by DNA hybridization and MLEE. Based on these phylogenetic relationships, serovars that are exclusively or predominantly diphasic (subspecies I, II, Illb, and VI) cluster apart from the monophasic subspecies (Ilia, IV, V, and VII). This suggests that, following the divergence of S. enterica and E. coli from a common ancestor, E. coli evolved as an commensal of mammals while Salmonella remained associated with reptiles, which are still the primary hosts of the monophasic subspecies. Salmonella serovars are typically classified as either monophasic or diphasic based on their ability to produce one or two forms of flagellin. Subsequently, Salmonella evolved as an intracellular pathogen through the acquisition of several pathogenicity islands, which conferred the ability to invade host epithelial cells and circumvent host defenses. The diphasic condition originated in the lineage ancestral to subspecies I, II, Illb, and VI is a mechanism to further evade the host
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THE EVOLUTION OF BACTERIAL PATHOGENS
25
immune system and is likely to have assisted in the exploitation of birds and mammals of potential hosts [181]. This scenario is supported by the phylogenetic distribution of pathogenicity islands in Salmonella [193]. The SPI-1 island, which confers the ability to invade nonphagocytic host cells, was acquired very early in the evolution of Salmonella and is present in all subspecies, whereas the SPI-2 island, which is necessary for intracellular proliferation, is absent from Salmonella bongori, which were originally recovered from nonmammalian hosts. This suggests that the evolution of Salmonella as a pathogen has been marked by the acquisition and/or generation of several genes that facilitate interactions with the host [9, 194].
J.
Staphylococcus
The are two notable cases where MLEE has been applied to uncover the evolutionary history of infective strains of Staphylococcus aureus. The first involves strains of S. aureus causing toxic shock syndrome (TSS) in young, healthy menstruating women. Almost all strains of S. aureus recovered from TSS patients express a toxin (designated TSST-1) [195, 196], which is now known to be encoded as part of a 15-kb pathogenicity island present only in TSST-1 -positive strains [17]. The analysis of genetic variation in 315 isolates of S. aureus expressing TSST-1 revealed that toxin production occurs in association with chromosomal backgrounds representing the full breadth of genotypic diversity in the species as a whole, as might be expected for genes encoded on a mobile element [197]. But despite the diversity among strains expressing TSST-1, a single distinctive clone causes the majority of cases of toxic shock syndrome. It is not known whether the present-day distribution of the TSST-1 gene in S. aureus reflects an evolutionarily old association or the independent acquisition of the TSST-1 pathogenicity island by multiple strains. However, these results suggest that the particular clone causing TSS has properties conferring strong affinity for human cervicovaginal surfaces [197]. A second case where MLEE has provided insights into the nature of staphylococcal infections involved strains of S. aureus resistant to the antimicrobial agent methicillin. Soon after methicillin entered clinical use, there were several hospital outbreaks caused by methicillin-resistant S. aureus (MRSA), and these organisms have now achieved global distribution [198]. Methicillin resistance is conferred by the expression of a modified penicillin-binding protein encoded by the mec gene. MLEE was employed to determine the extent of genetic diversity of MRSA strains and the relationships among strains from temporally and geographically separated outbreaks [199, 200]. The mec gene is harbored by many divergent lineages of S. aureus and has spread through multiple episodes of horizontal transfer. Many of the MRSAs recovered from Europe and Africa soon after methicillin was introduced into clinical use in the 1960s were identified as the
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same ET, suggesting that most early outbreaks were caused by dissemination of a single clone that had acquired the mec gene. However, the association of mec with genetically divergent strains contradicts the idea that all of the extant MRS As descended from a single methicillin-resistant clone [201].
K.
Streptococcus
A study on the diversity and population structure of Streptococcus pyogenes (Group A Streptococcus) was originally undertaken to determine the relationships among strains causing toxic-shock-like syndrome and other invasive diseases [202] (see Chapter 15 herein, by Caparon). Of the 108 strains analyzed by MLEE, there were 33 ETs; but nearly half of disease episodes (including more than 70% of the cases of toxic-shock-like syndrome) were caused by two related clones, designated ETl and ET2. The genetic structure of Streptococcus pyogenes has also been characterized with respect to the nucleotide sequence variation in several genes, including those encoding the scarlet fever exotoxin {speA) [202, 203], serotype M protein (emm) [204, 205] and complement inhibitor (sic) [206], streptokinase (ska) [205], pyrogenic enterotoxin B (speB) [205], C5a peptidase (scp) [205], superantigen SSA(ssa) [207], and hyaluronidase (hyl) [208]. In many cases, horizontal transfer and genetic exchange have contributed to the allelic diversity at these loci, and the phylogenies of strains produced from sequences of individual genes often do not match those based on MLEE. The level of polymorphism in the sic gene in M1 strains of Streptococcus greatly exceeds that observed at any other locus in this species. Moreover, virtually all nucleotide substitutions alter the amino acid sequence of the encoded protein, and all insertions and deletion are in frame [206]. Because the Sic protein functions to inhibit complement, this variability has been ascribed to selection acting to adapt this protein to host factors. Although it has long been known that certain genes of Streptococcus pneumoniae are subject to horizontal transfer and recombinational exchange [30, 209, 211], the population structure and genetic variation of this species has only recendy been assessed. Using MLST, the sequence diversity of strains of S. pneumoniae associated with disease was assessed by examining ~450-bp portions of seven housekeeping genes in nearly 300 isolates [212]. Despite relatively low levels of sequence variation, the numbers of alleles per locus ranged from 18 in aroE to 34 in xpt. Among the 143 STs, 34 contained more than one isolate and 12 included at least five invasive isolates. In 26 of the 34 STs containing multiple isolates, there was a perfect congruence between ST and serotype; however, strains of the same serotype could differ by as many as six of seven loci [212]. Moreover, there was evidence of recombination among loci, and the repeated recovery of identical isolates causing invasive disease in geographically distant regions suggests that certain STs define strains with increased capacity to cause disease.
1.
L.
THE EVOLUTION OF BACTERIAL PATHOGENS
27
Vibrio
Vibrio cholerae is a natural inhabitant of aquatic environments; however, some clones can cause severe diarrheal disease in humans (see Chapter 10 herein, by DiRita). Seven pandemics of cholera have been recorded since 1817, with the sixth pandemic subsiding in 1925 and the seventh beginning in Indonesia in 1961. Although V. cholerae, the causative agent of cholera, has been subdivided into nearly 200 serovars, the number of pathogenic serotypes is small, and all recorded pandemic and epidemic cases have been associated with the 01 serotype on a worldwide scale. Non-01 strains are more frequently isolated from environmental sources but have been implicated in cases of gastroenteritis, septicemia, and meningitis in humans. An outbreak of cholera in India that began in 1991 was caused by an 0139 strain. Toxigenic isolates of V. cholerae are also separated into Classical and El Tor types, which are differentiated on the basis of polymixin sensitivity, hemolysis and hemagglutination activity, phage resistance patterns, and the Voges-Proskauer reaction. The first six pandemics were thought to have originated from Classical strains, and the seventh from an El Tor strain. Based on MLEE, the seventh-pandemic clone is relatively homogenous worldwide. A large group of strains isolated over 30 years from several countries affected by the seventh pandemic constitute a single ET (ET3) [213-215], with isolates from Australia (ETl), the U.S. Gulf Coast (ET2), and Latin America (ET4) each representing a unique type. Among more recently isolated samples of V. cholerae from Latin America, ET4 was still prevalent, but about 10% of strains were of ET3, showing that the recent outbreaks of cholera in this region have originated from introduction of new toxigenic 01 strains. All seventh-pandemic clones have the same sequences for the asd (aspartatesemialdehyde dehydrogenase) gene and the ctxB (toxin) gene [216, 217], suggesting a single origin of this clone. The toxigenic 0139 strain is genetically closely related to the seventh-pandemic strains and presumably evolved from an early seventh-pandemic isolate [218]. But based on ribotyping, it was concluded that several clones, rather than one, caused the seventh pandemic, with one group of representing clones arising in 1961 and found only in Asia, and the other arising in 1966 and spreading worldwide [219]. However, the frequency of genetic exchange between rrn operons is high in seventh-pandemic clones—nine new ribotypes have been detected among 47 isolates sampled over a 33-year period [220]—and this degree of recombination could obscure the relationships among clones that are identified by this method. The original sequencing of asd in isolates of V. cholerae detected three homogeneous, but distantly related, clones representing the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones [218]. The subsequent sequencing of other loci [221] revealed virtually no variation in either the mdh (malate dehydrogenase) and hlyA (hemolysin) genes among sixth-pandemic, seventh-pandemic. Gulf Coast, and 0139 isolates, and these sequences were distinct from
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environmental, nontoxigenic non-Ol strains. The frequent occurrence of identical gene sequences among pathogenic isolates suggests that these clones are indeed closely related, and that the variation observed at other loci, and in the nontoxigenic isolates, has been generated, in part, by recombination.
IX. Conclusions Infectious diseases caused by microbial pathogens—ranging from plague to tuberculosis—are among the world's leading causes of death. And while investigations into the molecular bases of virulence are underway for numerous bacterial pathogens, all but the most rudimentary information pertaining to the extent and organization of genetic variation, the factors contributing to allelic diversity, and the genetic structure of their populations is available for the majority of these microorganisms. Aside from identifying the forces shaping the extent and organization of genetic diversity in those microorganisms, these findings are also applicable to the choice of procedures used for identification, epidemiology, and control of bacterial pathogens. The identification and classification of a pathogenic species could potentially influence analysis of its genetic diversity and population structure (e.g., if serovars of Salmonella were each considered a separate species, as originally determined on the basis of serotyping, the amount of variation in each would be very different than that presently detected in populations of Salmonella enterica). In addition, the wide variety of mechanisms that generate differences in pathogenic potential among related bacteria will affect the origin of virulence and, hence, the apportionment of genetic diversity in natural populations. Despite these factors, several generalizations have emerged from the population genetic analysis of bacteria. Foremost is that most species of bacteria are highly variable but clonal and consist of relatively small numbers of genotypes. Moreover, only a small proportion of clones cause most of the disease worldwide, implying that there are large differences in virulence among strains. Among the most productive approaches for future research will be the analysis of variation in virulence-associated loci within populations of pathogens since these genes are likely to respond to selective pressures presented by the host. Not only do such studies provide information about the genetic structure of the populations and the factors that contribute to genetic diversity, but also they can identify polymorphisms associated with particular clinical symptoms or disease pathologies. Such analyses will be essential for understanding the endemic and epidemic spread of pathogenic organisms, and for the development of vaccines to abate their effects on human populations.
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141. Baloga, A. O., and Harlander, S. K. (1991). Comparison of methods for discrimination between strains of Listeria monocytogenes from epidemiological surveys. Appl. Environ. Microbiol. 57, 2324-2331. 142. Norrung, B., and Skovgaard, N. (1993). Application of multilocus enzyme electrophoresis in studies of the epidemiology of Listeria monocytogenes in Denmark. Apj?!. Environ. Microbiol. 59,2817-2822. 143. Avery, S. M., Hudson, J. A., and Buncic. S. (1996). Multilocus enzyme electrophoresis typing of New Zealand Listeria monocytogenes isolates. Int. J. Food Microbiol. 28, 351-359. 144. Caugant, D. A., Ashton, F. E., Bibb, W. P., Boerlin, P., Donachie, W., Low, C, Gilmour, A., Harvey, J., and Norrung, B. (1996). Multilocus enzyme electrophoresis for characterization of Listeria monocytogenes isolates: Results of an international comparative study. Int. J. Food Microbiol. 32, 301-311. 145. Louie, M., Jayaratne, P., Luchsinger, I., Devenish, J., Yao. J., Schlech, W., and Simor, A. (1996). Comparison of ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis for molecular typing of Listeria monocytogenes. J. Clin. Microbiol. 34, 15-19. 146. Bibb, W. F, Schwartz, B., Gellin, B. G., Plikaytis, B. D., and Weaver, R. E. (1989). Analysis of Listeria monocytogenes by multilocus enzyme electrophoresis and application of the method to epidemiologic investigations. Int. J. Food Microbiol. 8, 233-239. 147. Piffaretti, J. C , Kressebuch, H., Aeschbacher, M.. Bille, J., Bannerman, E., Musser, J. M., Selander, R. K., and Rocourt, J. (1989). Genetic characterization of clones of the bacterium Listeria monocytogenes causing epidemic disease. Proc. Natl. Acad. Sci. U.S.A. 86, 3818-3822. 148. Harvey, J., and Gilmour, A. (1994). Application of multilocus enzyme electrophoresis and restriction fragment length polymorphism analysis to the typing of Listeria monocytogenes strains isolated from raw milk, nondairy foods, and clinical and veterinary sources. Appl. Environ. Microbiol. 60, 1547-1553. 149. Graves, L. M., Swaminathan, B., Reeves. M. W.. Hunter, S. B.. Weaver, R. E., Plikaytis, B. D., and Schuchat, A. (1994). Comparison of ribotyping and multilocus enzyme electrophoresis for subtyping of Listeria monocytogenes isolates. J. Clin. Microbiol. 32, 2936-2943. 150. Vines, A., Reeves, M. W., Hunter, S., and Swaminathan, B. (1992). Restriction fragment length polymorphism in four virulence-associated genes of Listeria monocytogenes. Res. Microbiol. 143, 281-294. 151. Rasmussen, O. F., Beck, T., Olsen, J. E.. Dons, L., and Rossen, L. (1991). Listeria monocytogenes isolates can be classified into two major types according to the sequence of the listeriolysin gene. Infect. Immun. 59, 3945-3951. 152. Rasmussen, O. E, Skouboe, P., Dons. L.. Rossen, L.. and Olsen, J. E. (1995). Listeria monocytogenes exists in at least three evolutionary lines: Evidence from flagellin, invasive associated protein and listeriolysin O genes. Microbiology 141, 2053-2061. 153. Wiedmann, M., Bruce, J. L., Keating, C , Johnson, A. E., McDonough, P. L., and Batt, C. A. (1997). Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65, 2707-2716. 154. Bloom, B. R., and Murray, C. J. L. (1992). Tuberculosis: Commentary on a reemergent killer. Science 251, 1055-1064. 155. Kapur, V., Whittam, T. S., and Musser, J. M. (1994). Is Mycobacterium tuberculosis 15,000 years old?y. Infect. Dis. 170, 1348-1349. 156. Cave, M. D., Eisenach, K. D., McDermott, P E, Bates, J. H., and Crawford, J. T. (1991). IS6110: Conservation of sequence in the Mycobacterium tuberculosis complex and its utilization in DNA fingerprinting. Mol. Cell. Probes 5, 73-80.
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157. Yang, Z., Barnes, P. F, Chaves, R, Eisenach, K. D., Weis, S. E., Bates, J. H., and Cave, M. D. (1998). Diversity of DNA fingerprints of Mycobacterium tuberculosis isolates in the United States, y. Clin. Microbiol. 36, 1003-1007. 158. Sreevatsan, S., Pan, X., Stockbauer, K. E., Connell, N. D., Kreiswirth, B. N., Whittam, T. S., and Musser, J. M. (1997). Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionary recent global dissemination. Proc. Natl. Acad. Sci. U.S.A. 94, 9869-9874. 159. Yeh, R. W., Ponce de Leon, A., Agasino, C. B., Hahn, J. A., Daley, C. L., Hopewell, R C , and Small, P. M. (1998). Stability of Mycobacterium tuberculosis DNA genotypes. J. Infect. Dis. 177,1107-1111. 160. Achtman, M. (1995). Epidemic spread and antigenic variability of Neisseria meningitidis. Trends Microbiol. 3, 186-192. 161. Achtman, M. (1997). Microevolution and epidemic spread of serogroup A Neisseria meningitidis—a review. Gene 192, 135-140. 162. Achtman, M. (1998). Microevolution and epidemic spread of Neisseria meningitidis. Electrophoresis 19, 593-596. 163. Caugant, D. A. (1998). Population genetics and molecular epidemiology of Neisseria meningitidis. Apmis 106, 505-525. 164. Peltola, H. (1983). Meningococcal disease: Still with us. Rev. Infect. Dis. 5, 71-91. 165. Caugant, D. A., Froholm, L. O., Bovre, K., Holten, E., Frasch, C. E., Mocca, L. P., Zollinger, W. D., and Selander, R. K. (1986). Intercontinental spread of a genetically distinctive complex of clones of Neisseria meningitidis causing epidemic disease. Proc. Nat. Acad. Sci. U.S.A. 83, 4927^931. 166. Caugant, D. A., Kristiansen, B. E., Froholm, L. O., Bovre, K., and Selander, R. K. (1988). Clonal diversity of Neisseria meningitidis from a population of asymptomatic carriers. Infect. Immun. 56, 2060-2068. 167. Olyhoek, T., Crowe, B. A., and Achtman, M. (1987). Clonal population structure of Neisseria meningitidis serogroup A isolated from epidemics and pandemics between 1915 and 1983. Rev. Infect. Dis. 9, 665-692. 168. Caugant, D. A., Mocca, L. F, Frasch, C. E., Froholm, L. O., Zollinger, W. D., and Selander, R. K. (1987). Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern. J. Bacteriol. 169, 2781-2792. 169. Morelli, G., Malomy, B., Muller, K., Seller, A., Wang, J. F, del Valle, J., and Achtman, M. (1997). Clonal descent and microevolution of Neisseria meningitidis during 30 years of epidemic spread. Mol. Microbiol. 25, 1047-1064. 170. Feil, E., Carpenter, G., and Spratt, B. G. (1995). Electrophoretic variation in adenylate kinase of Neisseria meningitidis is due to inter- and intraspecies recombination. Proc. Natl. Acad. Sci. U.S.A. 92, 10535-10539. 171. Feil, E., Zhou, J., Maynard Smith, J., and Spratt, B. G. (1996). A comparison of the nucleotide sequences of the adk and recA genes of pathogenic and commensal Neisseria species: Evidence for extensive interspecies recombination within adk. J. Mol. Evol. 43, 631-640. 172. Zhou, J., Bowler, L. D., and Spratt, B. G. (1997). Interspecies recombination, and phylogenetic distortions, within the glutamine synthetase and shikimate dehydrogenase genes of Neisseria meningitidis and commensal Neisseria species. Mol. Microbiol. 23, 799-812. 173. Maynard-Smith, J., and Smith, N. H. (1998). Detecting recombination from gene trees. Mol. Biol. Evol. 15, 590-599. 174. White, P. B. (1926). Further studies on the Salmonella group. Med. Res. Spec. Rep. Counc. (Great Brittain) Sen 103, 3-160.
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192. Nelson, K., Wang, F. S., Boyd, E. R, and Selander, R. K. (1997). Size and sequence polymorphism in isocitrate dehydrogenase kinase/phosphatase gene (acek) and flanking regions in Salmonella enterica and Escherichia coli. Genetics 147, 1509-1520. 193. Ochman, H., and Groisman, E. A. (1996). Distribution of pathogenicity islands in Salmonella spp. Infect. Immun. 64, 5410-5412. 194. Baumler, A. J., Tsolis, R. M., Ficht, T. A., and Adams, L. G. (1998). Evolution of host adaptation in Salmonella enterica. Infect. Immun. 66, 4579-4587. 195. Bergdoll, M. S., Crass, B. A., Reiser, R. R, Robbins, R. N., and Davis, J. R (1981). A new staphylococcal enterotoxin, enterotoxin F, associated with toxic-shock-syndrome Staphylococcus aureus isolates. Lancet 1, 1017-1021. 196. Schlievert, R M., Shands, K. N., Dan, B. B., Schmid, G. R, and Nishimura, R. D. (1981). Identification and characterization of an exotoxin from Staphylococcus aureus associated with toxic-shock syndrome. J. Infect. Dis. 143, 509-516. 197. Musser, J. M., Schlievert, R M., Chow, A. W., Ewan, R, Kreiswirth, B. N., Rosdahl, V. T., Naidu, A. S., Witte, W., and Selander, R. K. (1990). A single clone of Staphylococcus aureus causes the majority of cases of toxic shock syndrome. Proc. Natl. Acad. Sci. U.S.A. 87, 225-229. 198. Grubb, W. B. (1990). Molecular epidemiology of methicillin-resistant Staphylococcus aureus. In "Molecular Biology of the Staphylococci" (R. Novick and R. A. Skurray, eds.), pp. 595-606. VCH, New York. 199. Musser, J. M., and Selander, R. K. (1990). Brazilian purpuric fever: Evolutionary genetic relationships of the case clone of Haemophilus influenzae biogroup aegyptius to encapsulated strains of Haemophilus influenzae. J. Infect. Dis. 161, 130-133. 200. Musser, J. M., and Kapur, V. (1992). Clonal analysis of methicillin-resistant Staphylococcus aureus strains from intercontinental sources: Association of the mec gene with divergent phylogenetic lineages implies dissemination by horizontal transfer and recombination. / Clin. Microbiol. 30, 2058-2063. 201. Kreiswirth, B., Komblum, J., Arbeit, R. D., Eisner, W., Maslow, J. N., McGeer, A., Low, D. E., and Novick, R. P. (1993). Evidence for a clonal origin of methicillin resistance in Staphylococcus aureus. Science 259, 227-230. 202. Musser, J. M., Hauser, A. R., Kim, M. H., Schliever, R M., Nelson, K., and Selander, R. K. (1991). Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: Clonal diversity and pyrogenic exotoxin expression. Proc. Natl. Acad. Sci. U.S.A. 88, 26682672. 203. Nelson, K., Schlievert, R M., Selander, R. K., and Musser, J. M. (1991). Characterization and clonal distribution of four alleles of the speA gene encoding pyrogenic exotoxin A (scarlet fever toxin) in Streptococcus pyogenes. J. Exp. Med. 174, 1271-1274. 204. Whatmore, A. M., Kapur, V., Sullivan, D. J., Musser, J. M., and Kehoe, M. A. (1994). Non-congruent relationships between variation in emm gene sequences and the population genetic structure of group A streptococci. Molec. Microbiol. 14, 619-631. 205. Musser, J. M., Kapur, V., Szeto, J., Pan, X., Swanson, D. S., and Martin, D. R. (1995). Genetic diversity and relationships among Streptococcus pyogenes strains expressing serotype Ml protein: Recent intercontinental spread of a subclone causing episodes of invasive disease. Infect. Immun. 63, 994-1003. 206. Stockbauer, K. E., Grigsby, D., Pan, X., Fu, Y.-X., Perea Mejia, L. M., Cravioto, A., and Musser, J. M. (1998). Hypervariability generated by natural selection in an extracellular complementinhibiting protein of serotype Ml strains of gvou^ \ Streptococcus. Proc. Natl. Acad. Sci. U.S.A. 95,3128-3133.
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207. Reda, K. B., Kapur, V., Goela, D., Lamphear, J. G., Musser, J. M., and Rich, R. R. (1996). Phylogenetic distribution of streptococcal superantigen SSA allelic variants provides evidence for horizontal transfer of ssa within Streptococcus pyogenes. Infect. Immun. 64, 1161-1165. 208. Marciel, A. M., Kapur, V., and Musser, J. M. (1997). Molecular population genetic analysis of a Streptococcus pyogenes bacteriophage-encoded hyaluronidase gene: Recombination contributes to allelic variation. Microb. Pathogen. 22. 209-217. 209. Dowson, C. G., Hutchison, A., Brannigan, J. A., George, R. C , Hansman, D., Linares, J., Tomasz, A., Maynard Smith, J., and Spratt, B. G. (1989). Horizontal transfer of penicillin-binding protein genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 86, 8842-8846. 210. Dowson, C. G., Hutchison, A., Woodford, N., Johnson, A. R, George, R. C., and Spratt, B. G. (1990). Penicillin-resistant viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains of Streptococcus pneumoniae. Proc. Natl. Acad. Sci. U.S.A. 87, 5858-5862. 211. Coffey, T. J., Enright, M. C., Daniels, M., Morona, J. K., Morona, R., Hryniewicz, W., Paton, J. C., and Spratt, B. G. (1998). Recombinational exchanges at the capsular polysaccharide biosynthetic locus lead to frequent serotype changes among natural isolates of Streptococcus pneumoniae. Molec. Microbiol. 27, 73-83. 212. Enright, M. C., and Spratt, B. G. (1998). Amultilocus sequence typing scheme for Streptococcus pneumoniae: Identification of clones associated with serious invasive disease. Microbiology 144, 3049-3060. 213. Wachsmuth, I. K., Evins, G. M., Fields, R I., Olsvik, O., Popovic, T., Bopp, C. A., Wells, J. G., Carrillo, C, and Blake, P. A. (1993). The molecular epidemiology of cholera in Latin America. y. Infect. Dis. 161, 621-626. 214. Evins, G. M., Cameron, D. N., Wells, J. G., Greene, K. D., Popovic, T, Giono-Cerezo, S., Wachsmuth, L K., and Tauxe, R. V. (1995). The emerging diversity of the electrophoretic types of Vibrio cholerae in the Western Hemisphere. J. Infect. Dis. 172, 173-179. 215. Beltran, P, Delgado, G., Navarro, A., Trujillo, F., Selander, R. K., and Cravioto, A. (1999). Genetic diversity and population structure of Vibrio cholerae. J. Clin. Microbiol. 37, 581-590. 216. Olsvik, O., Wahlberg, J., Petterson, B., Uhlen, M., Popovic, T, Wachsmuth, L K., and Fields, P. L (1993). Use of automated sequencing of polymerase chain reaction-generated amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae 01 strains. J. Clin. Microbiol. 31, 22-25. 217. KaraoHs, D. K., Lan, R., and Reeves, P. R. (1995). The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-01 Vibrio cholerae. J. Bacteriol. Ill, 3191-3198. 218. Karaolis, D. K., Lan, R., and Reeves, P. R. (1994). Molecular evolution of the seventh-pandemic clone of Vibrio cholerae and its relationship to other pandemic and epidemic V. cholerae isolates. /. Bacteriol 176, 6199-6206. 219. Koblavi, S., Grimont, F, and Grimont, P A. (1990). Clonal diversity of Vibrio cholerae 01 evidenced by rRNA gene restriction patterns. Res. Microbiol. 141, 645-657. 220. Lan, R., and Reeves, P. R. (1998). Recombination between rRNA operons created most of the ribotype variation observed in the seventh pandemic clone of Vibrio cholerae. Microbiology 144, 1213-1221. 221. Byun, R., Elbourne, L. D., Lan, R., and Reeves, P. R. (1999). Evolutionary relationships of pathogenic clones of Vibrio cholerae by sequence analysis of four housekeeping genes. Infect. Immun. 67, 1116-1124.
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CHAPTER 2
Germ Warfare: The Mechanisms of Virulence Factor Delivery JILL REISS HARPER THOMAS J. SILHAVY
I. II. III. IV. V. VI. VII. VIII.
Introduction The General Secretory Pathway Autotransporters: Type V Two-Step Secretion: Type II ABC Transporters: Type I Conjugal Transfer Systems: Type IV Contact-Dependent Secretion: Type III Concluding Remarks References
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/. Introduction Pathogenic bacteria synthesize a diverse array of virulence determinants. These virulence proteins, which comprise the arsenal of bacterial weapons, have a wide variety of activities that require them to be targeted to specific locations. For example, proteins involved in attachment of the bacterium to the host cell must be localized to the bacterial surface, some bacterial toxins are secreted into surrounding fluids, and others are injected directly into the cytoplasm of the eukaryotic host cell. Thus, in pathogenic bacteria secretion itself is a virulence determinant; without the means to selectively target proteins, these bacteria are harmless.
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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The problem of targeting proteins to their correct location is not unique to pathogenic bacteria. All cells have subcellular compartments that are bound by lipid bilayers, and it is essential that each of these compartments contain a characteristic set of proteins. To maintain this organization, cells have specific mechanisms to target proteins from their site of synthesis in the cytoplasm to each noncytoplasmic location. This targeting, of course, requires the movement of proteins into and across the lipid bilayers. In most cases, proteins destined to leave the cytoplasm are tagged with a signal sequence at the amino terminus that serves to target them to the cellular secretion (Sec) machinery that includes a heterotrimeric complex of integral membrane proteins. Signal sequences and the components of this heterotrimeric complex have been conserved in all three domains of life [1]. While bacterial cells are not as complex as their eukaryotic counterparts, they do exhibit some compartmentalization. If we consider lipid bilayers to be compartments, then Gram-positive bacteria have three destinations to which proteins can be targeted: the cytoplasm, the membrane, and the extracellular environment. Gram-negative bacteria, on the other hand, can target proteins to five distinct locations: the cytoplasm, the inner membrane, the periplasm, the outer membrane, and the extracellular environment. In Gram-positive bacteria, the Sec machinery is sufficient for targeting to the extracellular environment since the secreted proteins must pass through only one lipid bilayer. The outer membrane of Gram-negative bacteria complicates protein secretion to the extracellular environment. At least five different mechanisms, termed Types I through V, appear to be conserved among the Gram-negative bacteria. (Unfortunately, this nomenclature is confusing and incomplete because it does not include the mechanisms responsible for secretion of certain colicins [2] and the process of pilus assembly [3], for which there is currendy no name. Nonetheless, this nomenclature system has caught on and will be used in this chapter.) The various Gram-negative secretion systems can be divided into those that utilize the Sec machinery for translocation across the inner membrane (Sec-dependent) and those that do not (Sec-independent). The Sec-dependent secretion systems include Types II and V. The Type II system involves a two-step mechanism in which proteins are first targeted to the periplasm by the Sec machinery and then secreted from the cell by a complex reaction requiring a dozen or so additional proteins. The Type V systems, also called the autotransporters, utilize the normal pathway for outer membrane targeting. Autoproteolysis releases a secreted protein domain into the environment. The Sec-independent secretion systems include Types I, III, and IV. The Type I systems utilize a complex of three proteins that span both the inner and outer membranes, and they secrete proteins directly into the media. The Type IV systems are not well characterized at this time; they appear to be closely related to systems used for the conjugal transfer of DNA from one bacterium to another. The Type III systems are fascinating
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devices that actually allow injection of bacterial proteins directly into the cytoplasm of the host cell. Sometimes they are called "contact-dependent" to reflect this capacity for injection. Altogether these systems provide a wide range of options for bacterial protein secretion and will be the focus of this chapter.
//. The General Secretory Pathway Protein export from the cytoplasm is a conserved pathway that was discovered and characterized in the Gram-negative bacterium Escherichia coli. As noted above, many proteins destined for noncytoplasmic locations are synthesized with a signal sequence that targets them for translocation. This amino-terminal signal is later cleaved during the export process [4]. The function of signal sequences was first demonstrated using gene fusions in which the amino-terminal end of an exported protein such as LamB, the receptor for bacteriophage X, is fused to the cytoplasmic enzyme (3-galactosidase, or LacZ (reviewed in [5]). Strains carrying gene fusions of this type exhibit several novel phenotypes that can be exploited to obtain mutations that alter the secretion process. First, LacZ is meant to fold in the cytoplasm; if it is located somewhere else, it folds improperly and exhibits no activity. Second, the attempted secretion of large amounts of LacZ lethally jams the secretion machinery. The original LamB signal sequence mutations were isolated as suppressors of this deadly jamming. Furthermore, these signal sequence mutations increased LacZ activity because they prevented enzyme export from the cytoplasm [6]. Further analysis of many signal sequences from both prokaryotic and eukaryotic proteins has shown that they consist of about 15-26 amino acids. There is no conserved sequence, but they do possess common features [7]. They contain a stretch of 10-12 hydrophobic amino acids preceded by one or two positively charged residues and followed at the carboxy-terminal end by a cleavage site for leader peptidase, which removes the signal sequence from the preprotein to yield the mature protein. This run of hydrophobic residues resembles a transmembrane domain. Mutations that block signal sequence processing do not prevent translocation, but leave the mutant precursor protein tethered to the inner membrane with the amino terminus of the signal sequence facing the cytoplasm [8]. To catalyze insertion into and translocation across the inner membrane, E. coli has a set of proteins—the Sec proteins—that were identified using two complementary genetic approaches. In one approach, suppressors of signal sequence mutations were selected based on their ability to restore localization of a mutant precursor protein. The genes identified by this approach were called/?r/ for protein localization [9]. In the complementary approach, mutations were sought that increased the LacZ activity of an exported fusion protein and simultaneously
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conferred a conditional lethal phenotype. Genes identified in this way were termed sec for secretion [10]. Subsequent molecular analysis revealed that three of the sec genes were also/7r/ genes: secA/prlD [10, 11], secE/prlG [12, 13], and secY/prlA [9, 12, 14]. It is these three proteins that form the essential core of the secretion machinery; protein translocation can be reconstituted in vitro with only Sec A, SecE, and SecY [15, 16]. Later genetic experiments identified three additional nonessential sec genes—secB [17], secD [18], and secF [19]—and biochemical studies added two others—secG [16, 20] and jo/C [21]. Interestingly, prl alleles of secG were identified in 1997 [22]. Biochemical approaches have elucidated the role of the Sec proteins in protein export from the cytoplasm [23]. The Sec machinery is comprised of soluble cytoplasmic proteins and peripheral and integral cytoplasmic membrane proteins (Fig. 1, see color plate). In order to be exported from the cytoplasm, a protein must first be recognized by the secretion machinery. Multiple, partially redundant mechanisms ensure the accuracy of precursor recognition. In the cytoplasm, SecA binds signal sequences directly [24]. The secretion-specific chaperone SecB binds to the mature portions of exported proteins such as LamB or MalE [25, 26] and maintains them in an unfolded, export-competent state [27-31]. This contributes to recognition because SecB also binds SecA [32, 33]. SecA functions as a dimer to direct the precursor to the membrane and energize the translocation reaction [34]. The complex of SecA, precursor protein, and SecB interacts on the cytoplasmic face of the heterotrimeric SecYEG complex in the inner membrane. The SecYEG complex, based on studies with its eukaryotic counterparts, likely forms a protein-conducting channel through the inner membrane [35-37]. Binding of ATP then induces a change in the structure of SecA that allows it to insert into the inner membrane [38]. Once SecA enters the membrane, SecB is released into the cytoplasm and the precursor is partially translocated through the SecYEG complex [39]. This translocation allows access of signal peptidase to the precursor, and signal peptidase then cleaves the signal peptide. SecD, SecF, and YajC seem to stabilize the membrane-bound form of SecA [40]. ATP hydrolysis causes SecA to release the partially translocated precursor and deinsert from the membrane. Additional ATP binding and hydrolysis by SecA will repeat the insertion/deinsertion process to catalyze the progressive threading of the translocating protein through the inner membrane [40-42]. Translocation may also continue using energy from the proton-motive force in a step that is poorly characterized. E. coli possesses another mechanism for targeting proteins to the secretion apparatus in the inner membrane—SRP, or Signal Recognition Particle. This targeting factor is a diminutive version of its eukaryotic counterpart. Prokaryotic SRP contains a protein, Ffh, that is homologous to eukaryotic SRP54, the subunit that recognizes the signal peptide, and an RNA, 4.5S RNA, that resembles eukaryotic 7S RNA. E. coli also has a protein, FtsY, that is similar to the a subunit of the mammalian SRP receptor. These proteins are functional in targeting; the
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bacterial proteins can replace their eukaryotic counterparts in vitro [43]. Genetic analysis of secB mutants and ffh-dtp\eied strains argues that there may be two separate subsets of exported proteins in E. coli—those targeted by SecB and those targeted by Ffh [44, 45]. Alternatively, or in addition, prokaryotic SRP may function in the insertion of inner membrane proteins [46-48]. As noted above, components of the secretion machinery such as signal sequences, SecYEG, and SRP are conserved throughout biology. SecA is found only in eubacteria and chloroplasts [1]. Apparently, eukaryotes use a different mechanism to energize translocation. SecB has only been found in the family Enterobacteriaccae [ 1 ].
///. Autotransporters: Type V The simplest of the Sec-dependent secretion systems are the Type V systems, also known as the autotransporters [49]. The prototype of these is the IgA protease from M gonorrhoeae [50], which has been studied extensively for quite some time because the proteolysis of IgA by IgA protease is a major contributor to virulence. Other examples of autotransporters include the immunoglobulin A (IgA) protease from Haemophilus influenzae [51], the serine protease from Serratia marcescens [52], and the vacuolating cytotoxin VacA from Helicobacter pylori [53]. Autotransporters are first exported to the outer membrane, where a proteolytic event releases the final product into the medium (Fig. 2, see color plate) [54]. The secreted protein is first expressed in the cytoplasm as a large multidomain protein consisting of an amino-terminal Sec-dependent signal sequence, the 106-kD mature portion of the protein, a 30-amino-acid y-protein, a 15-kD secreted a-protein, and a 45-kD carboxy-terminal (J-protein, which remains inserted in the outer membrane [55]. The amino-terminal signal sequence targets the protein for translocation from the cytoplasm by the Sec machinery. Concomitant with translocation across this membrane, the signal peptide is cleaved and the remainder of the protein is released into the periplasm. The carboxy-terminal p-domain is targeted to the outer membrane, where it forms a P-barrel pore or channel through which the rest of the protein can pass in its unfolded state through the outer membrane onto the cell surface. The protein then undergoes autoproteolysis to cleave the (J-domain from the rest of the protein. The (J-protein remains in the outer membrane, while the mature, a-, and y-domains are released into the extracellular environment. Subsequent autoproteolytic cleavages release the mature protein from the small a- and y-proteins [56]. Their function is unknown; however, they could play a role as chaperones.
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The signals that direct the secretion of IgA protease are twofold: first, the presence of an amino-terminal signal sequence to direct export from the cytoplasm via the Sec machinery and, second, the presence of the (i-domain for secretion from the periplasm across the outer membrane [57]. The P-domain contains the information necessary for insertion into the outer membrane, and this insertion is sufficient for the rest of the protein to pass onto the surface. The information in the ^-domain responsible for targeting to the outer membrane has been reduced to a minimal "core" region that retains the ability to translocate a passenger protein across the outer membrane. This core is conserved among the carboxy-terminal regions of the IgA p-domains of several species. Based on structural predictions, the p-core resembles the p-barrel motif conserved among Gram-negative outer membrane proteins, and presumably all of these proteins share a common mechanism for outer membrane insertion [56]. However, this important process remains poorly characterized. Little was known about the secretion of autotransporters until the discovery that IgA protease itself is the only protein, aside from the Sec machinery, required for its secretion into the extracellular environment. The key experiment was the isolation of a DNA fragment from A^. gonorrhoeae that allowed secretion of active IgA protease when expressed in Escherichia coli [58]. Because E. coli does not usually secrete proteins into the extracellular environment, this indicated that all the components for secretion were present on the plasmid. Sequencing revealed that the cloned fragment contained a single gene coding for a protein significantly larger than the secreted, active form of IgA protease. Subsequent work demonstrated that the mature IgA protease, a-protein, and p-protein are the result of autoproteolytic processing at cleavage sites similar to those on host proteins that are the target of IgA protease [55, 57]. Other important work has shown that fusion proteins consisting of a signal sequence, the Vibrio cholerae toxin B subunit, and the IgA p-domain are efficiently secreted by E. coli, demonstrating that the signal sequence and the p-domain are sufficient for secretion. This work also shows that sequences translocated through the p-domain are generally in an unfolded state and that the translocation occurs in a linear fashion [59]. That the passenger proteins are translocated through the p-core in their unfolded state is further supported by the lack of cysteines, and therefore disulfide bonds, in the translocated portions of the autotransporters. Disulfide bonds would presumably introduce structures that cannot pass through the P-core pore [60]. Work with S. marcescens serine protease has demonstrated that, at least for this IgA protease-like protein, part of the carboxy-terminal p-domain plays the role of a chaperone and assists in folding of the mature enzyme once located on the surface [61]. Other work involves the development of systems to facilitate kinetic and structural studies of autotransporter secretion [62].
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The simplicity of these systems is striking, especially in contrast to other systems we will discuss. Accordingly, these autotransporters have implications for biotechnology, and many are being examined for their potential ability to secrete heterologous proteins from E. coli.
IV. Two-step Secretion: Type II The Type II secretion systems provide a commonly used mechanism for the extracellular secretion of proteins from Gram-negative bacteria. The pal system of Klebsiella oxytoca is the founding member of this diverse family. This system directs the secretion of pullulanase, a starch-debranching enzyme. Pullulanase is a lipoprotein that is exported to the periplasm by Sec-dependent means, targeted to the outer membrane, and then secreted by a complex mechanism requiring more than a dozen other proteins [63]. Examples of Type II secretion systems include the out systems of Envinia dvysanthemi and Erwinia cawtovora [64, 65], the xcp system of Pseudomonas aeruginosa [66], exe from Aeromonas hydrophila [67], xps from Xanthomonas campestris [68], and eps from Vibrio cholerae [69]. Type II systems also show similarities to systems that catalyze biogenesis of type IV pili, assembly of filamentous phages, and competence for DNA uptake [70-72]. The Type II secretion systems consist of about 12-14 proteins that are encoded by a cluster of genes [72, 73] (Fig. 3, see color plate). Although there is no direct evidence to support it, one model suggests that some of the components of the Type II systems form a pilus-like structure in the envelope [71, 74, 75]. The presence of several proteins—PulG, H, I, and J—that have homology to type IV pilin subunits led to this suggestion. These proteins, the pseudopilins, contain consensus prepilin peptidase cleavage sites and are processed by the PulO protein, which is homologous to the PilD/XcpA prepilin peptidase involved in the biogenesis of type IV pili [76]. PilD/XcpA is a multifunctional protein from P. aeruginosa that processes and A^-methylates the subunits of the type IV pilus and also the components of the apparatus that secretes alkaline phosphatase, phospholipase C, elastase, and exotoxin A [77-83]. The purpose of this processing is not yet known; it does not seem to be involved in pseudopilin complex formation or in relocalization of the pseudopilins [73]. Furthermore, the pseudopilins in K. oxytoca have been shown to localize to the cytoplasmic membrane, which may not lend well to their role in a pilus-like complex [84]. Nonetheless, it would seem that the role of the O protein is to process the components of the pilus-like structure so that they can assemble properly. The pilus-like structure is thought to guide secreted proteins in their folded conformation to the secretin PulD, located in the outer membrane. The secretins
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are a conserved family of proteins that form pores in the outer membrane large enough to allow secreted proteins to pass into the surrounding medium [85-87]. Support for the PulD protein forming a pore in the outer membrane comes from its sequence similarity to gpIV, a filamentous phage protein, and PilQ proteins, involved in type IV pilus assembly [88]. It has been suggested that this similarity reflects similar mechanisms at work in the filamentous phage type IV pili, and Type II secretion systems. Indeed, PulD protein forms large stable complexes in the outer membrane that are similar to complexes formed by the gpIV and PilQ proteins [87, 89, 90]. The gpIV protein forms pores [91], and one of the PulD-like proteins, XcpQ from P. aeruginosa, has been shown to form multimeric ring complexes in the outer membrane that could also serve as pores [92]. PulD targeting requires the activity of PulS, a lipoprotein that is also found in the outer membrane [93]. PulS also acts as a chaperone to protect PulD from degradation [87, 94]. Additional components required for secretion include PulC, F, K, L, M, and N, all of which are located in the inner membrane and have unknown functions [84, 93, 95, 96]. The final component required for secretion is PulE, a cytoplasmic protein with a putative nucleotide-binding motif [96, 97]. PulE associates with the inner membrane only in the presence of the other Pul proteins [97]. The specific protein with which PulE interacts is unknown, but in V. cholerae the PulE homolog EpsE interacts with the PulL homolog EpsL [98]. The obvious role for PulE, because of its nucleotide-binding site, would be to act, along with the proton-motive force, as the energizer for the process of secretion. PulE could also play a role in signaling [98]. In any event, ATP binding by PulE is essential for secretion [97]. Perhaps the most surprising feature of the current model is that, with the exception of PulD, PulS, and PulE, all of the Type II components are found in the inner membrane [72]. Why a system that targets proteins from the periplasm through the outer membrane needs so many cytoplasmic membrane proteins and one cytoplasmic protein is not understood. Like Type IV autotransporters, secretion via Type II systems requires the presence of a classical signal peptide recognized by the Sec machinery and cleaved by signal peptidase I in most cases, or signal peptidase II in the case of lipoproteins like K. oxytoca pullulanase. This signal, of course, is necessary and sufficient to get the secreted protein into the periplasm. What is the signal that distinguishes a protein to be secreted via the Type II system from any other periplasmic protein? One approach to this question has been deletion analysis of secreted proteins to analyze the regions necessary and sufficient for secretion. From this work in P. aeruginosa, it appears that the first 30 amino acids of exotoxin A play an important role in extracellular secretion [99, 100]. There also seems to be a second secretion signal located in the carboxy terminus of the protein [100]. Another strategy has implemented the use of
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reporter fusions to proteins such as p-lactamase. The secretion of these fusion proteins is surprisingly resistant to large internal deletions [101, 102]. Two regions, each of about 80 amino acids and separated by about 600 amino acids, have been identified as putative extracellular targeting signals in pullulanase [102]. In exotoxin A of f! aeruginosa, the signal has been localized to amino acids 60-120 in the amino-terminal end of the protein [101]. Based on the ambiguity of these results, it seems unlikely that all Type II secretion systems will recognize a common stretch of amino acid sequence like the Sec machinery does. It is more likely that a combination of specific sequences and conformational information targets secreted proteins such as pullulanase to the Type II secretion machinery [103]. Some evidence already supports the idea that these proteins must be folded into a certain conformation in order to be secreted [104-109]. It seems likely that proper folding of the protein allows regions that are far apart in the primary sequence to come together and form a signal patch that is then recognized by the secretion machinery [103]. The history of concept development with the Type II secretion systems is interesting and informative (reviewed in [73]). It had been established that a large number of proteins secreted by Gram-negative bacteria were synthesized with classical signal peptides at their amino termini. However, viable mutants had been isolated in several species that were defective in the secretion of proteins to the extracellular surroundings. Presumably, the genes identified were specifically involved in the process of extracellular protein secretion (e.g., [110, 111-114]); this secretion system could not be essential. Based on the simplicity of certain systems, such as the recendy identified autotransporters, it was originally thought that few cellular components would be required for export across the outer membrane [73]. The discovery of the extremely complicated Type II systems was, therefore, quite surprising. The breakthrough occurred with the examination of pullulanase secretion. Previous attempts to express pullulanase in E. coli resulted in accumulation of enzyme in the membranes [115]. Finally, a large 23-kb DNA fragment from Klebsiella oxytoca was cloned that supported pullulanase secretion in E. coli [116]. Subsequent work showed that the cloned fragment contained 14 genes—pulC, D, E, F, G, //, /, y, K, L, M, A^, O, and S—that are required for the secretion of pullulanase [84, 117, 95, 96]. In the meantime, genes involved in the secretion of proteins in several pathogenic species were shown to be homologous to several of the pul genes [64, 66, 81]. Surprisingly, a functional secretion system that is homologous to the Pul system has been identified in E. coli K-12, which historically had not been thought to actively secrete proteins into the environment [118-120]. Understanding how all of these components interact to catalyze protein secretion will not be easy. It seems likely that all of these components make up
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a macromolecular machine. The isolation or visualization of such a machine would be an important advance.
V. ABC Transporters: Type I At least in terms of number of components, the Type I, or ABC, transporter systems are quite simple. In these systems, only three proteins are required for Sec-independent secretion. Examples of the proteins secreted by the Type I secretion systems include a-hemolysin from Escherichia coli, Proteus vulgaris, and Morganella morganii [121]; leukotoxin from Pasteurella haemolytica [122]; metalloprotease from Serratia marcescens [123]; alkaline protease from Pseudomonas aeruginosa [124]; and proteases A, B, and C from Erwinia chrysanthemi [125]. As their name implies, these systems include a component that is a member of the ATP-binding cassette, or ABC, family of transporter proteins (reviewed in [2]). ABC transporters are found in all organisms from bacteria to mammals and function in the import and export of a diverse array of molecules. This family of proteins can be subdivided into three groups—the bacterial importers, the eukaryotic transporters, and the bacterial exporters [2]—with the bacterial exporters comprising the largest subfamily. The first bacterial exporter to be identified was the transporter for the a-hemolysin secreted by uropathogenic E. coli (reviewed in [2]; see also Wandersman, 1996), and this system remains the paradigm. The Type I systems secrete proteins and other substrates via a mechanism in which transfer across both the inner and outer membranes occurs in a single step [126, 127] (Fig. 4, see color plate). The most surprising feature of these systems is that secretion across both the inner and outer membranes requires only three proteins. These components include an ABC transporter, a membrane fusion protein, or accessory factor, and an outer membrane component. The secreted protein, while still in the cytoplasm, is recognized by and binds to the ABC protein via a carboxy-terminal signal. Substrate binding inhibits the ATPase activity of the ABC protein [128] and promotes interaction of the ABC protein with the membrane fusion protein. This interaction then stimulates interaction of the membrane fusion protein with the outer membrane component, so that the secretion apparatus is assembled in an ordered fashion [129,130]. The mechanism by which the substrate passes from the cytoplasm to the environment is not clear. The most likely scenario is that the complex of the ABC protein, membrane fusion protein, and outer membrane factor forms a channel through which the proteins pass, in one step, from the cytoplasm to the external medium. This model, of course, depends on the pore-forming ability of the outer membrane factor, which is still under debate.
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The ABC transporter—HlyB, in the case of E. coli a-hemolysin—contains an ATP-binding domain that is homologous to ATP-binding cassettes found in many types of proteins in organisms from bacteria to mammals [131, 132]. These ATP-binding cassettes are absolutely required for secretion; mutations that block ATPase activity also block hemolysin secretion [128, 133]. These transporter proteins reside in the inner membrane [134] with the ABC cassette in the cytoplasmic carboxy-terminal domain of the protein. The transmembrane domain of HlyB does not perform any other functions other than the anchoring of HlyB. Replacement of the HlyB transmembrane domain with another unrelated domain does not affect secretion of hemolysin [135]. Several lines of evidence demonstrate that the ABC transporter proteins are also responsible for substrate specificity. Hybrid ABC transporter systems comprised of components from different bacteria tend to secrete ABC cognates [129]. In addition, mutations in the signal sequence of a-hemolysin have been used to identify compensatory mutations in the cytoplasmic domain of HlyB that alter the substrate specificity of the system [136]. Indeed, it is the recognition and binding of the substrate by the ABC transporter that promotes interactions between the ABC transporter and the other two components [130]. The second component of the Type I secretion systems is known as the membrane fusion protein, or accessory factor [2, 137]. These proteins share a common predicted topology consisting of an amino-terminal domain anchored in the inner membrane, a periplasm-spanning domain, and a (3-barrel carboxy-terminal domain that is thought to span the outer membrane [138, 139]. The predicted topology of these proteins is supported by fractionation studies. HlyD fractionates to both the inner and outer membranes, while truncated HlyD, missing its carboxy-terminal 10 amino acids, fractionates only to the inner membrane [140]. The third component of the a-hemolysin-like systems is the outer membrane factor, which in the a-hemolysin system is TolC. TolC is unusual because the structural gene is unlinked, and the protein performs other important functions in E. coli [141, 142]. Demonstration of TolC involvement in a-hemolysin secretion was an important advance that clarified a very confusing situation [143]. TolC and other members of the membrane fusion protein family are thought to form a pore in the outer membrane through which the secreted substrates pass into the external milieu, but the evidence for their pore-forming abilities is limited [144-146]. Structural characterization of TolC has demonstrated that the protein exists as a trimer in the outer membrane with extensive (3-barrel structures [145, 146]. The carboxy-terminal domain of TolC protrudes into the periplasm and most likely contacts the other components, thus providing a bridge between the inner and outer membranes [146]. Proteins secreted by the ABC transporters, of course, lack signal sequences [147, 148]. The first insight into the nature of the signal was that the carboxy-terminal 23-kDa fragment of the 107-kDa HlyA is secreted [126, 149], Gene fusion
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approaches further dehneated the portion of the carboxy terminus that is necessary and sufficient for hemolysin secretion to the last 48-60 amino acids [150-155]. Other members of this family show similar carboxy-terminal signal peptides [156-159]. Attempts have been made to identify individual amino acids within this signal, but none emerged [160,161]. In addition to possible "contact" residues [160], these carboxy-terminal sequences may have other structural features that are important in the recognition of the secretion machinery. An extended amphipathic helix as well as other structural features is found in the carboxy-terminal regions of many of the proteins secreted via Type I mechanisms [155]. Yet another region just upstream of the carboxy terminus may play a role in signal recognition as well. This region, which consists of a glycine-rich GGXGXD sequence that is repeated up to 36 times, is found in all types of cytotoxic proteins secreted by the ABC transporters [162]. This repeated sequence is clearly important for secretion, and may function as an internal chaperone to keep the secretion signal exposed [163, 164]. The cloning and sequencing of the chromosomal hemolysin gene cluster helped establish the novelty of the secretion system for a-hemolysin [165]. The hemolysin, HlyA, has no signal peptide, indicating that secretion is independent of the Sec machinery [147], and the sequence of HlyB revealed features of an ABC transporter [2, 138, 166]. As noted above, the third component of the a-hemolysin secretion system, the outer membrane protein TolC, was not identified until several years later [143]. In the meantime, other proteins from numerous bacteria were found that are secreted by similar systems. For example, the hemolysins from Proteus vulgaris, Proteus mirabilis, and Morganella morganii have gene clusters homologous to the E. coli hly cluster, and mutations in the secretion factors for these hemolysins can be complemented by the homologous E. coli genes [121]. Type I systems are also involved in the secretion of Pasteurella haemolytica leukotoxin [167], Erwinia chrysanthemi proteases [125], and Serratia marcescens Has A heme-binding protein [168]. The presence of a large number of Type I secretion systems that are highly homologous to one another has expedited the characterization of these systems. Characterization of other Type I systems such as the Has system in Serratia marcescens has provided important new information. HasA is an extracellular heme-binding protein. Perhaps the most surprising result is that interfering with SecB [169] can prevent HasA secretion. This provides the first example of SecB function in a different secretion system, and it suggests that proteins secreted by Type I systems must be maintained in an unfolded state. Further study of Type I systems may provide a means to attenuate the virulence of organisms that possess them. In addition, these systems have been demonstrated to be an efficient method of delivering antigens, such as the p67 sporozoite antigen of Theileria parva, for vaccination purposes [170-172].
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W. Conjugal Transfer Systems: Type IV Type IV secretion systems are used by the plant pathogen Agwbacterium tumefaciens for the transfer of oncogenic T-DNA and proteins to plants, and certain Type IV components are used by Bordetella pertussis for the secretion of pertussis toxin (reviewed in [173]). Based on sequence homologies, these secretion systems appear to be novel adaptations of the conjugal transfer system used for the horizontal transfer of plasmids. Identification of a similar type of system in Legionella pneumophila [174, 175] and of homologs in the pathogenicity island of Helicobacter pylori (reviewed in [176-178]) demonstrate that the Type IV systems may be more widespread than originally thought. The conjugal transfer systems in Gram-negative bacteria, which include the F plasmids and the IncN plasmids, are well characterized. These plasmids carry the tra genes, which are required for plasmid transfer into another bacterium. Over the years, it has become clear that the mechanism of T-DNA transfer in A. tumefaciens is quite similar to the mechanism of conjugal plasmid transfer in Gram-negative bacteria (reviewed in [173]). The ways in which the DNA is processed prior to transfer are similar [179-181]. Finally, sequence analysis has demonstrated that the virB genes from A. tumefaciens show striking similarity to the tra genes from the IncN, IncP, and other conjugative plasmids [182-184] and that the tra genes most likely encode the proteins that form the DNA-channeling pore in the outer membrane [185, 186]. It is reasonable that a system for transferring DNA between bacteria might evolve into a system for delivering bacterial DNA into host cells. More surprising, however, was the discovery that these conjugal transfer systems have also been adapted to promote the secretion of proteins, such as the Bordetella pertussis toxin. This finding makes more sense, though, if we look at DNA transport systems as systems that are designed to transport proteins. The DNA merely "hitches" a ride with the protein as the protein gets transported into the recipient cell, regardless of whether the recipient is another bacterium or a plant or animal host cell. Evidence for this model comes from early experiments that demonstrated that some A. tumefaciens proteins can be transported by themselves into plant cells. At least two proteins, VirE2 and VirD2, are secreted along with the T-DNA as a complex, the T-complex, into the recipient plant cells. Mixed infections of plant cells with one bacterial strain deleted for T-DNA and one mutant for the virulence protein VirE2 result in tumorigenesis in the host plant, indicating that the protein can be transported into the plant in the absence of the T-DNA [187]. This tumorigenesis is, however, dependent on the presence of the virB operon, which encodes the transport apparatus, and VirD2, which binds the T-DNA. These results indicate that the VirE2 protein forms complexes with the T-DNA once both are in the plant. This is further supported by the observation that plants with a virEl transgene undergo tumorigenesis when infected with a
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normally avirulent virE2 mutant strain [188]. In addition, VirF, which is also required for virulence, has been shown by similar experiments to be exported into plant cells independent of T-DNA [189]. The mechanism of protein transport from Agrobacterium tumefaciens is not yet clear. VirD4 and 11 different proteins encoded by the virB operon are required for transport of the T-complex and are proposed to form a pilus-like transport apparatus through which the proteins and DNA can pass (Fig. 5, see color plate). The VirB proteins have been fairly well characterized as to their subcellular locations (reviewed in [176]). The proteins appear to form a macromolecular complex that traverses the entire bacterial envelope, leading to the exterior of the cell. This huge complex is most likely a channel that allows secreted proteins to pass from the cytoplasm to the surface of the cell. Starting from the outside, this macromolecular complex is comprised of VirB9 and its companion lipoprotein VirB7. These two proteins form the bulk of the structure that spans the outer membrane and periplasm. VirB6, which is the main integral inner membrane protein, and several other proteins, VirB3, VirB8, and VirB 10, which are associated with the inner membrane, are proposed to form the channel through which the T-complex passes across the inner membrane. In addition, three ATPases, VirD4, VirB 11, and VirB4, are associated with the inner membrane and provide energy for both assembly of the transport complex and secretion of the T-complex. The final component, the VirBl protein, resides in at least two locations in the cell. First, it is located in the large complex between the two membranes. Second, it is cleaved and exported to the surface. VirBl has homology to transglycosylases and has been proposed to bore holes in the peptidoglycan layer and allow pilus assembly to occur. For quite some time, the idea of a pilus-like structure, much like the conjugative F pili encoded by a subset of the tra genes, has been favored as a mechanism of T-complex transport from the bacterial surface to the interior of the plant cell. Until recendy, however, there was no evidence to support this hypothesis. Lai and Kado have now shown that VirB2 actually does form pilus-like structures [190], with VirB5 suggested to be a minor pilin subunit. The signal that directs the VirE2, VirD2, and VirF proteins to the secretion apparatus is not currently known. These proteins do not contain the amino-terminal signal sequences that are recognized by the Sec machinery. Thus, the Type IV systems appear to be Sec-independent. However, pertussis toxin subunits, which may be secreted by a similar mechanism, are synthesized with classical signal sequences and are secreted to the periplasm by Sec-dependent mechanisms. Thus, while it is true that the B. pertussis Ptl proteins, which comprise the pertussis toxin secretion apparatus, are homologous to the Tra and Vir proteins of conjugal plasmid and T-DNA transfer systems, the actual mechanisms of toxin secretion and DNA secretion may be very different. Indeed, some of the proteins that are absolutely required for DNA transfer in the Tra and Vir systems are absent from
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the Ptl system. The Ptl proteins are probably responsible for secreting the pertussis toxin from the periplasm, while the Tra and Vir proteins are transferring molecules from the cytoplasm [173]. Perhaps characterization of the Legionella and Helicobacter Type IV systems will provide answers to this dilemma.
VIL Contact-Dependent Secretion: Type III Like the Type I systems, the Type III systems secrete proteins via a Sec-independent mechanism. However, these systems are much more complicated than the three-component ABC transporter systems. The Type III systems, which are sometimes called contact-dependent secretion systems, allow not only secretion but also injection of virulence factors directly into the cytosol of eukaryotic host cells. Type III systems are known in animal pathogens such as Yersinia pestis and other Yersiniae, Shigella flexneri, and Salmonella typhinnirium, as well as plant pathogens such as Pseudomonas syringae and Erwinia species, and they will likely be found elsewhere. Since the Yersinia Ysc system is the best characterized of these systems, it will be our primary focus. For more detailed discussions see [191]. Yersinia secretes a collection of virulence proteins that are inappropriately called Yops (yersinia outer proteins) for historical reasons. While these Yops were known to be essential for virulence for some time, their site of action has been elusive for several reasons (reviewed in [192]). For one, the Yops are not secreted under conditions in which Ca^^ is present at the millimolar levels; the absence of Ca^+ stimulates Yops secretion into the media. This observation was exploited to identify mutants that are secretion defective, and the genes identified were called ysc (Yop secretion) [193]. The Ysc proteins mediate Yop secretion in a Sec-independent manner despite the presence of YscC, a homolog of the Type II outer membrane secretin PulD [193]. Homologs of the Ysc proteins have been found in many pathogenic bacteria such as Salmonella typhimurium (the Inv/Spa proteins). Shigella flexneri (the Mxi/Spa proteins), and Pseudomonas solanacearum (the Hrp proteins) [194-199]. This is not the whole picture, though. Another set of observations complicates the story. The secreted Yops aggregate terribly in the extracellular medium, and adding them direcdy to eukaryotic cells has no cytotoxic effects. The real breakthrough in characterization of Yersinia secretion came with the observation that the cytotoxic effect of YopE is dependent on the presence of YopD protein. However, this requirement for YopD can be circumvented by direct microinjection of YopE into HeLa cells [200]. Yops must require both secretion out of the bacteria and injection or translocation into the host cell to exert their toxicity. This
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translocation of Yops is mediated by a subset of the Yops, YopD and YopB. This was further supported by two key experiments. In the first, YopE was shown by microscopy to be injected into the host cell in a YopD-dependent manner [201]. Second, a reporter gene (Cya) was fused to YopE, and the enzymatic activity of this hybrid protein was used to demonstrate YopB- and YopD-dependent injection into host cells [202]. Thus, the process of Yop injection occurs in two steps: secretion out of the bacterium and translocation into the eukaryotic host. These two steps require greater than 20 proteins to transfer toxins direcdy from pathogen to host (Fig. 6, see color plate). This direct transfer requires that the cell coregulate secretion and translocation. The bacterial cell senses contact with the host cell via the YopN protein. YopN (LcrE) appears to remain on the surface of the bacterial cell, where it plugs the secretion pore [201, 203, 204]. The TyeA protein, identified in 1998, may also play a role similar to that of YopN; it also localizes to the bacterial cell surface and seems to function in the negative regulation of secretion [205]. When the bacterium contacts a host cell or Ca^"*^ is removed, the secretion channel is unplugged. In Yersiniae, this allows the secretion of factors that negatively regulate the expression of Yops and toxin synthesis commences. It seems that Yersinia might also have a plug on the cytoplasmic side of the secretion apparatus. LcrG may function as a cytoplasmic YopN, with LcrV acting as its inhibitor [206, 207]. In other systems, such as Shigella Ipa secretion, the secreted toxins accumulate in the bacterial cytoplasm prior to the stimulation of secretion by contact with host cells. In the absence of Ca^"^, the fully folded Yops are secreted from the bacterial cytoplasm to the exterior of the cell, where they are retained on the surface or released into the external environment. Secretion probably occurs through a channel-like complex that traverses the entire bacterial envelope. LcrD and its homologs in other organisms function as the channel through the inner membrane [191, 208]. As mentioned earlier, YscC and related proteins show homology to the outer membrane secretins (PulD) from the Type II systems [193]. They localize to the outer membrane [197, 209] and form ringlike multimers through which the Yops could pass [209]. YscJ is a lipoprotein [193] and may function to bridge between the inner and outer membranes. Passage through the inner and outer membranes is likely energized by the cytoplasmic ATPase YscN [210]. Despite this progress, there remain many essential Ysc proteins about which little is known. YscD, J, Q, R, S, T, and U are inner membrane proteins that show homology to the components of the flagellar export apparatus [191, 193, 211]. Accordingly, these proteins, as well as YscO and YscG, are probably part of a macromolecular machine that spans the bacterial envelope [193, 211, 212]. Little is known about the components YscF, I, K, L, and P, except that the F, I, K, and L proteins may be cytoplasmic [191]. The next step, translocation, is facilitated by the formation of a translocator through which the effector Yops are injected into the eukaryotic cell. YopB and
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YopD are thought to form a channel through which the effector Yops can enter the host cytosol. Indeed, these proteins look like transmembrane proteins in contrast to most Yops, which are soluble proteins [213]. Furthermore, YopB has been shown to exhibit a pore-forming hemolytic activity consistent with its role as the pore [214]. More recent evidence, however, suggests that YopD is translocated into the cytosol of the host, which seems inconsistent with its role in forming the translocation pore [215]; the role for YopD in the translocation is, therefore, unclear at this time. LcrV, mentioned earlier as having a role in regulation of secretion, may also have an important role in the translocation process. LcrV has been shown to facilitate the secretion of YopB and YopD and, therefore, may be important for assembhng the translocation machinery [207, 216]. Similarly, TyeA appears to have dual roles since it has been found to participate in translocation in a subset of the Yops [205]. Another factor that is important in translocation of proteins into the host cytosol is YopK. Mutants defective in yopK exhibit an increased cytotoxicity resulting from increased levels of translocated YopE, while overexpression of YopK causes decreased translocation [217]. YopK is not translocated but remains associated with the bacteria from which it is secreted, which supports the hypothesis that YopK acts as a modulator of translocation by negatively controlling the size of the pore through an association with the pore-forming proteins [217]. Another group of proteins important in translocation of Yops into host cells is the chaperones (reviewed in [218]). These chaperones, several of which have been identified, are bacterial cytoplasmic proteins that demonstrate specificity for one or two substrates and are usually named Syc for specific Yop chaperone. SycE, also called YerA, acts as a chaperone for YopE; it binds but does not target YopE to the secretion machinery in the inner membrane [219, 220]. Unlike general chaperones, SycE and the other Syc chaperones bind to a specific sequence on the target protein, and these sequences coincide with the domain required for efficient translocation into eukaryotic cells [221]. The implication is that by binding to these translocation domains the Syc proteins prevent premature interaction of their respective Yops with components of the translocator, YopB and YopD [221]. The Type III chaperones include SycE, SycH (for YopH), SycD (for YopD), and YscB (for YopN) [218, 222]. In Salmonella typhimurium, the Type III secretion system involved in eukaryotic cell invasion has been isolated and visualized [223]. These "needle complexes" clearly resemble the flagellar basal body, and this strengthens the idea that the two systems have a common ancestor [223]. Undoubtedly, the isolation of similar macromolecular structures from organisms with other Type III systems will be forthcoming. Yops were isolated from culture supernatants in the early 1980s [224-226], and later work revealed the genes encoding the Yops and hinted at the mechanism of their regulation [227, 228]. Further examination of the sequence information
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available for the Yops showed that these proteins do not contain typical Sec-dependent amino-terminal signal sequences, nor do they contain the carboxyterminal signals required for secretion by the Sec-independent Type I secretion systems, which was the only known Sec-independent secretion mechanism at the time [229-232]. The use of gene fusion technology narrowed down the Type III signal sequence to the amino-terminal 48 amino acids of the YopH protein; this sequence, fused to a reporter protein, directed the secretion of the reporter by the Yersinia export system [232]. Later studies with other yo/?//and v6>/7£'gene fusions located the signal for secretion in the amino-terminal 11-17 amino acids of the Yops, while the signal for translocation into the eukaryotic cytoplasm requires at least the first 50-70 amino acids [233, 234]. This suggests that the Yops contain several domains—a secretion domain, a translocation domain, and an effector domain [233]. Surprisingly, the secretion domains show very little similarity at the amino acid level [231, 233, 234]. A gene fusion approach reported in 1997 provided insight into the signal recognized by the Ysc secretion machinery [235]. As expected, hybrid proteins containing the first 15 amino acids of YopN fused to neomycin phosphotransferase are efficiendy secreted. Single point mutations that alter any of the YopN amino acids cause no significant reduction in the secretion of the fusion protein. Very unexpected, however, was the observation that frameshift mutations, which alter the entire YopN sequence, have little effect either. These results imply that the signal for Yop secretion lies in the mRNA and that Yop secretion may be coupled with translation [235]. Future work is necessary to determine the nature of the mRNA signal for Yop secretion and to identify the factors that recognize this signal. However, these striking findings present a paradox. Remember that many of the Yops require a cytoplasmic Syc chaperone to be translocated. If, in fact, Yop secretion occurs cotranslationally, there should be no protein in the cytoplasm for the Syc proteins to bind. One possible explanation for these two very different signals is that perhaps Yersinia switches between the signals during different stages of pathogenicity. The mRNA signal facilitates YopE secretion under conditions of Ca^"^ deprivation, yet YopE, in addition to having its first 15 codons intact, must bind to its cognate chaperone, SycE, for translocation into eukaryotic cells [236]. Perhaps secretion into the medium requires different signals than translocation, and Yersinia uses different modes of substrate recognition for these processes. It will be interesting to see if knowledge gained from the Yersinia system applies generally to all Type III systems. For example, is the mRNA signal universal? Does this apply even to flagellar subunits? Certain mechanistic steps must be conserved because selected components from one system can be exchanged with the homologous components from another system [237-240]. Obviously, the characterization of these secretion systems have important clinical applications, but these systems, which so efficiendy inject proteins, also have important biotechnological implications for the delivery of proteins to eukaryodc
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cells, such as their utility as antigen delivery systems for vaccine development [241].
VIIL Concluding Remarks At first glance, the five mechanisms for protein secretion that are conserved among Gram-negative bacteria appear quite diverse. Some work independently of the Sec apparatus—Types 11 and V—and some do not—Types I, III, and IV. The various systems require anywhere from one special component in the case of the autotransporters, to 20 or more components, in the case of the contact-dependent secretion systems. Despite this diversity, however, at least two major themes are emerging. First, whether it is a secretin such as PulD or other proteins such as the P-protein domain of the autotransporters, these secretion systems all possess a putative pore-forming protein that allows passage of the secreted substrate through the outer membrane. Mounting evidence supports a channel for the signal-sequence directed passage of proteins through the bacterial cytoplasmic membrane (SecYEG) or the endoplasmic reticular membrane of eukaryotic cells (the Sec61p complex), and YopBD seems to form a channel that allows Yop passage into the eukaryotic cytosol. Mother Nature seems to have a favored solution to the barrier problem posed by lipid bilayers. Second, we predict that the multiple components of complex secretion systems form a macromolecular machine that spans the Gram-negative cell envelope, and that such structures will likely resemble surface structures such as pili or flagella. This is most strikingly demonstrated by the Type III secretion system in Salmonella. In this case "needle complexes" have been isolated, visualized, and shown to resemble the flagellar basal body. We anticipate that similar techniques applied to the other systems will be fruitful as well.
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189. Regensburg-Tuink, A. J. G., and Hooykaas, R J. J. (1993). Transgenic N. glauca plants expressing bacterial virulence gene virF are converted into hosts for nopaline strains of A. tumefaciens. Nature 363, 69-1 \. 190. Lai, E. M., and Kado, C. I. (1998). Processed VirB2 is the major subunit of the promiscuous pilus of Agrobacterium tumefaciens. J. Bacterial. 180, 2711-2717. 191. Hueck, C. J. (1998). Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol. Mol. Rev. 62, 379-433. 192. Cornelis, G. R., and Wolf-Watz, H. (1997). The Yersinia Yop virulon: A bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23, 861-867. 193. Michiels, T., Vanooteghem, J.-C., Lambert deRouvroit, C., China, B., Gustin, A., Boudry, R, and Cornelis, G. (1991). Analysis of virC, an operon involved in the secretion of Yop proteins by Yersinia enterocolitica. J. Bacteriol. 173, 4994-5009. 194. Galan, J. E., Ginocchio, C , and Costeas, P. (1992). Molecular and functional characterization of the Salmonella invasion gene invA: Homology of InvA to members of a new protein family. J. Bacteriol. 174, 4338-4349. 195. Allaoui, A., Sansonetti, P. J., and Parsot, C. (1992). MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins. J. Bacteriol. 174, 7661-7669. 196. Venkatesan, M. M., Buysse, J., and Oaks, E. V. (1992). Surface presentation of Shigella flexneri invasion plasmid antigens requires the products of the spa locus. J. Bacteriol. 174, 1990-2001. 197. Allaoui, A., Sansonetti, P. J., and Parsot, C. (1993). MxiD, an outer membrane protein necessary for the secretion of the Shigella flexneri Ipa invasins. Mol. Microbiol. 7, 59-68. 198. Bergman, T, Erickson, K., Galyov, E., Persson, C , and Wolf-Watz, H. (1994). The IcrB (yscN/U) gene cluster of Yersinia pseudotuberculosis is involved in Yop secretion and shows high homology to the spa gene clusters of Shigella flexneri and Salmonella typhimurium. J. Bacteriol. 176,2619-2626. 199. Huang, H. C , Lin, R. H., Chang, C. J., Collmer, A., and Deng, W. L. (1995). The complete hrp gene cluster of Pseudomonas syringae pv. syringae 61 includes two blocks of genes required for harpin Pss secretion that are arranged colinearly with Yersinia ysc homologs. Mol. Plant Microbe Interact. 8, 733-746. 200. Rosqvist, R., Forsberg, A., and Wolf-Watz, H. (1991). Intracellular targeting of the Yersinia YopE cytotoxin in mammalian cells induces actin microfilament disruption. Infect. Immun. 59, 4562-4569. 201. Rosqvist, R., Magnusson, K. E., and Wolf-Watz, H. (1994). Target cell contact triggers expression and polarized transfer of Yersinia YopE cytotoxin into mammalian cells. EMBO J. 13, 964-972. 202. Sory, M.-P, and Cornelis, G. R. (1994). Translocation of a hybrid YopE-adenylate cyclase from Yersinia enterocolitica into HeLa cells. Mol. Microbiol. 14, 583-594. 203. Forsberg, A., Viitanen, A. M., Skurnik, M., and Wolf-Watz, H. (1991). The surface-located YopN protein is involved in calcium signal transduction in Yersinia pseudotuberculosis. Mol. Microbiol. 5, 977-986. 204. Persson, C , Nordfelth, R., Holmstrom, A., Hakansson, S., Rosqvist, R., and Wolf-Watz, H. (1995). Cell-surface-bound Yersinia translocate the protein tyrosine phosphatase YopH by a polarized mechanism into the target cell. Mol. Microbiol. 18, 135-150. 205. Iriarte, M., Sory, M.-P, Boland, A., Boyd, A. P, Mills, S. D., Lambermont, I., and Cornelis, G. R. (1998). TyeA, a protein involved in control of Yop release and in translocation of Yersinia Yop effectors. EMBO J. 17, 1907-1918.
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206. Nilles, M. L., Williams, A. W., Skrzypek, E., and Straley, S. C. (1997). Yersinia pestis LcrV forms a stable complex with LcrG and may have a secretion-related regulatory role in the low-Ca-^ response, y. Bacterial. 179, 1307-1316. 207. Nilles, M. L., Fields, K. A., and Straley, S. C. (1998). The V antigen of Yersinia pestis regulates Yop vectorial targeting as well as Yop secretion through effects on YopB and LcrG. J. Bacterial. 180,3410-3420. 208. Piano, G. V., Barve, S. S., and Straley, S. C. (1991). LcrD. a membrane-bound regulator of the Yersinia pestis low-calcium response. / Bacterial. 173, 7293-7303. 209. Koster, M., Bitter, W., de Cock, H., Allaoui, A., Cornelis, G. R., and Tommassen, J. (1997). The outer membrane component, YscC, of the Yop secretion machinery of Yersinia enterocalitica forms a ring-shaped multimeric complex. Mol. Microbiol. 26, 789-797. 210. Woestyn, S., Allaoui, A., Wattiau, P., and Cornelis, G. R. (1994). YscN, the putative energizer of the Yersinia Yop secretion machinery. J. Bacterial. 176, 1561-1569. 211. Piano, G. V., and Straley, S. C. (1995). Mutations in yscC. yscD, and >\srG prevent high-level expression and secretion of V antigen and Yops in Yersinia pestis. J. Bacterial. Ill, 3843-3854. 212. Payne, P. L., and Straley, S. C. (1998). YscO of Yersinia pestis is a mobile core component of the Yop secretion system. J. Bacterial. 180, 3882-3890. 213. Hakansson, S., Bergman, T., Vanooteghem, J. C, Cornelis, G., and Wolf-Watz, H. (1993). YopB and YopD constitute a novel class of Yersinia Yop proteins. Infect. Innniin. 61, 71-80. 214. Hakansson, S., Schesser, K., Persson, C, Galyov, E. E., Rosqvist, R., Homble, P., and Wolf-Watz, H. (1996). The YopB protein of Yersinia pseudotuberculosis is essential for the translocation of Yop effector proteins across the target cell plasma membrane and displays a contact-dependent membrane disrupting activity. EMBO J. 15, 5812-5823. 215. Francis, M. S., and Wolf-Watz, H. (1998). YopD of Yersinia pseudotuberculosis is translocated into the cytosol of HeLa epithelial cells: Evidence of a structural domain necessary for translocation. Mol. Microbiol. 29, 799-813. 216. Sarker, M. R., Neyt, C, Stainier, 1., and Cornelis, G. R. (1998). The Yersinia Yop virulon: LcrV is required for extrusion of the translocators YopB and YopD. J. Bacterial. 180, 1207-1214. 217. Holmstrom, A., Pettersson, J., Rosqvist, R., Hakansson, S., Tafazoli, R, Fallman, M., Magnusson, K.-E., Wolf-Watz, H., and Forsberg, A. (1997). YopK of Yersinia pseudotuberculosis controls translocation of Yop effectors across the eukaryotic cell membrane. Mol. Microbiol. 24, 73-91. 218. Wattiau, P., Woestyn, S., and Cornelis, G. R. (1996). Customized secretion chaperones in pathogenic bacteria. Mol. Microbiol. 20, 255-262. 219. Wattiau, P., and Cornelis, G. R. (1993). SycE, a chaperone-like protein of Yersinia enterocalitica involved in the secretion of YopE. Mol. Microbiol. 8, 123-131. 220. Frithz-Lindsten, E., Rosqvist, R., Johansson, L., and Forsberg, A. (1995). The chaperone-like protein YerAof Yersinia pseudotuberculosis stabilizes YopE in the cytoplasm but is dispensable for targeting to the secretion loci. Mol. Microbiol. 16, 635-647. 221. Woestyn, S., Sory, M.-P, Boland, A., Lequenne, O.. and Cornelis, G. R. (1996). The cytosolic SycE and SycH chaperones of Yersinia protect the region of YopE and YopH involved in translocation across eukaryotic cell membranes. Mol. Microbiol. 20, 1261-1271. 222. Jackson, M. W., Day, J. B., and Piano, G. V. (1998). YscB of Yersinia pestis functions as a specific chaperone for YopN. J. Bacterial. 180. 4912-4921. 223. Kubori, T, Matsushima, Y, Nakamura, D., Uralil. J., Lara-Tejero, M., Sukhan, A., Galan, J. E., and Aizawa, S.-I. (1998). Supramolecular structure of the Salmonella typhinmriiun Type III protein secretion system. Science 280, 602-605.
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224. Portnoy, D. A., Moseley, D. A., and Falkow, S. (1981). Characterization of plasmids and plasmid-associated determinants of Yersinia enterocolitica pathogenesis. Infect. Immun. 31, 775-782. 225. Straley, S. C , and Brubaker, R. R. (1981). Cytoplasmic and membrane proteins of yersiniae cultivated under conditions simulating mammalian intracellular environment. Proc. Natl Acad. Sci. U.S.A. 78, 1224-1228. 226. Bolin, I., Portnoy, D. A., and Wolf-Watz, H. (1985). Expression of the temperature-inducible outer membrane proteins of yersiniae. Infect. Immun. 37, 506-512. 227. Comelis, G., Vanooteghem, J.-C, and Sluiters, C. (1987). Transcription of the yop regulon from Y. enterocolitica requires trans acting pYV and chromosomal genes. Microb. Pathogen. 2, 367-379. 228. Mulder, B., Michiels, T., and Comelis, G. (1989). Identification of additional virulence determinants on the pYVe plasmid of Yersinia enterocolitica. Infect. Immun. 57, 2534-2541. 229. Forsberg, A., and Wolf-Watz, H. (1988). The virulence protein Yop5 of Yersinia pseudotuberculosis is regulated at transcriptional level by plasmid pIBl-encoded trans-diCimg elements controlled by temperature and calcium. Mol. Microbiol. 2, 121-133. 230. Michiels, T., and Comelis, G. (1988). Nucleotide sequence and transcription analysis of yop51 from Yersinia enterocolitica W22703. Microb. Pathogen. 5, 449^59. 231. Michiels, T., Wattiau, P, Brasseur, R., Ruysschaert, J.-M., and Comelis, G. (1990). Secretion of Yop proteins by Yersiniae. Infect. Immun. 58, 2840-2849. 232. Michiels, T., and Comelis, G. (1991). Secretion of hybrid proteins by the Yersinia Yop export system./ Bacteriol. 173, 1677-1685. 233. Sory, M.-P, Boland, A., Lambermont, I., and Comelis, G. R. (1995). Identification of the YopE and YopH domains required for secretion and internalization into the cytosol of macrophages, using the cyaA gene fusion approach. Proc. Natl. Acad. Sci. U.S.A. 92, 11998-12002. 234. Schesser, K., Frithz-Lindsten, E., and Wolf-Watz, H. (1996). Delineation and mutational analysis of the Yersinia pseudotuberculosis YopE domains which mediate translocation across bacterial and eukaryotic cellular membranes. J. Bacteriol. 178, 7227-7233. 235. Anderson, D. M., and Schneewind, O. (1997). An mRNA signal for the type III secretion of Yop proteins by Yersinia enterocolitica. Science 278, 1140-1143. 236. Lee, V. T., Anderson, D. M., and Schneewind, O. (1998). Targeting of Yersinia Yop proteins into the cytosol of HeLa cells: One-step translocation of YopE across bacterial and eukaryotic membranes is dependent on SycE chaperone. Mol. Microbiol. 28, 593-601. 237. Groisman, E. A., and Ochman, H. (1993). Cognate gene clusters govem invasion of host epithelial cells by Salmonella typhimurium and Shigella flexneri. EMBO J. 12, 3779-87. 238. Hermant, D., Menard, R., Arricau, N., Parsot, C , and Popoff, M. Y (1995). Functional conservation of the Salmonella and Shigella effectors of entry into epithelial cells. Mol. Microbiol. 17, 781-9. 239. Ginocchio, C. C , and Galan, J. E. (1995). Functional conservation among members of the Salmonella typhimurium InvA family of proteins. Infect. Immun. 63, 729-732. 240. Rosqvist, R., Hakansson, S., Forsberg, A., and Wolf-Watz, H. (1995). Functional conservation of the secretion and translocation machinery for vimlence proteins of yersiniae, salmonellae, and shigellae. EMBO J. 14, 4187-4195. 241. Russmann, H., Shams, H., Poblete, E, Fu, Y, Galan, J. E., and Donis, R. O. (1998). Delivery of epitopes by the Salmonella type III secretion system for vaccine development. Science 281, 565-568.
CHAPTER 3
Regulation of Virulence Gene Expression in Bacterial Pathogens CHARLES J. DORM AN STEPHEN G. J. SMITH
I. II. III. IV. V. VI. VII. VIII. IX. X. XL XII. XIII. XIV. XV. XVI. XVII. XVIII. XIX. XX. XXI. XXII.
Introduction Transcription Initiation Regulatory Protein Families Covalent Modification of Transcription Factors Regulatory Networks The Oxidative Stress Response The Modular Nature of Bacterial Regulatory Proteins The Overlap between Genome Structure and Gene Regulation Other Classes of Protein Regulators DNA Structure and Gene Regulation Stereotypical and Stochastic Events in the Control of Gene Expression The Switch Controlling Type 1 Fimbrial Expression in E. coli Pap Pilus Gene Transcription Contact-Dependent Gene Regulation The Virulence Gene Regulatory Cascade of S. flexneri A Thermometer Protein from the Salmonella Virulence Plasmid Cell-Density-Dependent Regulation Adaptive Mutation Rare tRNAs and Translation Modulation Protein Splicing AntisenseRNA Perspective References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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76 77 79 82 86 87 89 92 94 94 97 101 103 105 106 109 110 114 114 115 115 116 117
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/. Introduction As with other bacterial genes, the expression of most virulence genes seems to be regulated. This is usually rationalized as a mechanism that avoids inappropriate expression of virulence traits while ensuring their rapid expression when they are required. It implies that bacteria have the means to know when to express the genes and when not to. Consequently, those wishing to understand the control of gene expression must consider both the mechanisms of gene regulation and the means by which bacteria interpret their internal and external environments. Most of the pioneering work on bacterial gene regulation was carried out in Escherichia coli K-12 and its near relatives, using as model systems genes coding for carbohydrate utilization and other traits with no obvious role in bacterial virulence. Nevertheless, the lessons learned from these studies have proved to be extremely valuable and in many cases are applicable to virulence genes. In general terms, genes can be regulated positively (activated) or negatively (repressed), and in E. coli repression seems to be the chief method by which gene expression is controlled [1]. Genes can be regulated at transcription or translation, or posttranslationally. Regulation at the transcriptional level, and in particular at the level of transcriptional initiation, is the most efficient method for the obvious reason that it is better to control a complex process at its point of origin rather than further downstream. It is clear that bacteria have invested heavily in transcriptional control mechanisms, and many of these are emerging as control elements in virulence genes. Thinking about bacterial gene regulation has been influenced very strongly by the operon model, in which a dedicated regulatory protein controls simultaneously the expression of a number of sequential genes that are transcribed as a polycistronic message. The operon represents a simple mechanism for coordination of gene expression, and this concept is critical for useful insights into the means by which the transcriptional profile of the entire cell is modulated as the bacterium experiences environmental change. Grouping several operons or individual genes under the command of a common regulatory protein produces a regulon, with all of the members being coregulated in response to a common signal (Fig. 1). Allowing regulon members to belong to more than one regulon produces a networking of regulons, with genes responding to distinct, yet overlapping signals. The challenge of understanding the complexities of these higher levels of coordination is emerging as a key issue in the new era of microbiology that follows determination of the genome sequences of many bacteria. It is within this regulatory complex that virulence genes are located. Many possess dedicated regulators and also display sensitivity to regulatory inputs that are shared with many of the ''housekeeping" genes of the cell. For this reason, an appreciation of bacterial gene regulatory mechanisms in general is a prerequisite for understanding how virulence gene expression is controlled.
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•^..^;^2^i f-^:s^-.Wi4^m'M~
Fig, 1 Coordinated control of transcription. Four separate genetic loci are shown that respond to a common stimulus. The stimulus acts through two unrelated regulatory proteins, RegA and RegB. This grouping is referred to as a stimulon. Proteins RegA and RegB both control their own regulons of genes by binding to an operator sequence near the promoter (P). In the case of RegA, the regulon consists of independent gene 1, and operon I. which is composed of three cotranscribed genes. In the case of RegB, the regulon consists of operons 2 and 3. composed of two and three cotranscribed genes, respectively. The stimulon shown here consists of all of the genes responding to the stimulus, a total of nine genes.
//. Transcription Initiation The first step in transcription involves recognition and binding of the promoter DNA by RNA polymerase (Fig. 2). The ''standard" bacterial promoter consists of four elements whose locations are given with respect to the transcription start site, + 1. These are the -10 and -35 boxes to which the sigma factor of RNA polymerase binds, the spacer between these boxes, and a recently discovered UP element, which consists of an AT-rich sequence in the region ^ 0 to -60 [2, 3]. For recognition by sigma-70, the ideal sequences for the -10 and -35 boxes are TATAAT and TTGACA, respectively, and the ideal length of the spacer is 17 bp. In general, promoter strength is a function of similarity to these ideal sequences and this spacer length. Differences in the structure of the promoter result in different basal levels of expression. Regulatory features can be imposed to increase or decrease expression with respect to the intrinsic, basal level. Promoter selection is influenced strongly by the type of sigma factor carried by RNA
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
A+T-rich UP element
17 bp spacer TGN motif
Fig. 2 RNA polymerase and a sigma-70-dependent promoter. The key elements of the promoter are shown, and consist of the A+T-rich UP element extending from ^ 0 to -60, the -35 and -10 canonical sequences, the 17-bp spacer, and the transcription start site at position +1. The TGN motif, lying immediately to the left of the -10 box, is also shown. The five protein subunits of RNA polymerase are alpha (two copies), beta, beta prime, and sigma. The sigma factor contacts the promoter at the -10 and -35 boxes, while the alpha subunits contact the promoter via their carboxy-terminal domains (CTDs). These domains also contact transcription factors bound upstream of the promoter, while other regulatory protein contacts can be made on polymerase at the beta and the sigma subunits (see text). Alpha NTD represents the amino terminal domain of this protein.
polymerase. Sigma-70 (RpoD) recognizes most bacterial promoters, while other sigma factors such as sigma-32 (RpoH, heat shock), sigma-38 (RpoS, stress and stationary phase), or sigma-54 (RpoN, nitrogen limitation) direct polymerase to other promoters. Selection is made on the basis of the DNA sequence of the promoter. Thus, a requirement by a group of genes for a particular form of RNA polymerase represents a level of transcriptional networking; one can speak of heat shock genes, stationary phase genes, nitrogen-regulated genes, etc. Recruitment of RNA polymerase to the promoter may depend on factors additional to the DNA sequence of the sigma factor recognition motif [4]. These factors may include upstream and downstream DNA sequences, and regulatory proteins. The involvement of regulatory proteins provides a further level of coordinated control, since copies of the same protein may operate at multiple promoters in the genome. Interactions between RNA polymerase and transcription factors usually involve protein-protein contact [5]. Within RNA polymerase, contact is usually made with the carboxy-terminal domain of the alpha subunit or with the sigma-70 subunit, although contact with the P' subunit can also occur [5, 6] (Fig. 2). The nature of the contact depends on the type of regulatory protein and the distance its binding site lies upstream of the promoter. Transcription activator proteins assist RNA polymerase with the formation of an open complex in which the DNA helix at the promoter melts (see below). Some repressor proteins can act by preventing successful transcriptional initiation and elongation through direct allosteric interaction with RNA polymerase; they may achieve
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similar results by altering the local structure of the DNA at the promoter. Other repressor proteins can act simply by interfering sterically with the ability of polymerase to bind to the promoter. In this way, the processes of both closed- and open-complex formation can be regulated positively and negatively, although not every promoter displays each type of regulation [7]. Transcription factors can act on polymerase by recruiting it to the promoter, and the well-characterized cyclic-AMP receptor (CRP) protein works in this way. Recruitment assists polymerase in establishing a closed complex, but it can also play a key role in assisting isomerization to an open complex. Thus, CRP works to recruit RNA polymerase to the lac promoter [8], but it both recruits polymerase and drives formation of an open complex at the gal promoter [9]. In contrast, the unrelated transcription factor NtrC is not involved in recruitment; it instead acts on prebound polymerase. This ATP-dependent enhancer-binding transcription factor activates bacterial promoters recognized by the RpoN (sigma-54) form of RNA polymerase, and it exerts its effect at a distance through DNA looping [10, 11] (Fig. 3). In addition to its much-studied role as a transcription activator, CRP also has the ability to act as a transcription repressor; for example, it can repress the cya gene, encoding adenylate cyclase [12]. It can also exert effects beyond the family of sigma-70-dependent promoters with which it is usually associated; for example, CRP has been shown recently to be able to repress the sigma-54-dependent dctA promoter from Rhizobium meliloti [13]. After an open complex has been created, the template strand can begin to direct polymerization of the transcript. The binding of the initiating NTP can stabilize the open complex. For example, ATP is the initiating NTP in the PI promoter of the rrnB ribosomal RNA gene in E. coli, and the availability of ATP controls the stability of the open complex at this promoter. In this way, the size of the ATP pool, which is determined by the metabolic activity of the cell and is a function of growth rate, dictates the rate at which the rniB PI promoter functions, and hence the rate of synthesis of stable RNA. This provides a mechanistic link between stable RNA synthesis and growth rate [14].
///. Regulatory Protein Families Bacterial transcription factors are usually classified by amino acid sequence comparison with prototypic members of families of DNA-binding proteins, such as the LysR-like and AraC-like protein families [15, 16]. The most common DNA-binding motif in prokaryotic transcription factors is the helix-turn-helix (HTH) structure, and this is utilized by both the AraC and LysR families. A strong correlation has been reported between the position of the HTH motif, and the function of the protein. In repressors, the HTH is usually associated with the amino terminus, while it occurs in the carboxy terminus in activators; the LysR family forms a distinct group with a repressor-like HTH location. The functional significance of the HTH location remains obscure and may simply reflect the evolutionary origins of the proteins [17].
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
sigiiia*54 form of RNA polymerase bound to promoter
|>hospho-NtrC uligomer
.|4<>
.24
408
-12
clos^ complex
enhancer
DMA loop
+ATP
iiiRNA
open complex
Fig. 3 DNA looping and NtrC-dependent transcription initiation. The phospliorylated NtrC protein forms an oligomeric structure at an upstream enhancer sequence between positions -108 and -140. RNA polymerase containing sigma-54 is bound to the promoter in a closed complex, DNA looping allows physical contact to occur between NtrC and the bound polymerase, and ATP consumption accompanies isomerization of the closed to an open transcription complex. The sigma factor is lost from the polymerase once transcription is initiated.
Environmental sensing by members of these families often occurs through binding of specific ligands. For example, the LysR protein binds diaminopimelate, a component of the bacterial cell wall [18], while AraC binds arabinose [19]. Ligand binding alters the behavior of these proteins in terms of their interaction with the genes they control. It is believed that the change in protein conformation that results from ligand binding is responsible for conversion of the transcription factor to an active form, either as an activator or repressor [20]. This model of gene regulation in response to a chemical signal is appealing, not least because it is easy to understand. Less accessible are mechanisms by which changes in the physical environment act through such proteins to alter transcription, since no chemical signal may
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be apparent. For example, the SpvR protein of Salmonella typhimurium and related serovars is an LysR-like positive regulator of plasmid-linked spv virulence genes [21-23]. The spv genes are induced during infection of mice, and they are required for full virulence and establishment of a systemic infection [24-26]. Yet, how SpvR detects and transmits to the spv promoters signals associated with adaptation to the environments encountered during mouse infection is still unclear. In some cases, LysR-like proteins have been found to exhibit a two-stage activation of gene promoters. The first stage involves binding of one protein dimer to a promoter-distal high-affinity binding site, and the second involves the recruitment of another protein dimer to a second, lower-affinity promoter-proximal binding site. This second stage may be cofactor dependent and involve a ligand-induced conformational change in the protein-DNA complex that allows transcription to be initiated [27, 28]. Although the SpvR protein interacts with the promoter of the spv structural genes in a way that is consistent with this two-stage model [23], no ligand has been identified that is responsible for its activation of ^/j>v transcription as the bacterium enters the stationary phase of growth. On the other hand, the involvement of the RpoS sigma factor at the SpvR-activated promoters may fulfill the regulatory requirements that ligand-binding normally achieves at sigma-70-dependent promoters controlled by other LysR-like proteins. The AraC-like VirF proteins of enteroinvasive E. coli (EIEC, [29]) and Shigella flexneri [30], both of which regulate homologous sets of invasion genes located on a high-molecular-weight virulence plasmid, the related protein of the same name found in Yersinia species that regulates plasmid-encoded yop virulence gene expression [31], and the VirF homologs Rns [32] and CfaD/CfaR [33] from enterotoxigenic E. coli that control adhesin expression, all activate virulence gene expression in response to increases in temperature. By analogy with AraC, these are probably homodimeric proteins. No ligand has been identified as being required for this process to occur, and, in most cases, few details are available on the mechanism by which activation is achieved in response to the thermal signal. Another important family of transcription factors is related to the proteins CRP and FNR. CRP binds cAMP and functions as a broad-domain regulator of gene expression in bacteria, while FNR has a redox-sensitive iron-binding center and controls expression of many genes under anaerobic conditions. Significandy, from the point of view of the theme of this volume, the cAMP-CRP protein is required by S. typhimurium for virulence in mice [34]. It also contributes to virulence gene regulation in Vibrio cholerae, where it is involved in repression of the genes coding for cholera toxin and the toxin-coregulated pilus [35]. In contrast, the FNR protein is not required by S. typhimurium for mouse virulence [36]. The CRP/FNR family is growing and now includes the dimeric PrfA protein, a major regulator of virulence gene expression in the facultative intracellular pathogen Listeria monocytogenes. In L. monocytogenes, PrfA regulates virulence gene transcription in response to temperature, and once again this regulatory protein does not appear to require a ligand for activadon [37]. The Fur protein is responsible for regulating many genes concerned with iron metabolism in bacteria, and it is highly conserved from species to species. Bacterial
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
pathogens of mammalian hosts usually find that available iron levels are extremely low, and they have evolved regulatory circuits that respond to iron concentrations and use these to control the expression of genes that can assist the bacteria in iron acquisition. In addition to iron-uptake systems, bacteria use Fur or a related ironregulatory protein to control the expression of toxin and hemolysin genes. Examples include the Fur-regulated bacteriophage-encoded shiga-like toxin of some pathogenic strains of £. coli, and the DtxR-regulated bacteriophage-encoded diphtheria toxin of Corynebacterium diphtheria [38, 39]. In S. typhimurium, an intact fur locus is required for virulence in mice and for resistance to acid pH stress [40]. On binding Fe^"^, these proteins become proficient for binding at specific operator sequences associated with the promoters they control. In the case of Fur, the binding sites are termed "iron boxes". Fur and its relatives normally act as transcription repressors [41]. As with the AraC and LysR families, the CRP/FNRlike proteins and the iron regulator proteins are active as dimers. This is a general feature of transcription factors in prokaryotes, as it is in eukaryotes. These proteins recognize and bind to specific operator sequences, and these frequently display dyad symmetry (hence the dimeric nature of the proteins). The possession of the appropriate operator sequence by a gene admits it to membership of the regulon controlled by the regulatory protein that binds to that sequence.
IV. Covalent Modification of Transcription Factors Protein phosphorylation is now recognized as an important mechanism by which transcription factor activity in bacteria is modulated in response to environmental cues. The usual arrangement involves an environmental sensor protein with histidine protein autokinase activity that becomes phosphorylated in response to a signal or group of signals. The phosphorylated sensor serves as a substrate, or phosphate donor, for a second protein that elicits a response. This second protein is called a response regulator. In addition to its kinase activity, the sensor often possesses a phosphatase activity that can dephosphorylate the response regulator. These "two-component" regulatory protein partnerships are found in many bacteria, and the chemistry of the phosphorylation and dephosphorylation reactions seems to be highly conserved among members of the family. Many, but by no means all, of the histidine protein kinase sensor proteins are associated with the cytoplasmic membrane, where extracellular domains are believed to be involved in signal reception. Two-component systems regulate environmental responses by the bacterium usually, but not exclusively, at the level of transcription. An exception to the transcriptional response mode involves the use of the phosphotransfer systems in controlling swimming behavior in enteric bacteria [42]. Examples in which the details of two-component regulation have been elucidated most clearly include control of OmpC and OmpF porin expression in E. coli and 5. typhimurium by the EnvZ/OmpR partnership, and the control of nitrogen assimilation in enteric bacteria by the NtrB/NtrC proteins (Fig. 4).
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—C)yter oienibiane-
iicnv-- dnd oihc? tncinbct^ oi the
Ssgtna-54 dependent promoter ol',i?l«4 gene and oilier members of the NtrC reaiilon
Fig. 4 The EnvZ/OmpR and NtrB/NtrC two-component regulatory systems. The EnvZ histidine protein kinase is shown associated with the cytoplasmic membrane, where it detects changes in osmotic pressure. The NtrB histidine protein kinase is a cytosolic protein, and it responds to changes in the level of combined nitrogen in the cell. Both kinases become phosphorylated in response to their cognate signals at conserved histidine residues within the carboxy-terminal domains. Their response regulator partners then remove the phosphates, becoming phosphorylated on aspartate residues within their amino-terminal domains. Phosphorylated OmpR and NtrC are then proficient enough to activate transcription of genes possessing the appropriate binding sites at their promoters for these proteins. Each response regulator has a DNA-binding motif within its carboxy terminal domain. C = carboxy terminus; N = amino terminus.
Two-component regulatory systems, with their distinctive phospho-relay mechanism, are at the heart of several virulence gene networks. These include the PhoP/PhoQ system in S. typhimiiriunu which plays a critical role in survival of the bacterium in macrophages, and the OmpR/EnvZ system, which is crucial for successful infection of mice by the same bacterium using the oral route. Initially, neither of these two-component regulatory systems was linked to the control of virulence genes [43^5]. Now each is seen as a component of a complex regulatory network that includes factors playing a direct role in bacterial virulence. The PhoP/PhoQ system is central to the virulence of S. typhimurium
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[46, 47], while the OmpR/EnvZ system has been shown to influence virulence in Salmonella and Shigella [48, 49]. The PhoQ protein is a membrane-associated histidine protein kinase, while PhoP is a transcription regulator that is involved in activating some genes (collectively called pag, for PhoP-activated genes) and repressing others (prg, PhoP-repressed genes) (Fig. 5). The periplasmically located sensing domain of PhoQ responds to magnesium ions, and the magnesium concentration has a profound modulatory influence on the whole PhoQ/PhoP regulon [50, 51]. The divalent cations bind to an acidic cluster in the sensing domain of the PhoQ protein and apparently lock it into a conformation that cannot signal. This occurs without affecting the oligomeric state of the protein [52, 53]. Following the identification of PhoP/PhoQ as a regulator of virulence genes in S. typhimurium [47], it was discovered that it interacts with a second two-component system to exert a further layer of control on virulence gene transcription [54, 55]. ThtpmrAB locus encodes a two-component system that mediates resistance to the peptide antibiotic polymyxin B and modifications to lipopolysaccharide (LPS). This locus forms an operon with the PhoP-activated gene pagB (also called pmrC), giving PhoP a role in pmrAB gene activation [54] (Fig. 5). PmrB is probably involved in sensing pH. The PmrA protein is the response regulator, and it is in turn responsible for the expression of several genes known to be activated by PhoP This PmrA-mediated activation of pag genes is dependent on the PhoP/PhoQ proteins for induction in response to Mg^"^ limitation but not for pH-mediated induction [55], Despite the overlap between the two systems, PhoP/PhoQ has a domain of influence that is distinct from that of PmrA/PmrB. For example, the PhoP-dependent resistance of S. typhimurium to the NP-1 defensin peptide is not affected by PmrA/PmrB [54]. Thus, the PhoP/PhoQPmrA/PmrB cascade provides a nice illustration of the way in which two phospho-relay systems can be networked. It has been proposed that this network has evolved to assist S. typhimurium to adapt to low Mg^"^ environments, such as that within the phagolysosome of macrophages. Here, S. typhimurium may be forced to use the Mg^^ from its own LPS, with PmrA-dependent modifications allowing the bacterium to maintain LPS integrity [56]. Networking of the type illustrated by PhoP/PhoQ and PmrA/PmrB is distinct from the concept of crosstalk, in which the histidine protein kinase of one partnership can phosphorylate the response regulator of another. While examples of such crosstalk have emerged from work with purified systems in vitro [57, 58], the in vivo significance of crosstalk may not be very great, and in vivo crosstalk of this type may even be detrimental [59]. The role of OmpR/EnvZ in controlling the invasive phenotype of S. flexneri is not well characterized. There are no data showing a direct role for OmpR in the control of specific virulence genes, and it seems likely that OmpR acts indirectly on the invasion genes through its ability to modulate the porin profile of the cell [60]. In S. typhimurium, porin control is a large part of the contribution of OmpR/EnvZ to virulence [61], but other factors are also involved. S. typhimurium
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FmrA*l*
kesi^hiiHH- tii c;itionic
Modilk-alioii ofl IN
pmrii
pm? \
puiiH
Fig. 5 The PhoP/PhoQ-PmrA/PmrB regulatory cascade in S. typhimuriiim. The PmrB and PhoQ proteins are located in the cytoplasmic membrane, where they detect environmental signals and transmit them to their regulons via their cognate response regulators. PmrA and PhoP, respectively. The response regulators are activated by phosphorylation, and PhoP has a role in the positive control of the genes that code for PmrA and PmrB. making them members of the pag (PhoP-activated gene) regulon. C = carboxy terminus: N = amino terminus: prg = PhoP-repressed gene. A possible role for PmrB in pH sensing is indicated by "pH?".
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mutants deficient in OmpR protein expression cannot induce filament formation in HeLa cells [62]. This indicates that OmpR and EnvZ are required for filament induction while the bacterium is inside host cells. This defect is independent of the OmpR-controlled porins, and may reflect a direct role for OmpR in controlling the S. typhimurium genes responsible for filament induction [62]. The ompR locus is also required for Salmonella-pvomoiQd apoptosis of macrophages [63]. OmpR is also involved in controlling interactions between different S. typhimurium cells. This aggregative phenotype involves the agf locus (known as csg in E. coli) coding for curli fimbriae and its normal expression is OmpR-dependent. This locus is also subject to control by the sigma-38 form of RNA polymerase and by the histone-like protein H-NS (see below) [64]. In Salmonella typhi, a pathogen of humans, the EnvZ/OmpR system is required for expression of the Vi polysaccharide; OmpC and OmpF porin expression is also controlled by EnvZ/OmpR in this pathogen [65]. Evidence now points to the use of tyrosine protein phosphorylation in E. coli as a mechanism for achieving pleiotropic effects on gene expression. A gene called typA, which codes for a protein subject to tyrosine phosphorylation, is required for the normal expression of the H-NS nucleoid-associated protein and other proteins [66]. The S. typhimurium equivalent of typA is called bipA, and this gene has been associated with pathogenesis [67]. Further research is required to assess the importance of this form of posttranslational protein modification for global control of bacterial gene expression and virulence.
V. Regulatory Networks Networking of genes and operons is in part a reflection of the distribution of transcription factor-binding sites (operators) throughout the bacterial genome. It also permits overlaps to occur between regulons. For example, the spv virulence genes already referred to depend on the LysR-like protein SpvR for their activation (see the preceding). They also depend on the sigma-38 form of RNA polymerase [68, 69]. Thus, they belong to a large group of coregulated genes (those dependent on sigma-38) and to a much more exclusive group (those controlled by SpvR, i.e., spvR itself and the spvABCD operon). The lac operon of E. coli is repressed by the Lad repressor, a protein with no other known function in the cell. In contrast, the lac operon is activated by the cAMP-CRP complex, which also regulates expression of very many other promoters in the cell. Included in this group is the araBAD operon, which is under the control of the AraC protein. Thus, lac and ara both belong to the cAMP-CRP regulon, while retaining individual regulatory features imposed by the locally acting Lad and AraC proteins, respectively. Among virulence genes, membership of complex regulatory networks is proving to be very common.
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The OmpR/EnvZ and NtrB/NtrC two-component systems also provide examples of overlap with other regulatory pathways in the cell. The OmpR/EnvZ-dependent OmpC and OmpF control pathway is governed primarily by changes in osmolarity, linking regulation of the ompC and ompF genes to other osmotically sensitive genes in the cell, such as the proU operon that encodes an uptake system for compatible solutes required to survive osmotic shock. However, the EnvZ/OmpR two-component system does not control proU, indicating that ompC, ompF, and proU do not constitute a regulon [70]. Instead, these are part of a stimulon, that is, a group of genes influenced by the same environmental signal not necessarily acting through a common regulatory protein. The NtrB/NtrC system controls transcription of the gene coding for glutamine synthetase, and this gene possesses a promoter recognized by the alternative sigma factor, sigma-54 [42]. Thus, this NtrB/NtrC-dependent gene is networked with other sigma-54-dependent genes.
W. The Oxidative Stress Response The networking concept is illustrated nicely by the response of bacteria to oxidative stress. This stress arises when molecular oxygen undergoes reduction to form toxic products such as hydrogen peroxide and superoxide radicals, and these are processed to harmless products by catalase and superoxide dismutase, respectively. In E. coli, there are three forms of superoxide dismutase (CuZnSod in the periplasm, and the cytoplasmically located MnSod and FeSod) and two catalases, HPI, encoded by katG, and the starvation-induced HPII, encoded by the katE gene [71]. The expression of genes encoding factors protective of cells undergoing oxidative stress involves several regulatory proteins. The SoxR and SoxS proteins are two of these, and they control the expression of approximately 10 genes involved in responding to superoxide-generating agents. SoxS is an AraC-like protein that regulates target genes (including sod A, the gene for MnSod), while SoxR is related to the metal-binding mercury regulator MerR, and is both a repressor and activator of transcription. The SoxR protein is homodimeric, and each monomer possesses a redox-sensitive [2Fe-2S] cluster [72]. Oxidation and reduction of this redox center activates or represses SoxR activity as a transcription factor, respectively. SoxR binds to a single regulatory site between the divergently transcribed soxR and soxS genes. This site lies in the unusually long (19 bp) spacer between the -10 and -35 boxes of the soxS promoter, and just downstream of the soxR promoter. Here it represses soxR transcription, whether stimulated by oxidative stress or not. However, on stimulation, SoxR stimulates soxS transcription by more than 30-fold by altering the local conformation of the
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DNA in a way that compensates for the nonstandard length of the spacer element [73, 74]. In addition to SoxR and SoxS, several proteins control sodA expression: this gene is under positive control of the Fnr anaerobic regulator, and under negative control of the ArcA/ArcB aerobic respiration two-component regulatory system and the Fur iron regulatory protein [71, 75]. Therefore, the multiplicity of inputs at the sodA promoter provides an example of the complexity of transcriptional regulatory networking in bacteria. Interestingly, SoxRS has also been found to crossregulate OmpF porin expression in E. coli. This involves SoxRS-mediated activation of the micF antisense RNA, which blocks translation of the ompF mRNA [76]. In this way, the SoxRS system can influence the diffusion of small hydrophilic molecules across the outer membrane of the cell. The case of micF is even more relevant to the topic of networking when one realizes that it is also controlled negatively by the leucine-responsive regulatory protein (LRP) and the nucleoid-associated protein H-NS [77, 78]. These two pleiotropic regulators are discussed in more detail in what follows. The OxyR protein of E. coli and related organisms belongs to the LysR family of transcription regulators, and it activates, among others, the genes coding for HPI catalase and the Dps DNA-binding protein, both of which protect the cell from hydrogen peroxide damage, and it regulates its own gene negatively [79]. Both HPI and Dps are also under the positive transcriptional control of the RpoS sigma factor, making their genes simultaneously members of both the OxyR and RpoS regulons [80, 81]. OxyR is regulated by oxidative stress posttranslationally. It is bound to its operator sites both in the active and the inactive configuration, and it is both the sensor of the signal and regulator of the response. OxyR is activated on oxidation through the formation of a disulfide bond, and it is inactivated when reduced by glutaredoxin, an enzyme whose gene is under OxyR control [82]. OxyR is also responsive to nitrosative stress due to exposure of the bacterium to 5-nitrosothiols [83]. These compounds are found in high concentrations in infectious and inflammatory sites, leading to speculation that OxyR may assist in pathogenesis by helping the bacterium to adapt to 5-nitrosothiols as well as to oxidative stress [83, 84]. Nevertheless, oxyR null mutants of S. typhimurium are fully virulent in Balb/C mice [85]. The oxyS gene lies upstream of oxyR, and the genes are transcribed divergently and coregulated. The product of oxyS is a stable and abundant 109-nucleotide RNA that is induced by oxidative stress in an OxyR-dependent manner and has pleiotropic effects in the cell [86]. One of its functions is to inhibit RpoS in exponentially growing cells undergoing oxidative stress. In stationary phase cells, OxyR is not activated by oxidative stress, and it is thought that RpoS can induce transcription of the katG (HPII catalase) and dps genes to protect the cell in the absence of OxyR activation [86]. In this way, the oxyS small RNA contributes to integration of the oxidative and stationary phase regulons.
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VIL The Modular Nature of Bacterial Regulatory Proteins The two-component systems provide a clear illustration that bacterial regulatory proteins are composed of modules and have been adapted to perform specific functions. In the case of the EnvZ/OmpR pair, EnvZ has an environmental signal-sensing domain in its amino terminus and a signal-transmission domain in its carboxy terminus. The two are divided by a transmembrane domain, which may also function in signaling. The OmpR protein has a signal receiver domain in its amino terminus and a DNA-binding domain in its carboxy terminus. In EnvZ, the signal transmitter is centered around the histidine residue that becomes phosphorylated in response to environmental signals (in this case, an increase in periplasm osmolarity) (Fig. 4). In OmpR, the signal-receiver domain has at its core the aspartic acid residue that will be phosphorylated by EnvZ. These transmitter and receiver domains are found in all examples of the two-component family studied to date. Despite this strong conservation of structure and function, the different family members do very different jobs in the cell. This diversity reflects variety in the structure and function of the signal-reception domains of the histidine protein kinases, and in the regulatory domains of the response regulators. The modular nature of the proteins is confirmed by the results of domain-swapping experiments. By fusing the signal-reception domain of the Tar chemotaxis receptor protein to the signal-transmitter domain of EnvZ, a hybrid protein, Tazl, was generated. Tazl responds to the presence or absence of aspartate (a trait of Tar but not EnvZ) but not to osmolarity (an EnvZ trait), and it regulates porin gene expression in response to aspartate [87]. Some histidine protein kinases possess signal-receiver domains in their carboxy termini. Examples include the BvgS protein of the whooping cough bacterium Bordetella pertussis and the VirA protein of the plant pathogen Agrobacterium tumefaciens [88, 89] (Fig. 6). It is possible that the addition of this module allows signaling to the response regulator proteins to be damped. Thus, signal-receiver domains can be attached to response regulators to modulate a biological function (DNA binding in the case of the Bordetella and Agrobacterium systems), or they can be added to the signal transmitters to modulate signal-to-noise discrimination and signal amplification. The B. pertussis BvgS protein possesses a further module at its carboxy terminus that resembles a transmitter domain, and it seems that both signal transmitter and the receiver domain must be phosphorylated before phosphotransfer to the DNA-binding BvgA response regulator is possible [90] (Fig. 6). Sensor proteins with both transmitter and receiver domains are involved in cell-density-dependent gene regulation (sometimes called quorum sensing) in the marine bacterium Vibrio harveyii (see the below). Here, the bacterium responds to two autoinducers called HAI-1 and HAI-2 through specific interactions with
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N
N TMl
peoplasmic domain
TM! periplasmic domain
TM2
Imker
kjfiase
TM2 linker
iHl kinase domain
domain
receiver domain
D
VirA hislidine prolein kinase of Agrohavterlum
receiver domain
carboxyterminal domain
iHl
D
H
tumefat tens
BvgS histidine protein kinase of Bordetelki pertmsis
Fig. 6 The modular nature of histidine protein kinases and response regulators. The proteins illustrated are Wrkirom Agrobacteriiim tumefaciens (left) and BvgS from Bordetellapertussis (right). Both proteins are associated with the cytoplasmic membrane, and each has two transmembrane sequences (TMl and TM2). The histidines at which the proteins are phosphorylated in the kinase domains are represented by H; BvgS also has such a domain in its carboxy terminus (see text). The aspartate residues that receive phosphate groups are represented by D within the receiver domains. C = carboxy terminal; N = amino terminal.
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the membrane-located sensor proteins LuxN and LuxQ, respectively. Once phosphorylated, these proteins transfer the signal to the DNA-binding protein LuxO, which then activates genes coding for bioluminescence [91]. The response regulators also display a modular structure. For example, proteins that are not involved in phosphotransfer regulatory relationships have been described as having DNA-binding domains similar to that of OmpR. The ToxR protein regulator of cholera toxin gene expression in V. cholerae is one such protein. It is unusual in being a membrane-located DNA-binding protein [92]. Its OmpR-like DNA-binding domain is located in the amino terminus. In contrast, the corresponding domain is located within the carboxy terminus of OmpR. The DNA-binding domain of OmpR is the founding member of a new class of DNA-binding motifs. Called winged-helix-turn-helix transcription factors, OmpR and its relatives bind DNA and interact productively with RNA polymerase [93]. Presumably, ToxR interacts with the transcription machinery in V. cholerae through a similar mechanism, with the proviso that this protein is tethered to the cytoplasmic membrane. ToxR also differs from OmpR in forming heteromeric complexes with the cytoplasmic membrane protein ToxS [94]. Another protein exhibiting similarity to the DNA-binding domain of the OmpR family of transcription factors is the HilA protein of S. typhimurium [95]. Like ToxR, the HilA protein has its DNA-binding domain within its amino terminus. However, this is not a membrane-associated DNA-binding protein. The hilA gene lies within a pathogenicity island, and its product activates transcription of invasion genes located in the same island [95]. Expression of invasion genes is under environmental control, and HilA is believed to play a role in the activation of invasion genes in response to environmental cues. HilA is under the transcriptional control of the SirA (i.e., UvrY) protein, which is a response regulator of the two-component family [96]. Yet, both the sensor responsible for SirA phosphorylation and the signal that controls its phosphorylation remain unknown. ToxR is not unique in being a membrane-associated transcription factor. The CadC protein of E. coli is involved in pH and lysine signaling, and it is a membrane-located DNA-binding protein with an OmpR-type DNA-binding domain [97]. Interestingly, ToxR-regulated genes also respond to pH [92]. Thus, protein modularization has permitted the OmpR winged-helix-turn-helix DNAbinding domain to be utilized in several different settings to influence the expression of both virulence and housekeeping functions in a variety of bacterial species. The MviA protein of S. typhimuhiim (also called RssB and SprE in E. coli) shows homology to response regulators, but only at its amino terminus [98]. The carboxy terminus does not resemble any known DNA-binding motif. This raises the possibility that a signal-reception module common to response regulators has been employed to modulate the activity of MviA in processes other than DNA binding. The MviA protein influences the stability of the RpoS sigma factor, probably by modulating the susceptibility of RpoS to degradation by the ClpXP protease [99-101]. This is consistent with the known role of the mviA gene as a determinant of S. typhimurium virulence in mice [98]. This theme of protein
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modularization will be revisited in the discussion of the nucleoid-associated proteins H-NS and StpA (see below).
VIIL The Overlap between Genome Structure and Gene Regulation Many of the elements that impose structure on the bacterial genome are emerging as regulators of gene expression. While the bacterial nucleoid lacks the highly organized structure of eukaryotic nucleosomes, there is a degree of structure, and this is imposed partly by the histone-like proteins [102]. Protein H-NS is of particular interest because of the number of virulence systems in which it plays a part. When H-NS influences transcription, it usually acts as a repressor. It forms higher-order multimers on DNA but is not thought to bind a ligand or undergo covalent modification. An example of H-NS as a repressor of virulence gene expression may be found in S. typhimurium, where it regulates spv virulence gene expression [103], or in S.flexneri, where it regulates invasion gene expression [60]. H-NS is a 15.6-kDa polypeptide present in about 18,000 copies per cell. It does not have a consensus sequence for its binding site in DNA; instead it seems to bind to particular structures, especially those showing DNA curvature. H-NS can itself influence the structure of DNA by its ability to constrain supercoils. In this way it can contribute to the compaction of the nucleoid that is essential for its efficient packaging within the confines of the bacterial cell [104, 105]. In E. coli and at least some related organisms, H-NS is partnered by the StpA protein (see below). This protein is 58% identical to H-NS in its amino acid sequence but is present in only about 200 copies per cell. It differs from H-NS in having a strong RNA-binding activity, and it serves a chaperone function in group I intron splicing reactions [106, 103, 108]. These two proteins crossregulate each other's genes negatively [109], and in rich growth media stpA transcription is confined to a narrow window in exponential phase [110]. Like H-NS, StpA can form homomeric higher-order structures. In addition, the proteins can form heteromeric structures with one another, and this may allow a primitive form of regulation if the heteromeric and homomeric structures have distinct biological functions (see below). This is in contrast to the "conventional" transcription factors such as the AraC-, LysR-, CRP-, and FNR-like proteins, which form only homodimers. H-NS and StpA each consist of two domains, connected by a linker sequence [106]. Genetic analyses and protein crosslinking studies show that the amino-terminal domains of these proteins are required for oligomerization, while the carboxy-terminal domains have DNA-binding activity [111-115], and in the case of StpA, RNA-modulating activity [106]. Thus, H-NS and StpA have a modular
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Structure, and individual modules have now been discovered to form components of proteins otherwise unrelated to these two. For example, the pMKlOl plasmidencoded KorB regulatory protein consists of two H-NS-like DNA-binding domains arranged in tandem, while the MdbA protein from plasmid p24-2 has an H-NS-related amino terminus linked to a carboxy-terminal domain with no relationship to H-NS [106]. This raises the possibility that the MdbA protein may form heteromeric complexes with H-NS or StpA, modulating the activities of these proteins as a result. The LEE pathogenicity island of enteropathogenic E. coli (EPEC) encodes an H-NS-like protein [116]. This protein, encoded by Orfl within the pathogenicity island, possesses an H-NS-like DNA-binding domain. The distribution of H-NS-like proteins among bacteria is variable. E. coli and its close relatives Klebsiella pneumoniae, S. flexneri, S. typhimurium, and Serratia marcescens, and the plant pathogen Envinia chrysanthemi all possess an H-NS homolog, as does B. pertussis, Proteus vulgaris, Pseudomonas aeruginosa, and Yersinia enterocolitica [105, 117-122]. The stpA gene has been cloned from E. coli, and S. typhimurium and been detected in E. chrysanthemi and S. flexneri [107, 123, 124]. There is no evidence for either gene in Aeromonas, Bacillus, Clostridium, or Listeria species [105]. The complete genome sequence of Haemophilus influenzae has revealed only one gene coding for an H-NS-like protein [125]. The HU protein of E. coli forms homo- and heterodimers from its alpha and beta subunits. It is an abundant histone-like protein and contributes to both structural and regulatory processes in the genome. HU does not have a consensus DNA sequence for binding; instead, it appears to be a general DNA-binding protein. It is closely related to integration host factor IHF, which in E. coli forms heterodimers exclusively from its alpha and beta subunits [126]. IHF binds to a consensus DNA sequence and can bend DNA by angles up to 180° [127]. This bending activity is central to its biological function, and it plays both structural and regulatory roles in the cell. It can perform the role of a transcription factor and has been shown to influence virulence gene transcription in Neisseria gonorrhoeae, where it controls expression of the pilE pilin gene, and in S. flexneri, where it controls transcription of dedicated virulence gene regulators [128-130]. Like HU, it does not bind a low-molecular-weight ligand. The factor for inversion stimulation, or FIS, is another protein with histone-like properties. FIS forms homodimers exclusively and is involved in both the determination of local nucleoid structure and gene regulation. It plays an important role in site-specific inversions of DNA and in transcriptional activation of stable RNA promoters [131-133]. It can also act as a repressor of transcription [134, 135]. Part of its activity derives from an ability to modulate the transitions in DNA structure that arise as a result of changes in the growth phase of the culture [136]. FIS does not bind a low-molecular-weight ligand, but its concentration is high in cells in the early exponential phase and very low in the stationary phase of growth. This allows FIS to influence gene expression and other genetic events in response to
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growth phase. In contrast, IHF levels are higher in the stationary than in the exponential phase of growth, while HU forms heterodimers mainly in mid-exponential phase, with homodimers occurring outside of this range. These fluctuations in histone-like protein availability and oligomeric state allow this group of proteins to modulate the gene expression profile of the cell in a very dynamic manner. Several genes are influenced by more than one of these proteins, allowing for quite complex regulatory patterns to be achieved. These include the genes coding for the histone-like proteins themselves. For example, H-NS is encoded by the hns gene and its transcription is repressed by H-NS and StpA but activated by PIS. Crossregulation of this form ties the genes of the cell together in tight regulatory networks.
IX. other Classes of Protein Regulators The cAMP-CRP protein might be regarded as lying between the histone-like proteins and conventional transcription regulators. It binds to a well-conserved consensus DNA sequence, is active as a homodimer, and binds a low-molecularweight ligand (cAMP). In addition, it alters DNA structure at the binding site, by bending the DNA by angles of approximately 90°. Another example of such a difficult-to-classify protein is LRP, the leucine-responsive regulatory protein [137]. This DNA-binding protein has a consensus binding site (albeit a rather degenerate one), is active as a homodimer, can bend DNA, and has a ligand-binding capacity. Interestingly, it responds to several nonpolar amino acid ligands (L-leucine, L-alanine, L-valine, and L-isoleucine), and its activity can be potentiated, antagonized, or unaffected by this ligand interaction. The nature of the ligand effect seems to be determined by the system being regulated by LRP. Thus, this protein is a sophisticated control element capable of influencing both DNA structure and gene expression, and doing so in a manner that can link it to amino acid metabolism. The LRP protein is a very prominent regulator of adhesin gene expression in E. coli, as are cAMP-CRP, IHF and H-NS (see below).
X. DNA Structure and Gene Regulation Since promoter function is likely to be influenced by local DNA structure, the influence of DNA structuring elements such as the histone-like proteins is perhaps unsurprising. In addition, one must take account of factors directly influencing the flexibility of the DNA, and the facility with which it may form an open
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transcription complex. Negative supercoiling increases the free energy of the DNA and can assist in isomerization of a closed to an open complex. Environmental stimuli such as osmotic stress can alter the supercoiling level of the DNA, and this can modulate the efficiency of transcription initiation of many genes (see below). In this way, supercoiling variations represent a crude form of regulation, upon which more specific regulatory factors can be superimposed. Supercoiling is controlled by topoisomerases, with DNA gyrase (unique to prokaryotes) introducing negative supercoils while DNA topoisomerase I removes them. Some relaxing activity is also provided by DNA topoisomerases III and IV [138-140]. DNA gyrase is an ATP-dependent enzyme, and this dependency links its activity to the [ATP]/[ADP] ratio in the cell, and hence to metabolic activity. In this way, changes in environmental parameters such as osmolarity, temperature, oxygen availability, and pH, as well as growth phase, which alter the [ATP]/[ADP] ratio, can influence the degree to which bacterial DNA is supercoiled [141-147]. It is well established that transcriptional activity can alter the level of supercoiling of plasmids, and this is likely to be true of the chromosome [148]. These are usually short-range effects caused by perturbation of local DNA topology by the movement of RNA polymerase. The movement through DNA of other complexes, such as the DNA replication machinery, exerts similar effects. This movement can introduce simultaneously positive and negative supercoils into DNA, and these are then relaxed by DNA gyrase and topoisomerase I, respectively [149-151] (Fig. 7). Sensitivity to variations in DNA supercoiling is a function of the promoter sequence and, in many cases, is strongly context-dependent. The normal pattern involves activation by increases in negative supercoiling (or underwinding of the DNA duplex). However, there are examples of promoters that are inhibited by negative supercoiling, and which require a relaxed template for activation. The gyrA and gyrB genes, encoding the subunits of gyrase, have promoters of this type [138]. Since increased negative supercoiling enhances the efficiency of DNA unwinding, the mechanism of supercoiling-driven promoter activation is easy to understand. The mechanism by which DNA relaxation assists promoter activation is less clear. The G-C content of a promoter might be expected to influence the facility with which it can be opened, and hence make it susceptible to the influence of changes in DNA structure, such as supercoiling, that modulate DNA strand separation. Mutations that increase the number of G-C basepairs in a promoter can make it more difficult to melt, and this can be overcome by increased negative supercoiling of the DNA [152]. The G-C-rich discriminator region of stable RNA promoters such as the S. typhimuriwn hisR. a tRNA operon, makes the promoter a member of the stringently controlled regulon. These are genes controlled in response to the alarmones guanosine tetra- and pentaphosphate, and they are only expressed at high levels when the cell is growing rapidly [153]. The G-C-rich discriminator also imposes a well-characterized sensitivity to DNA supercoiling
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highly neijalivel)' %up^r^ml^
Fig. 7 Creation of two domains of supercoiling during transcription. A plasmid with two converging transcription units is divided into domains of relaxed and highly negatively supercoiled DNA when the genes are expressed. Dashed arrows show the directions the transcription complexes (TCs) will take on initiation. Coupled transcription and translation helps to establish the differentially supercoiled domains. Anchoring the ends of the DNA to nonrotatable supports, preventing supercoils from escaping by rotational diffusion, would achieve similar results in the case of single transcription units (see text).
on the promoter [154]. Thus, the nucleotide composition of the promoter can make a major contribution to its sensitivity to changes in DNA supercoiling. It has been suggested that the length of the spacer between the -10 and -35 boxes of the promoter might impose a sensitivity to DNA twist [155]. In one case where this has been carefully investigated, that of the proU promoter of S. typhimurium, which has a 16-bp spacer, DNA twist does not seem to be an issue in the supercoiling response. Instead, supercoiling imparts greater flexibility to the promoter DNA, presumably favoring productive interactions with RNA polymerase [156, 157]. In addition to supercoiling, other aspects of DNA structure contribute to control of gene expression. DNA cruciform extrusion contributes to the complex regulation of the bgl locus in E. coli, which also involves a H-NS protein, DNA gyrase,
3.
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and cAMP-CRP [158]. Loop formation promotes long-range interactions in the DNA, such as contact between regulatory proteins bound to remote sites and RNA polymerase at promoters [159, 160]. This type of action-at-a-distance is central to regulation of transcription by proteins such as the enhancer-binder NtrC [11] (Fig. 3). Such interactions are influenced by the intrinsic flexibility of the DNA, a feature that can be modulated by supercoiling. DNA curvature, a property determined by the base sequence of the DNA, can also influence long-range interactions [161, 162], and serve as a recognition site for the binding of proteins such as H-NS [105] (see above). Protein-induced DNA bending can serve a function similar to curvature, with the added benefit that, because the alteration in the trajectory of the DNA is protein dependent, it can be regulated by the availability of the protein and the accessibiHty of its binding site [163]. IHF is an excellent example of a protein that performs such a reversible DNA bending function, promoting long-range interactions in the genome [127].
XL Stereotypical and Stoctiastic Events in ttie Controi of Gene Expression The individual members of a clonal population of bacteria in a homogeneous environment might be expected to respond identically to a newly perceived stimulus. For example, if a pure culture of E. coli K-12 is subjected to a sudden drop in temperature, one might anticipate that the cold shock-inducible genes will be activated in every cell to a similar level. This is an example of a stereotypical response at the level of the gene to environmental stress. Tools for analyzing the expression of a given gene in individual cells within the population (as opposed to aggregate measurements of the entire population) have only become available recently, and there is little hard information on just how uniform such stereotypical responses are through the population. Nevertheless, data from studies of the survival of bacteria under stress indicate that in apparently homogeneous populations some cells are more prepared than others to deal with the environmental assault. Presumably, some of this inequality reflects variation at the level of the gene in the population, and may be influenced by the existence of microenvironments that alter gene expression sufficiently to give some population members an advantage (or place them at a disadvantage) in comparison with their apparendy identical neighbors. It may also reflect the operation of mutagenic processes within the population (see below). Variation could be achieved by exerting conventional gene regulatory mechanisms to differing extents in different cells of a clonal population. A role for microenvironmental influences is easy to imagine in such a scenario. For example, in a static liquid culture, cells at the liquid-air interface would experience a
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
different set of external stimuli to those within the body of the liquid. Thus, the gene expression profile of the interfacial cells will differ from those in the bulk liquid environment. Cells moving between these environments might display a third profile of gene expression, and so on for each subcategory of environment that may exist in this apparently homogeneous world. Of course, the environments encountered by bacterial pathogens during infection are much more complex than those experienced during laboratory growth and probably result in a very wide range of variation in the levels to which particular genes are expressed in the infecting population. In addition to conventional gene regulatory mechanisms, bacterial cells also possess the means to generate phenotypic diversity through altering the structure of the genome. These alterations can be extremely modest, as in methylation of a critical residue at a regulatory motif in the DNA, or more extensive, involving rearrangements of large stretches of DNA [164, 165]. Processes of this type that operate in a reversible manner are described as genetic switches. This distinguishes them from mutations, that is, small or large changes to the genome that are irreversible, or that reverse very rarely. An example of a reversible mutation is the insertion in a gene of a transposable element that subsequently leaves by precise excision, restoring gene function to normal. Genetic switches underlie the process of phase variation, in which a gene can exist in an expressed or nonexpressed state, with the state varying from cell to cell in the population. Switching between states can appear random, and phase variation is often regarded as an example of a stochastic genetic process. However, in some of the better-studied examples, clear roles are emerging for environmental stimuli in determining the rates at which such switches operate, and even in determining the directionality of the switch (i.e., favoring one phase or the other). Furthermore, regulatory features are being discovered that control the operation of the switch, and these may be under environmental control. Thus, these switches may not operate so randomly after all. Examples of genetic switches controlling phase and antigenic variation include inversion systems where a promoter is alternately connected and disconnected from the gene it transcribes, e.g., the fim switch controlling type 1 fimbrial expression in E. coli (Fig. 8) [166]; the tfp inversion system controlling adhesin expression in Moraxella lacunata [167]; shutde systems that move portions of genes into and out of expression sites in the genome, using an archive of silent gene copies as a source of alternative versions of a gene (such as the RecA-dependent pilus variation in N. gonorrhoeae [168]; Fig. 9); small RecA-independent sequence deletions or additions that involve slipped-strand mispairing among short directly repeated DNA sequences, altering the reading frame of genes coding for surface-expressed proteins (e.g., the opacity protein genes of A^. gonorrhoeae and A^. meningitidis', [169, 170]); the lipopolysaccharide loci of //. influenzae [171] (Fig. 10); alterations in the number of residues making up polymeric repeats that change the transcription or translation of the gene affected. This mechanism can influence transcription by altering the spacing of the elements of the gene
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REGULATION OF VIRULENCE GENE EXPRESSION IN BACTERIAL PATHOGENS
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F/g. ^ The switch controUing expression of type 1 fimbriae in Escherichia coli. ThtfimA gene codes for the major subunit of type 1 fimbriae, and its promoter is carried on a 314-bp segment of invertible DNA (the//m switch). GQUtsfimB and fimE encode recombinases for inverting the switch between ON and OFF phases. In the ON phase, the /•//??A promoter (PfimA) is directed toward the fimA gene; in the OFF phase, it is directed away. In the expanded views of the switch, binding sites for the recombinases are shown, and these overlap the inverted repeats IRL and IRR, which undergo recombination during DNA inversion. Also shown are binding sites for the accessory proteins integration host factor (IHF, two sites) and the leucine responsive regulatory protein (LRP, three sites).
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
pilE gene mciy
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F/g. 9 RecA-dependent pilin gene antigenic variation in Neisseria gonorrhoeae. The structures of the pilE pilin gene, and a silent gene, pilS, are shown. The coding sequence of the gene is composed of highly conserved and variable regions, the latter called minicassettes (MCs). RecA-dependent reciprocal recombination between the conserved sequences (represented by the crossovers to the left of mc4 and the right of mc2 in the figure) permits genetic information from the silent locus to be shuttled into the expressed locus. The result is a novel pilE gene sequence, coding for a new form of the PilE pilin protein.
REGULATION OF VIRULENCE GENE EXPRESSION IN BACTERIAL PATHOGENS
N
(CTCTT)n
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101
full-length protein
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(CTCTT)7or8 C out-of-frame; premature termination
Fig. 10 Phase variation in the opacity protein genes of N. gonorrhoeae and N. meningitidis. The opa genes have pentameric repeats of CTCTT within the region coding for the amino-terminal domain of the protein. Variations in the numbers of these repeats result in phase variation due to the effects on the reading frame of losing or gaining repeats. Repeat numbers vary during DNA replication as a result of slipped-strand mispairing, and this may involve the transient creation of novel DNA structures (see text).
promoter (e.g., the hifA and hifB genes of H. influenzae that encode the major subunit protein and the chaperone protein required for fimbrial expression [172]; Fig. 11), by altering the spacing between a promoter and an adjacent regulatory protein-binding site (such as in the case of the //Vn genes of B. pertussis [173]; Fig. 11), or it can alter the reading frame of the gene, interfering with its translation (e.g., iht pi IC pilus assembly gene of N. gonorrhoeae [174]; Fig. 11), the ope outer membrane protein gene of N. meningitidis [175], or the bvgS virulence gene of B. pertussis [176]). Switches of such diversity operate via a number of molecular mechanisms. All involve changes to the structure of the genome, and the rate at which these changes occur can be modulated by several factors. Examples of modulatory influences include the formation of unusual DNA structures, such as triple-stranded H-DNA, or the influence of transcription in the case of neisserial opa genes [177, 178]. These can serve to integrate the operation of the switch with other processes occurring in the genome, and may overrule its apparently random mode of operation.
XIL The Switch Controlling Type 7 Fimbrial Expression in E. coli The genetic switch controlling expression of type 1 fimbrial gene expression in E. coli is proving to be a very valuable guide to the principles by which a random genetic event can be brought under control and, perhaps, made to operate according to a program. The switch consists of a promoter carried within a short
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
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Fig. 7/ Phase variation based on homopolymeric DNA tracts. Top: the spacer common to the promoters of the hi/A and hifB genes of H. influenzae contains a poly-TA tract. Loss of TA dinucleotide repeats results in a reduction in promoter efficiency or complete inactivation. Center: the coding region of the pilC gene of A^. gonorrhoeae includes a poly-G tract. Loss of a G residue from this tract results in a shift in the reading frame of the gene and premature termination of translation. Bottom: the spacer between the promoter and binding site for a positive regulator in iht fimS gene of Bordetella pertussis contains a poly-C tract. Loss of C residues from this tract alters the spacing between these elements and makes the promoter nonfunctional.
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stretch of invertible chromosomal DNA. The promoter is required for transcription of the//mA gene, encoding the type 1 fimbrial subunit. The invertible element is flanked by 9-bp inverted repeats, and these form part of the binding sites for the site-specific recombinases that operate the switch. These proteins, FimB and FimE, are encoded by nearby genes {finiB and fimE, respectively), and are members of the integrase family of recombinases [179, 180]. They are very closely related, and show 48% identity in amino acid sequence. The FimB protein can invert the switch in both directions with approximately equal efficiency, whereas the FimE protein has a marked tendency to turn the switch off. Therefore, the switch is subject to directional bias, depending on which recombinase is acting at the inverted repeats at any time. The FimB and FimE recombinases do not act alone. They have requirements for two accessory proteins, IHF and LRP, both of which have been described earlier herein. The IHF protein binds to two sites, one in the switch and one just outside [181] (Fig. 8). The site within the switch is bifunctional since it is also required for operation of the fimA promoter. Without occupancy of this internal site by IHF, the switch ceases to function and the power of the promoter is reduced sevenfold [179]. The contribution of IHF to operation of the switch seems to be architectural; it probably promotes interactions between the inverted repeats and their associated recombinases that are favorable for recombination. A similar role has been described for LRP, which has three binding sites within the switch [182]. LRP interaction with the switch is enhanced by leucine, isoleucine, valine, and alanine, linking switch operation to nonpolar amino acid pools in the cell. The switch is also subject to modulation by H-NS. This protein binds to and represses the promoters of the recombinase genes and may also interact with the switch direcdy, retarding the rate of inversion. The latter possibility is consistent with the preference of H-NS for curved DNA since the fim switch includes DNA elements displaying a strong potential for curvature. In addition to the influences of two recombinases and three accessory proteins, the fim switch is also highly sensitive to the degree of supercoiling of its DNA. Departures from optimal levels of supercoiling can either inactivate the switch (too supercoiled), or cause it to become heavily biased toward one outcome (too relaxed) [183]. All of these influences serve to constrain the freedom of the switch to operate randomly. Instead, it seems to display several dependencies simultaneously. These may serve to direct the switch in ways that benefit the cell during its interacdons with a host, or while it is in a free-living state.
XIII. Pap Pilus Gene Transcription Pap pili assist uropathogenic E. coli in attachment to uroepithelial cells [184]. The pili are expressed at 37°C but not at 23°C, and are subject to phase variadon at the higher temperatures [185]. It has been suggested that low temperature is a cue that the bacteria are outside the host, and so it is unnecessary to express the pili [186]. The control of transcription and phase variation in the pap system is complex and multifactorial. Similar circuitry governs expression of the E. coli F1845 and S pili.
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
encoded by the daa and sfa operons, respectively [187]. Phase variation is a function of the differential methylation by the Dam methylase of two GATC sites in the pap regulatory region called GATC-I and GATC-II [188] (Fig. 12). This methylation, involving just two basepairs, represents the modification to the genome that underlies phase variation in the pap system. In Phase-ON cells, GATC-I is unmethylated while GATC-II is methylated; the converse is true in Phase-OFF cells [ 189]. Methylation at these sites is blocked by the LRP protein. In Phase-OFF cells LRP binds cooperatively to three sites (LRP sites 1, 2, and 3) in the pap regulatory region and so blocks methylation of GATC-II (which is part of LRP site 2). In Phase-OFF cells, GATC-I remains available for methylation by Dam. Methylation of GATC-I reduces the affinity of LRP for sites 4 and 5. Failure of LRP to bind at sites 4 and 5 prevents/7(3/?^/transcription, locking cells in the OFF phase until after the next round of DNA replication [188, 190]. The Papl protein collaborates with LRP to block methylation of GATC-I. The proteins form a complex that has enhanced LRP affinity for sites 4 and 5 at GATC-I. Shifting LRP binding to GATC-I frees GATC-II for methylation by the Dam methylase. The LRP protein is a transcription activator when bound to GATC-I but is a repressor when bound to GATC-II [191, 192] (Fig. 12). Transcription of papl is activated by the PapB protein, and the cAMP-CRP complex activates both papB and papl, with cAMP-CRP playing the role of an
^ papB
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antirepressor [193-195]. The nucleoid-associated protein H-NS is involved in thermoregulation of pap gene expression, where it acts as a repressor of transcription at low temperatures [196]. The binding of H-NS to the pap regulatory sequences containing the GATC-I and GATC-II sites prevents methylation, although the mechanism by which this occurs is not understood [186]. Thus, the regulation of pap gene expression occurs at many levels. On top of phase variation, expression of pap responds to the metabolic state of the cell (via cAMP-CRP) and to temperature via a mechanism that involves H-NS.
XIV. Contact'Dependent Gene Regulation The pap genes of uropathogenic E. coli have been implicated in a form of gene activation that is triggered by contact with the host. Induction requires the presence of the PapG adhesin protein that recognizes a specific carbohydrate receptor on uroepithelial cells, and the presence of this receptor. Among the targets of the induction is barA (or airS), a gene required for production of siderophores (iron-chelators) and their receptors, and without which E. coli cannot grow in urine. BarA/AirS shows homology to histidine protein kinases, raising the possibility that it senses Pap-mediated contact and transmits the signal to virulence genes via an unidentified response regulator [197]. In the human and rodent pathogens Yersinia pseudotuberculosis (which causes adenitis and septicaemia), Y. enterocolitica (gastrointestinal infections), and Y. pestis (bubonic plague), contact with host cells results in export of a bacterial transcriptional repressor, allowing previously repressed virulence genes on a plasmid to become activated [198, 199]. The repressor is the LcrQ protein (equivalent to the 57% identical YscMl and YscM2 proteins in K enterocolitica, encoded by closely related genes [200]), and is exported from the bacterial cytosol into the external medium via a plasmid-encoded type III secretion system that is triggered by host-microbe interaction. Plasmid-linked yop virulence genes that are subject to LcrQ repression are then transcribed. LcrQ is more accurately described as an anti-activator, since it inhibits the ability of the LcrF protein to activate yop gene transcription. (LcrF, called VirF in Y. enterocolitica, is an AraC-like transcription factor that responds to temperature.) The LcrQ protein has not been shown to bind to yop DNA, and it may exert its negative regulatory effects indirecdy [198, 201]. This regulatory mechanism is reminiscent of one employed in the control of flagellar biosynthesis in S. typhimurium. There, the counterpart of LcrQ is an anti-sigma factor called FlgM, which is secreted via the type III secretion system used to assemble flagellae and an integral component of the flagellum [202].
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
XV. The Virulence Gene Regulatory Cascade of S. flexneri S. flexneri causes bacillary dysentery in humans, and is a facultative intracellular pathogen. The bacteria enter and replicate within the cells of the colonic epithelium, and move between cells. The process involves M-cell entry, basolateral invasion of epithelia, actin-dependent intra- and intercellular movement, and macrophage killing. The genes required for expression of the pathogenic phenotype are located on a high-molecular-weight plasmid. They occupy 31 kb and consist of ipa genes coding for secreted proteins needed for invasion of host cells, the mxi and spa genes encoding components of a type III secretion system required for Ipa protein secretion [203, 204], and regulatory elements that govern the expression of the virulence factors (see below). The S. flexneri virulence gene regulatory system illustrates many points that are of general relevance in bacterial pathogens (Fig. 13). The key genes are grouped in operons under the collective control of a regulatory protein. The specific regulators and all of the structural genes are located on a plasmid, but chromosomally encoded proteins with wide-ranging effects in the cell also influence expression of the virulence genes. Gene expression is under tight control and responds to particular environmental influences. In the S. flexneri system, a considerable amount of molecular detail is available [60], and a very similar system is found in enteroinvasive E. coli (EIEC) [29, 205]. The control regime takes the form of a cascade, at the top of which is an AraC-like protein called VirF. This plasmid-encoded protein binds to and activates transcription of a gene coding for a subordinate regulator called VirB [206]. This protein is presumed to have DNA-binding activity (it shows homology to DNA-binding proteins involved in plasmid maintenance) and is required for transcriptional activation of the structural genes. These are organized in large, divergendy transcribed operons (Fig. 13). The virB regulatory region is the key control element in the cascade. It seems to be occupied by the VirF protein under both activated and nonactivated conditions. Activation requires a temperature of 37°C and physiological levels of osmolarity, together with a pH of 7.4 [207-209]. The pH control is exerted positively via the CpxA/CpxR two-component regulatory system, and CpxR has been shown to bind directly to the upstream region of the v/rFgene [210]. The organization of the genes into a regulatory cascade allows for very tight control of expression coupled with rapid activation of the system once appropriate environmental signals are received. The cascade displays a gearing effect in which the gene at the top, virF, is regulated over only a twofold range, while the next gene, virB, is controlled over a 10-fold range, and the structural genes at the bottom of the cascade experience a 100-fold range of regulation. Control of virF expression is loose at the level of transcription, while virB is more tightly
3.
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REGULATION OF VIRULENCE GENE EXPRESSION IN BACTERIAL PATHOGENS
i
.^
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w w Fig. 13 The virulence gene regulatory cascade of Shii^ella flexueri. The virulence regulon occupies approximately 33 kb of a 230-kb virulence plasmid. Large gaps between genes have been deleted for convenience, and are represented by short diagonal lines through the genetic map shown in the center of the figure. The virF and virB genes are illustrated in more detail at the top and bottom of the figure, respectively. Positive regulatory inputs are represented by downward vertical arrows above, and negative influences by T-shaped symbols below the promoter regions. See text for details.
controlled, with the structural genes being under the tightest control of all [211]. This regime seems to ensure that energetically wasteful expression of the structural genes under inappropriate conditions is avoided while allowing for sufficient expression of the regulatory proteins under nonpermissive conditions to facilitate a rapid activation of the structural genes when inducing conditions
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
arise. The system also has the capacity to permit fine-tuning of individual structural operon promoters once activation has occurred [211]. The virB promoter provides one of the clearest examples of a role for DNA supercoiling in the environmental activation of a virulence gene [212, 213]. Although the VirF protein is required absolutely for virB promoter activation, no activation occurs at 30°C (i.e., in the absence of a thermal signal) unless the level of supercoiling of the virB promoter is increased by artificial means [213]. Since increases in temperature cause a general increase in negative supercoiling in bacterial DNA, the simplest interpretation of these data is that the change in DNA structure resulting from a transition to growth at 37°C helps either to drive open complex formation or to improve the interaction between bound VirF and RNA polymerase, or both. It is also a possibility that the multimerization of VirF on DNA may be driven by DNA structural changes at the binding site. Other AraC-like proteins, such as the E. coli MelR protein, display DNA-dependent multimer formation that results in a change in their biological activities [214]. The dependence of the virB promoter on DNA structure for activation is also consistent with a requirement for a normal complement of DNA topoisomerases in the cell [212, 215, 216]. In addition, the promoter is repressed by the H-NS nucleoid-associated protein and requires the IHF protein for full activity in stationary phase [118, 130, 212]. Both H-NS and IHF contact the virB regulatory sequences directly. H-NS levels in the cell are maintained at a near-constant value of about 20,000 copies per cell [217], except in cold-shock, when the level increases by about three- to fourfold [218, 219]. One of the tasks of VirF may be to overcome the repression imposed by H-NS at the virB promoter, a task it carries out in collaboration with thermally induced increases in the negative supercoiling of the DNA [60, 220]. As cells enter stationary phase, their DNA becomes relaxed, a situation that would inhibit supercoiling-dependent promoters such as that of the virB gene [141]. The function of IHF in stationary phase may be to offset this negative effect by maintaining the virB promoter in an active conformation [130]. The virF gene is under posttranscriptional control and provides a good example of the power of translational regulation to influence the expression of a virulence gene regulon. Mutations in the miaA gene, coding for the tRNA A^-isopentyladenosine (i^ A37) synthetase, which is required for the synthesis of the modified nucleoside 2-methyl-A^-isopentyladenosine, a component of tRNA, have a negative effect on the translation of virF mRNA, and hence on the expression of the other genes in the regulatory cascade [221]. Normal expression of the virF gene also requires a functional tgt gene (called vacC in S. flexneri) that codes for tRNA-guanine transglycosylase, required for the synthesis of modified bases in tRNA. Mutations in tgt {vacC) interfere negatively with translation of virF mRNA, and hence with expression of the virulence genes that depend on VirF [222]. The location of the S. flexneri virulence genes on a plasmid is also significant. In EIEC, this plasmid has been shown to integrate at a specific location on the chromosome. This integration event is accompanied by shutting down of vim-
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lence gene expression, caused by an H-NS-dependent repression of virB transcription [223]. The plasmid also displays significant instability, being lost from the cell when expression of the virulence genes is induced. The instability depends on the functioning of the virF and virB genes and suggests that expression of the structural gene operons makes the plasmid difficult to maintain [224]. The virulence genes are also prone to inactivation by insertion sequences and to DNA rearrangements. Inactivation of virF and virB prevents virulence gene expression and stabilizes the plasmid, although the significance of this in vitro observation is not understood fully. In the case of insertion sequence mutations, these may reverse precisely in the future, restoring virulence gene expression. Thus, the inactivating mutations may serve as a means of ensuring that the plasmids are maintained in the absence of selective pressure [224]. The S. flexneri virulence gene control network also illustrates the point that both specific and general regulators of gene expression can contribute to control of expression of a particular phenotype. In this case, the phenotype is invasiveness, and the specific control elements (VirF and VirB) are encoded by the virulence plasmid, while the general control elements (H-NS, IHF, the DNA topoisomerases, and the tRNA modification systems) are encoded by the chromosome. The general regulatory elements key the virulence genes into the global gene expression program of the cell, while the specific elements operate at a local level to optimize and fine-tune virulence gene expression.
XVL A Thermometer Protein from ttie Salmonella Virulence Plasmid The tlpA gene on the large virulence plasmid of S. typhimiiriiim encodes an autoregulatory repressor protein called TlpA [225]. This protein responds to changes in temperature by shifting ixom an inactive unfolded monomer to an active folded coiled-coil dimeric protein. Its ability to bind to the tlpA gene promoter and repress it is a function of the intracellular concentration of TlpA (which influences protein-protein interactions) and the thermally determined stmcture of the protein. At 22°C, the promoter is repressed, and derepression occurs as the temperature increases. The shift in the structure of the protein is reversible; in other words, TlpA does not become denatured permanently by increases in temperature. No accessory proteins are required for TlpA action at the tlpA promoter, and thermally induced changes in DNA conformation do not appear to be an issue. It will be interesting to see if more examples of this type of regulatory protein emerge in the future. It has been pointed out that a DNA sequence motif characteristic of the tipA promoter is found in several virulence genes, including spvA on the plasmid, and the PhoP/PhoQ-regulated genes prgH
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CHARLES J. DORMAN AND STEPHEN G. J. SMITH
and pagC on the chromosome [225]. It will be important to determine which genes, if any, in addition to tlpA are under the control of the TlpA protein.
XVII. CelI'Density'Dependent Regulation Although bacteria are unicellular, they can collaborate to perform certain functions, some of which are of benefit to pathogens during infection of a host. At the heart of these processes Hes a mechanism by which the bacteria sense their own population density. Various forms of bacterial group behavior are regulated by small diffusible self-produced regulatory molecules. Individually, bacteria are incapable of producing the signal in sufficient quantities to elicit a response, but as a population increases in density a critical threshold is crossed and signaling results in a response, usually at the level of transcription, that alters bacterial behavior [226-228]. Early research on cell-density-dependent regulation focused on the bioluminescent phenotype in certain marine bacteria, such as Photobacterium fischeri (formerly Vibrio fischeri). When free-living, and at a low culture density, these bacteria do not emit light. When the population density increases, as during growth in the specialized light organ of the fish with which they have a symbiotic relationship, the bacteria become bioluminescent and emit a blue-green light. This is caused by accumulation of a low-molecular-weight A^-acyl-L-homoserine lactone (AHL) that is synthesized by the bacteria, and sensed by the population. The particular AHL made by P. fischeri is A^-(3-oxo)-hexanoyl-L-homoserine lactone (OHHL). The sensor/regulator is the autoregulatory membrane-associated LuxR protein, and, on binding OHHL, this activates transcription of the bioluminescence genes, which are organized as an operon on the bacterial chromosome (Fig. 14). Also in this operon is the gene for Luxl, which is involved in production of OHHL. Thus, upregulation of the lux operon involves increased transcription of the gene for signal production. It is proposed that downregulation occurs because the LuxR protein is unstable and is subject to rapid turnover by the Lon protease [228]. The population-density-dependent system used by another marine bacterium, V harveyii, has already been discussed in the context of the modular nature of bacterial regulatory proteins (see above). Here, the signal-reception and -transmission system is of the type used by the "two-component" regulators and is based on a phosphotransfer mechanism [91]. Among Gram-negative pathogens, cell-density-dependent regulation based on AHL signaling has been characterized in P aeruginosa, where two systems operate to control expression of a wide array of virulence genes. One system is composed of LasI, a signal generator, and LasR, a response regulator, and is sensitive to A^-(3-oxo)-dodecanoyl-L-homoserine lactone (OdDHL). This controls
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Fig. 14 Cell-density-dependent gene regulation in Photohactehiim {Vibrio) fischeri. Expression of lux genes is controlled by a diffusible autoinducer (OHHL, see text for details). This is in low concentrations at low culture densities, and the luxICDABEG operon is expressed at a basal level. Autoinducer is synthesized by the Luxl protein, and no single cell can produce enough to induce bioluminescence. At high culture densities, autoinducer levels are sufficient to activate luxICDABEG transcription. This occurs via the LuxR sensor protein, which also acts a repressor of its own gene.
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the expression of alkaline protease, elastase, exotoxin A, exoenzyme S, neuraminidase, and hemolysin. The second system is composed of VsmI (or Rhll), a signal generator, and VsmR (or RhlR), a response regulator. It is sensitive to A^-butanoyl-L-homoserine lactone (BHL), and controls expression of chitinase, elastase, hydrogen cyanide, pyocyanin, rhamnolipid, lectins, staphylolytic activity, hemolysin, and alkaline protease [229, 230]. Cell-density-dependent regulation also contributes to the ability of P. aeruginosa to form biofilms, an important feature of the pathogen in colonization of catheters, and in growth in the lungs of cystic fibrosis patients [231]. Interestingly, the RhlR protein and the BHL signal are required for activation of the rpoS gene, encoding the sigma-38 component of RNA polymerase [232]. This points to a very wide network of effects exerted by this cell-density-dependent regulatory system in the cell, extending beyond dedicated virulence genes to include genes involved in general adaptation by the cell to changes in growth phase and environmental composition. Detailed information on cell-density-dependent regulation and bacterial virulence is also available for the Gram-negative plant pathogens A. tumefaciens, Erwinia carotovora, and Erwinia stewartii [233]. In A. tumefaciens, the cause of crown gall tumors in plants, A^-(3-oxooctonoyl)-L-homoserine lactone (OOHL) regulates transfer of the tumor-inducing plasmid from one bacterial cell to another by conjugation. In E. carotovora, OHHL regulates production of carbapenem antibiotics. It also regulates the expression of the virulence factors protease, cellulase, and pectinase in the same organism, and exopolysaccharide production in E. stewartii [233]. In the case of E. carotovora, OHHL coordinates the expression of an antimicrobial agent (the carbapenem antibiotic) and the expression of three virulence factors required for successful aggression against a plant host. This may be a way in which the pathogen can eliminate bacterial competitors while ensuring that it undertakes an attack on the host only when the bacterial population is of sufficient size. Among Gram-positive bacteria, similar population density regulatory strategies are in use, albeit with different types of signaling molecules. Typically, a complex phenotype is controlled by a two-component regulatory system that responds to a peptide signal. In the case of Staphylococcus aureus, several virulence traits are controlled by the accessory gene regulatory (agr) locus. In the laboratory, as the culture enters the late exponential phase of growth, agr coordinates repression of cell-surface-associated factors (e.g., protein A, fibronectin binding protein, and coagulase) and induction of secreted components (such as alpha toxin, betahemolysin, toxic-shock syndrome toxin, enterotoxin, lipases, and proteases). The signal is an 8-amino acid peptide called AgrD, which is derived from a 45-amino acid precursor. AgrD production requires AgrB, with which it is translationally coupled. These factors are translated from RNAII (Fig. 15). Transcribed divergently from RNAII is RNAIII, and this is under positive transcriptional control. The AgrD pheromone is one of three signals that activates RNAIII production; the others are the genetic loci sar, xpr, and the RNAIII-activating protein, or RAP [234-237].
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Fig. 15 The Agr regulatory system of Staphylococcus aureus. The effector molecule of the agr locus is RNAIII. It is under the control of the AgrD pheromone. whose synthesis requires the agrB and agrD genes. The agrA and agrC genes code for a two-component signal transduction system that responds to AgrD levels. AgrA-P probably operates on the <:/^i,'/- locus indirectly.
The AgrD peptide binds to a histidine protein kinase encoded by the agrC gene, leading to phosphorylation of the AgrA response regulator [238]. Direct evidence that AgrA is a DNA-binding protein is lacking, but it may exert its effect through interaction with SarA, which has been shown to bind to the RNAIII promoter [239]. Once expressed, RNAIII can influence either transcription or translation of the genes it controls. The mechanisms by which RNAIII influences transcription, positively or negatively, remain obscure. It can facilitate translation of the alpha toxin by binding to mRNA and inhibiting formation of a stem-loop that sequesters the ribosome-binding site [240]. In this way, it performs a function that is the opposite of an antisense RNA. Cell-density sensing systems in Gram-positive bacteria control several properties, including plasmid transfer in Entewcoccus faecalis\ expression of the competence phenotype in Streptococcus pneumoniae, S. sanguis, and Bacillus subtilis; nisin production in Lactobacillus lactis; and antibiotic production in Streptomyces griseus. With the exception of the actinomycete S. griseus, where a y-butyrolactone is used, all of these population-densitydependent regulatory systems involve signaling peptides, and several require two-component systems to transduce the signal [224].
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XVIIL Adoptive Mutation When a population of bacteria is subjected to environmental stress, a subpopulation undergoes hypermutation, allowing novel genetic combinations to emerge that result in bacteria that are more fit in the stressful environment [241-243]. This capacity to bring forth improved strains under stress operates in parallel with the conventional regulatory mechanisms that have evolved to enhance the survivability of every cell in the population. Regulation operates (almost) universally in the population and is reversible; the hypermutable state operates in a subpopulation, and its products cannot revert easily to their previous genetic state. In the main, the subject of adaptive mutation has been treated as a laboratory phenomenon. In part, this reflects the model systems that have been used to study it. Typically, these have been mutations in carbohydrate utilization operons in E. coli [244-246]. The concept of adaptive mutation in relation to infection and the microevolution of a population of bacteria in association with the host deserves to receive detailed attention [247]. This is a difficult research topic and needs more work. Results from one study indicate that mutator strains occur at a very high frequency (over 1 %) in populations of pathogenic E. coli and Salmonella species [248]. The mutators are defective in methyl-directed mismatch repair, typically a defect in the mutS gene, allowing rapid genetic variation to generate derivatives with enhanced virulence characteristics and antibiotic resistance. Presumably, these strains have been selected for by the environments in which these pathogens live. Under laboratory growth conditions, such mutator strains are destined for extinction, but in the wild they clearly have an advantage and so are maintained in the population at a high frequency.
XiX, Rote tRNAs ond Tronslotion Modulotion It has been suggested that there is a bias toward the use of rare codons in genes for regulatory proteins [249], although there seems to be litde evidence to support this view [250]. Instead, it seems that codon usage is a passive reflection of accumulation of random mutations rather than a mechanism for modulation of gene expression. Rare codon bias and its associated slow translation rates do not necessarily lead to lower levels of gene expression [251]. Strong modulation requires a coincidence of rare codon usage with sites prone to mishaps in translation. An important site for such events is the ribosome-binding site [252-254]. When rare codons are grouped near here, strong negative effects on gene expression may result due to premature termination of translation or queuing of ribosomes [255-259]. Elsewhere in the gene, the use of rare codons can result in frameshifting and premature termination and decoupling of transcription and translation. Such effects are subsidiary to slow translation of the rare codons, and can be overcome by increasing expression of the rare tRNA species [256, 259].
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The integrase protein of bacteriophage lambda is subject to translational modulation by the rare arginine codons AG A and AGG [260]. This protein is the site-specific recombinase that allows lambda to enter and leave the E. coli chromosome at the lambda attachment site, and it is related to the recombinases that operate the //m switch in E. coli [179, 180] (see above). Here, the FimB recombinase has also been found to be subject to modulation by a rare codon, in this case a leucine codon that is decoded by the product of the leuX gene. The fimB gene ofE. coli K-12 contains five of these codons, and the levels of the leuX gene product are thought to govern the rate at which fimB mRNA is translated, thus creating a dependency on the leiiX gene for normal expression of type 1 fimbriae [261]. The leuX gene can be inactivated, with concomitant loss of type 1 fimbrial expression, in uropathogenic E. coli that undergo deletion of their copy of the Pai II pathogenicity island. This island is inserted immediately adjacent to the leuX locus, and it deletes at a high frequency. Disruption of the leuX sequence appears to be a result of the deletion event [262]. This relationship between//mi5 and leuX is more accurately described as one of dependency rather than a regulatory one.
XX. Protein Splicing Protein splicing represents an unusual form of regulation. A small number of bacterial genes, including some from pathogens, code for proteins that must undergo splicing in order to become active [263]. By analogy with RNA splicing, one refers to "inteins" and "exeins" when describing the spliced and flanking structures, respectively. Examples include the RecA proteins of Mycobacterium tuberculosis and Mycobacterium leprae, and the GyrA protein of some mycobacteria [263, 264]. In each case, a single "intein" insertion resides within the protein, and this must be excised for the protein to become active. Excision is thought to be by an autocatalytic mechanism and proceeds via a branched protein intermediate [265]. Work with the 440-amino acid RecA intein of M. tuberculosis shows that this is a mobile genetic element that invaded the recA gene using a site-specific endonuclease activity [266]. The process by which an intein invades an intein-less gene with an appropriate insertion site is referred to as "intein homing" [263].
XXI. AntisenseRNA Antisense RNA is so called because its sequence is complementary to that of the transcript of the gene it is regulating. Regulation by antisense RNA is widely used in bacteria, especially by plasmids and phage [267, 268]. There are some instances of chromosomal gene regulation by this mechanism, and some of them involve genes with a proven role in pathogenesis. For example, the crp gene is inhibited at the level of transcription by an antisense RNA species called tic that complexes
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with the 5' end of crp mRNA [269]. Upstream of the ompC gene, and transcribed on the opposite strand, is a gene for the m/cFantisense RNA. This molecule shows complementarity to the 5' end of ompF mRNA, and it can complex with this to inhibit translation. It is thought that micF contributes to the shutting down of ompF gene expression in response to temperature increases that favor expression of the OmpC porin [270]. Sensitivity to micF aho extends the range of regulatory inputs influencing ompF gene expression. In addition to the direct inputs of the OmpR response regulator partner of the EnvZ histidine protein kinase, OmpF is also controlled (via micF) by LRP, H-NS and SoxRS [76-78]. In this way, micF may be regarded as an integrator of regulatory circuits in the cell.
XXIL Perspective Our understanding of gene regulation in bacteria has reached a very high level. Much of our information has been obtained from experiments with bacteria grown under laboratory conditions, often involving highly focused investigations of individual genes or operons. This approach has carried us a long way. The complexity of the regulatory circuits in use by bacteria can seem daunting at times, and certainly exposes the naivete of the view that bacteria are "simple" organisms. However, despite the current breadth of knowledge and the depth of understanding of many aspects of the regulatory processes in prokaryotes, for those interested in bacterial pathogens and their interactions with the host and with the external environment, the present levels of knowledge and understanding remain insufficient. At the time of this writing, the prokaryotic gene regulation field is in a state of flux due to the explosion of new knowledge from whole genome projects [271]. These projects provide us with catalogues of the genes in cells but tell us little about the manner in which expression of the genetic information is controlled. Nevertheless, because of the predictive power of genomics, many new regulatory proteins and other control elements are being discovered, and these must be fitted into the current gene regulation circuit diagrams. This process will require much experimental work. Knowledge of virulence gene regulation is being expanded through the application of recent technologies such as signature-tagged mutagenesis [25] and in vivo expression technology [24], the use of reporters such as the green fluorescent protein [272], and the exploitation of DNA chip technology [273]. These give a more true-to-life impression of which genes are important to bacteria during infection of the host, or for survival in tissue culture. They also provide important insights into what are the key signals provided by the host environment for gene control in pathogens. By combining the data from such studies with information from whole genome sequences, a more integrated view of bacterial gene regulatory circuits is in prospect.
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A renewed awareness of the importance of mutation (changes in the DNA sequence of the genes in the cell) to bacterial adaptation to the host is likely to be an essential component of a truly holistic view of the gene expression options available to the pathogen [231]. An important task for the immediate future will be to determine the mechanisms by which environmental changes can influence molecular processes that lead to sequence diversity within apparently clonal populations of bacteria [274]. This information must then be integrated with knowledge of "conventionaf gene regulation to give a more complete picture of the bacterial cell in action during infection. It will also be important to extend understanding at the molecular level beyond the individual bacterial cell, to take account of the role of intercellular signaling in the successful establishment of an infection. This movement is already underway in the field of cell-density-dependent regulation, and illustrates the importance of appreciating that the bacterium is not simply an entity within an environment, but is itself a component of that environment. It seems that the future of this very reductionist, molecular field must include many elements that have been regarded traditionally as aspects of ecology. This is a strong indication that molecular biology in prokaryotes is becoming truly integrative.
Acknowledgments This work was supported by grants from the WeUcome Trust and the European Union.
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CHAPTER 4
Strategies to Identify Bacterial Pathogenicity Factors ANDREW CAMILLI D. SCOTT MERRELL JOHN J. MEKALANOS
I. Introduction II. Biochemical Strategies A. Classical Approaches B. Chemical Modification Screens C. Zymography D. Receptor/Ligand Affinity Screens E. Immunological Methods F Subtractive Hybridization G. Differential Display H. Reverse Genetics III. Genetic Screens A. In Vitro Screens B. In VMY; Screens IV. Genetic Selections A. Direct Selections B. Complementation Approaches C. Selection for Nongrowing Bacterial Mutants D. //? V/IY; Expression Technology V. Genomic Approaches A. Genome Walking B. Genomic Analysis and Mapping by /// Vitro Transposition C. Computational Screens D. Transcriptional Profiling and the Use of Microarrays VI. Concluding Remarks References
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/. Introduction Since Pasteur, Lister, Koch, and their contemporaries first proved the germ theory of disease in the second half of the nineteenth century, humans have been striving to understand the pathogenic mechanisms of the many reprehensible microbes that plague humans. This desire to understand the microbial causation of many human diseases has led to many epidemiological studies and to increased knowledge of the diverse environmental reservoirs occupied by pathogenic bacteria. The information garnered has been extremely helpful in combating many diseases and has shed light on the persistence of pathogenic bacteria in the absence of a human host. For example, an understanding of the ability of Vibrio cholerae to survive within an aqueous environment led to the development of water treatment strategies that have proven successful in the prevention of cholera and similar diarrheal diseases. Two additional lines of predominantly empirical research—vaccine development and the discovery of antimicrobial compounds—have also had tremendous impact on the prevention and treatment of many bacterial diseases. However, there is now considerable evidence that science has begun to exhaust these empirical lines of research, placing the scientific community at the dawn of an era when detailed knowledge of the cellular and molecular aspects of host-pathogen interactions will seed the future development of novel vaccines and novel antimicrobial compounds. Essential to the establishment of a complete understanding of the host-pathogen interactions that are necessary for the manifestation of disease is the identification and detailed characterization of virulence factors produced by pathogens at each stage of the infection process. The other chapters of this book detail the progression of the understanding of many host-pathogen interactions that have been elucidated during the past century. In contrast, the present chapter focuses on the various biochemical, immunological, and genetic strategies that have been successfully employed by microbiologists to identify these bacterial pathogenicity factors. In addition, speculation on the utilization of several postgenomic strategies that are currently being developed is presented in an attempt to illuminate future methodologies, which should prove useful in further discoveries. A listing of each technique/strategy along with a brief description of some of their advantages and disadvantages is shown in Table I. Bacterial pathogenicity is a multifactorial and dynamic process that requires coordinate production and repression of many bacterial gene products. Though each bacterial pathogen interacts with the human host in a unique way, common events occur in many infections. These common events involve: (1) primary attachment to host cells or tissue, (2) invasion into host cells or tissues (in the case of invasive pathogens) or increased adherence by extracellular pathogens, (3) avoidance of and/or resistance to host immune defenses, (4) acquisition of nutrients, (5) multiplication, and, lasdy, (6) evacuation from the host to either a new host or an environmental reservoir. Invariably, at one or more of these stages.
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bacterial-produced toxins and/or the host immune response result in damage to host tissues or organs and give rise to pathology. Although a tremendous amount of knowledge has been amassed on the identity and function of toxins and other secreted or surface factors produced by bacterial pathogens, it is becoming more and more evident that the investigation of bacterial pathogenicity must be broadened to include an understanding of cytosolic factors that are involved in processes as diverse as production of metabolites, multiplication, and adaptation to dynamic and stressful environments. The reason for this is simple: any factor produced by a pathogen that plays a role in survival or multiplication in the host environment is, a priori, suitable as a target for inclusion in vaccine formulations or for development of antimicrobial drugs. Accordingly, in some sections of this chapter the reader may note the use of a broader definition of "pathogenicity factor" that includes any factor produced by a pathogen that plays a role in its survival, multiplication, or spread to new hosts.
//. Biochemical Strategies A.
Classical Approaches
Prior to the dawn of the recombinant DNA era, microbiologists seeking to identify bacterial pathogenicity factors primarily focused their efforts on the biochemical purification of macromolecules that could be associated with disease symptoms and could be produced by the pathogen during laboratory growth. This classical approach is still utilized today, although usually in conjunction with immunological and/or genetic methods that increase the breadth of the approach. For example. Western blot analysis using human convalescent serum could be used to identify proteins secreted by a pathogen during in vitro growth. Once the protein was identified, it could be purified by two-dimensional gel electrophoresis, an amino-terminal portion of the protein sequenced, degenerate polymerase-chain reaction (PCR) primers synthesized, a portion of the gene amplified by PCR, and the gene encoding the protein identified in the pathogen genome using the PCR product as a probe. This series combines techniques from several sections of this chapter and highlights the value of understanding and utilizing multiple techniques to identify pathogenicity factors. An essential element of the initial, purely biochemical purifications was that an activity had to be associated with the pathogenicity factor to allow its purification. The range of activities of the pathogenicity factors that could be isolated were therefore limited to the presence of enzymatic activity that could be assayed in vitro, or toxic activity that could be assayed using cultured host cells or whole animals. For this reason, many of the first pathogenicity factors
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ANDREW CAMILLI, D. SCOTT MERRELL, AND JOHN J. MEKALANOS
Techniques and Strategies for Identifying Pathogenicity Factors"
Technique or strategy
Advantages
Disadvantages
Classical biochemical approaches
Doesn't require genetic manipulation of pathogen. Can identify factors essential for viability.
Requires knowledge of enzymatic activity or function. Can be laborious.
Chemical modification screens
Doesn't require genetic manipulation. Can identify essential factors. Not necessary to know enzymatic activity or function.
Targets only a subset of pathogenicity factors (e.g., those exposed on the outer surface).
Zymography
Doesn't require genetic manipulation. Can identify essential factors. Direct detection of purified or partially purified factor.
Requires knowledge of enzymatic activity for which a substrate is available. Enzymatic activity must be stable and in most cases, renaturable. Can be expensive.
Receptor/ligand affinity screens
Doesn't require genetic manipulation. Direct purification of factor.
Requires knowledge of either ligand or receptor.
Immunological methods
Doesn't require genetic manipulation. Targets factors expressed during infection.
Targets only factors that are immunogenic, many of which may not be pathogenicity factors.
Subtractive hybridization
Doesn't require genetic manipulation. Can identify essential factors. Doe.sn't require knowledge of function.
Lacks sensitivity and can be laborious. Only a subset may encode pathogenicity factors.
Differential display
Doesn't require genetic manipulation. Can identify essential factors.
Lacks sensitivity, and can be laborious. Only a subset may encode pathogenicity factors.
Reverse genetics
Doesn't require genetic manipulation of pathogen. Can identify essential pathogenicity proteins.
Requires some amino acid sequence of protein factor.
Large-scale screenings
Can be comprehensive.
Is usually laborious. Usually requires an in vitro model of infection (e.g., tissue culture). Cannot identify redundant pathogenicity factors.
Targeting exported proteins
Many pathogenicity factors are exported. Simultaneously generates a null mutation in pathogenicity gene.
Only a subset of pathogenicity factors are exported proteins. Some protein-reporter fusions will be toxic.
Coordinate regulation screens
Directly identifies pathogenicity genes coregulated with known ones.
Requires knowledge of in vitro growth conditions that induce expression of pathogenicity genes.
Host mimicry
Directly identifies pathogenicity genes and may point to a role during infection.
Requires knowledge of in vitro conditions that mimic a host parameter(s).
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Technique or strategy
Advantages
Disadvantages
Recombinationbased in vivo expression technology
Identifies pathogenicity genes that are transiently induced or induced at a low level in host tissues.
Not all pathogenicity genes are transcriptionally induced in host. Only a subset of infection-induced genes encode pathogenicity factors.
Signature-tagged mutagenesis
Targets pathogenicity genes that play essential roles during infection. Allows multiple mutant strains to be screened per host animal.
Can be laborious. Requires large infectious dose in some disease models. Cannot identify redundant pathogenicity factors.
Direct selections
Directly identifies strains with mutations in pathogenicity genes.
Requires knowledge of a phenotype associated with the pathogenicity factor. For some direct selections only a subset of mutations will be in pathogenicity genes.
Complementation approaches
Directly identifies genes necessary and in some cases sufficient to confer a pathogenic property.
Depending on strategy, requires efficient means of transformation into pathogen, or a simple in vitro model of disease (e.g., adherence to tissue culture cells).
Selection for nongrowing bacterial mutants
Directly identifies genes necessary for multiplication in vitro under host-like conditions (e.g.. within tissue culture cells).
Only a subset of mutations will be in genes specifically required for in vivo growth.
In vivo expression technology
Directly identifies pathogenicity genes that are expressed in host tissues.
Pathogenicity genes that are transiently expressed during infection or are expressed at low levels may not be identified.
Genome walking
Rapid and simple method of identifying pathogenicity genes.
Requires mutagenesis studies to confirm roles in pathogenicity.
Genomic analysis Targets pathogenicity genes that play and mapping essential roles during infection. Allows by in vitro multiple mutant strains to be screened transposition per host animal.
Laborious. Requires prior genome sequence information. Cannot identify redundant pathogenicity factors.
Computational screens
Rapid and simple method of identifying pathogenicity genes.
Requires prior knowledge of pathogenicity factors to serve as query sequences. Requires mutagenesis studies to confirm roles in pathogenicity.
Transcriptional profiling and the use of microarrays
Rapid and comprehensive method of identifying pathogenicity genes.
Requires prior genome sequence information. Expensive. Requires mutagenesis studies to confirm roles in pathogenicity.
"This table is meant to list most of the techniques and strategies discussed in the text and is not all-inclusive. Only a brief description of some of the major advantages and disadvantages of each is given. Please read the text for more detailed discussion, it is up to the reader to thoroughly investigate and carefully weigh the advantages and disadvantages of each technique or strategy before embarking on a study to identify virulence factors.
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identified were bacterial exotoxins that possess dramatic activities whose effects can be easily monitored. For example, the Streptococcus pneumoniae pore-forming protein pneumolysin (Ply) lyses mammalian red blood cells (RBCs), producing clear, hemolytic zones around colonies grown on media containing RBCs. This activity was noted early in this century [72], and it provided a convenient assay to monitor purification of Ply to homogeneity as lysis of RBCs in solution could be easily monitored. A purification strategy that included acetic acid precipitation, ammonium sulfate precipitation, ion-exchange chromatography, gel filtration, and preparative acrylamide gel electrophoresis led to purification of Ply [108]. With purified Ply in hand, researchers were then able to convincingly show that Ply was sufficient to reproduce the toxic lethal effect of 5*. pneumoniae culture filtrates [108]. A second example of a purely biochemical identification of a pathogenicity factor is the isolation of cholera toxin, which built on the prior observations of De [27] and Dutta and colleagues [33] that the profuse watery diarrhea of cholera could be reproduced by enteral administration of sterile culture filtrates to animals. This observation, along with the ability to monitor purification by bioassay in infant rabbits and by immunoassay using polyclonal antiserum, led to the isolation of cholera toxin in a pure form using a purification strategy similar to that described above for Ply [38]. This purification precipitated many elegant studies (reviewed in [37]) that revealed cholera toxin's A-B5 subunit structure, its ganglioside GMj host cell receptor, and its primary mode of action—activation of host cell adenylate cyclase. The application of these purely biochemical approaches to other pathogens has been particularly successful in the identification of factors such as diphtheria toxin, pertussis toxin, and shiga toxin, and unquestionably validates the usefulness of such approaches. The potential for application of biochemical isolations is infinite and is Umited only by the ability of the researcher to measure the specific activity of a factor during purification. Potential applications could utilize prior understanding of enzymatic differences between pathogen and host. For instance, phosphorylated protein tyrosine residues are rare in bacteria, yet prevalent in signaling pathway components in human cells. Therefore, the Yersinia YopH protein, which was in actuality identified by other methods [12], could instead have been identified by a search for protein tyrosine phosphatase activity in whole cell bacterial extracts. The presence of such activity in a bacterial pathogen that is known to possess few examples of protein tyrosine phosphorylation could indicate a potential mode of interaction with the host by modification of host protein phosphorylation patterns. Understanding differences between host and pathogen enzymatic functions has many implications for application of biochemical strategies to isolate bacterial factors that specifically affect the host cell. For instance, though they themselves do not produce actin, many bacterial pathogens utilize host cell F-actin for attachment and intracellular spread. Therefore, one could conceivably design screens for bacterial factors that possess the ability to nucleate host cell G-actin
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or modify F-actin in vitro. Additionally, targeted biochemical screens could be designed to identify factors that proteolytically cleave key host proteins, phosphorylate or dephosphorylate protein serine and/or threonine residues, or covalently modify in other ways certain host proteins. Knowledge of differences between host and pathogen activities can also be used to screen for bacterial factors that alter normal host responses. For example, a recent study found that the Mycobacterium tuberculosis pathogenicity factor, lipoarabinomannan, stimulates the activity of a macrophage tyrosine phosphatase both in vivo and in vitro. This activity possibly inhibits proper macrophage function to provide a means of avoidance of this component of the host immune response [63]. Similar searches for factors that affect host enzymatic functions could also be conducted and could prove beneficial in the elucidation of heretofore undiscovered bacterial pathogenicity factors. Purely biochemical strategies for the discovery of bacterial pathogenicity factors have several advantages: (1) Many pathogenic bacteria are not easily genetically manipulatable and therefore are not easy targets for screens that employ large-scale mutagenesis strategies. Biochemical strategies to identify pathogenicity factors are therefore much more advantageous in these organisms. (2) Some pathogenicity factors are essential for bacterial growth in vitro and thus would never be isolated using genetic strategies that rely on mutagenesis. Therefore, biochemical isolations that do not involve disruption of normal bacterial processes allow isolation of these essential factors. (3) The activities of some pathogenicity factors are known, and this knowledge can facilitate design of strategies specific for the factor responsible for the activity. Despite these strengths, purely biochemical strategies for pathogenicity factor discovery also possess some disadvantages: (1) The advantage presented above as #3 can also serve as a major disadvantage when employing biochemical strategies to look for pathogenicity factors. The isolation and purification of factors that possess unknown functions is impossible since the ability to assay for function is critical to all of the classical biochemical strategies. (2) Biochemical purifications are often laborious as they rely on multiple purification steps that need to be designed or empirically determined to preserve specific activity of the factor one is attempting to isolate. For these reasons, genetic strategies are often more amenable for identification of pathogenicity factors having novel or unknown functions.
B.
Chemical Modification Screens
The first step in bacterial pathogenicity commonly involves adherence of the infectious agent to host cells or tissue. Because most of the host-pathogen interactions that allow this attachment to occur involve intimate contact between the bacteria and host cells, the outer surface of the bacterial cell has served as a
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prime hunting ground for factors involved in this aspect of pathogenicity. This section focuses on a few of the numerous biochemical strategies that have been used to dissect apart the intimate interactions that occur on the outer surface of bacterial pathogens. Although not a biochemical technique per se, direct visualization of bacterial surfaces by microscopy, in conjunction with fixation or biochemical staining procedures, can be used to identify surface-exposed pathogenicity factors. For example, light microscopy of bacterial cells stained with india ink and electron microscopy of polycationic ferritin-stained thin sections of bacterial cells have allowed visual detection of polysaccharide capsules surrounding some pathogens. Although much is already known about the roles of bacterial polysaccharide capsules in pathogenicity [6, 32], application of microscopy techniques and the ability to directly visualize capsule around newly isolated pathogens is useful in that it can provide an immediate advance in one's understanding of the potential pathogenicity of that organism. For example, two reports in 1994 demonstrated that a new epidemic strain of V cholerae, serotype 0139, expressed a polysaccharide capsule. This finding was novel in that the presence of a polysaccharide capsule had never before been seen in epidemic strains [57, 125]. Although the precise role of the capsule in the pathogenicity of this intestinal pathogen is not readily clear, it is possible that the capsule might be involved in adherence to the mucosal layer of the small bowel, or involved in resistance to certain host bactericidal mechanisms such as complement-mediated lysis. To this end, mutants that have lost the ability to express capsule have been shown to be attenuated for pathogenicity in an animal model of cholera [120]. This finding underscores the usefulness of visualization of bacterial surfaces as a means of detection of pathogenic factors. Electron microscopy has many orders of magnitude greater resolving power than light microscopy, and has been used to visualize the very long (0.5-15 |im) and thin (1-11 nm) adhesive fibers, called pili, that extend from the surfaces of many Gram-negative bacteria. Although it should be noted that not all pili expressed by pathogens play a role in human disease (e.g., [112]), the discovery of pili on the surface of a suspected or known pathogen provides a good starting point for further investigation, as some pili have been shown to be essential factors for establishment of infections. Direct application of both electron microscopy and light microscopy are therefore attractive in that they can provide immediate knowledge about surface structures such as pili or capsules. It should be noted, however, that other methods are required to assess the putative roles of these surface structures in pathogenicity. In addition to direct visualization of surface structures, electron microscopy and light microscopy have also proven themselves valuable tools for indirectly monitoring the presence of many bacterial surface adhesins—for example, surface factors involved in such diverse activities as in vitro autoagglutination [113], attachment to cultured host cells [117], and invasion into cultured cells [56]. Once
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again, though, other methods are ultimately needed to identify these pathogenicity factors, highlighting the importance of combining multiple techniques to gamer a greater understanding of host-pathogen interactions. Direct chemical modification of bacterial surface macromolecules using reagents that are unable to cross the bacterial outer membrane can facilitate identification of pathogenicity factors. The inability of these reagents to be internalized not only facilitates specific tagging of surface-exposed molecules but also often facilitates the purification and subsequent characterization of surfaceexposed pathogenicity factors. Two frequently used methods of surface modification involve radioiodination and biotinylation of intact cells followed by purification and electrophoretic separation of membrane proteins. Surface molecules that have been radioiodinated can be directly visualized by autoradiography following separation by gel electrophoresis, hi contrast, biotinylated molecules can only be visualized after binding of a secondary molecule such as streptavidin that possesses a high binding affinity for biotin. The secondary molecule is usually visualizable due to the fact that it has been either radiolabeled or conjugated to a colorimetric moiety such as alkaline phosphatase. Though these two surface modification techniques differ chemically, both have proven themselves useful for identification of surface factors involved in pathogenicity. Most recendy, these two labeling methods have been utilized to identify Bartonella henselae surface proteins, and to then further identify the subset of these surface proteins that function as adhesins for human endothelial cells [16]. Biochemical screens for pathogenicity factors that utilize chemical modification have advantages and disadvantages that force the investigator to consider the usefulness of the procedures. A primary advantage of chemical modification is that litde or no information need be known about the pathogenicity factors present on the cell surface other than the fact that they reside there. This allows the investigator to construct broadly based screens, which increases the chances of discovery of important pathogenicity factors. The primary limitations of these types of screens include the fact that many bacterial pathogenicity factors are indeed not expressed on the outer membrane and thus would be missed in screens that exclusively looked at this subset of molecules. In addition, isolation of surface-exposed molecules on a gel generally only provides a starting point for identification of putative pathogenicity factors, and must be followed up by other methodologies. For example, in order to assess a protein's role in pathogenicity, one might wish to mutate the gene encoding the surface protein by using reverse genetics.
C.
Zymography
Since some bacterial pathogenicity factors are enzymes, identification strategies that directly assay for enzymatic activity are useful in that they provide a more
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direct method of identification. For example, many bacterial proteases are involved in cleavage of host cell extracellular matrix proteins or host cellular proteins. Cleavage of these host proteins often assists the pathogen in the establishment of an infection and sometimes aids the bacterium in avoidance of the host cell immune response. One strategy for identification of bacterial enzymatic pathogenicity factors is zymography. This strategy combines electrophoretic separation of bacterial proteins with the subsequent visual detection of enzymatic activity in situ, that is, in the gel itself. The most common zymographs target identification of proteases, as the construction of the substrate matrix is simple. For example, a denaturing acrylamide gel can be embedded with a substrate polypeptide such as casein. This gel would then be loaded with a protein preparation from a pathogen of interest, and electrophoresed to separate the secreted or membrane proteins. The separated proteins would then be renatured by subsequent removal of denaturants from the gel. Next, an incubation period would allow enzymatic proteolysis to occur. Finally, the gel can be stained with coomasie blue, and the presence of a small clearing zone within the casein matrix indicates that the protein species present at that location possesses proteolytic activity. This methodology relies on the fact that the embedded casein molecules do not migrate appreciably during electrophoresis due to their lack of substantial net charge. Candidate proteases can then be purified and further studied biochemically, or purified and subjected to reverse genetics to identify the encoding gene. Various zymography modifications that involve utilization of secondary gels impregnated with additional polypeptide substrates have been made (reviewed in [67]) and then utilized for the study and/or identification of several putative pathogenicity factors from a variety of different pathogens [22, 34, 41]. The usefulness of zymography in identification of pathogenic factors is not limited to proteases. In fact, many other enzymatic activities have also been identified by utilization of zymographic techniques. Examples of the diverse activities and factors that have been identified include a secreted S. sobrinus dextranase inhibitor believed to be involved in colonization of the oral cavity [111], a Staphylococcus aureus murein hydrolase that is under the regulation of the agr and sar pathogenicity gene regulators [44], and a Prevotella melanginogenica hemolysin that is produced by this periodontal pathogen [4]. Indeed, zymography is a method that can be adapted for identification of additional types of enzymes, and is advantageous in that the potential applications for identification of enzymatic factors are as limidess as the imagination of the investigator. It should be noted, however, that limitations to zymography do exist. First, embedding a large gel matrix with some substrates can be very costly. Second, many bacterial enzymes that are involved in pathogenicity may not be produced during normal laboratory growth or may not be present in an active form. Third, bacterial proteases often exhibit high substrate specificity, and the identity of the substrate may not be known. Fourth, detection of an enzymatic
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activity requires that the enzyme be not only stable but renaturable after electrophoretic separation. The latter problem may be solved by running a nondenaturing gel, but then information concerning the molecular weight of the enzyme may be lost. And, fifth, biochemical methods (such as zymography) assume that the activity being pursued is important for pathogenesis; however, this can only be assessed by side-by-side comparison of the corresponding mutant and wild-type strains, or by reproduction of the disease symptoms by the purified protein in the case of toxins.
D. Receptor/Ligand Affinity Screens Intimate interaction between host and pathogen occurs on many levels. Not only do most bacterial pathogens have to adhere to host cells or tissues, but often they produce polypeptides that subsequendy interact with host cell components. These interactions are often key steps in the development and progression of pathogenicity. Because of this, many biochemical screens specifically target pathogenicity factors that show the ability to bind to host molecules with high affinity. Receptor/ligand screens are one such type of screen and have proven beneficial in identification of a number of interacting components. Receptor/ligand screens in the most straightforward form involve the following steps. First, total protein from the pathogen of interest is prepared and then separated on a denaturing acrylamide gel. Following separation, protein constituents are renatured and subsequently transferred and bound to a solid membrane support. This membrane can then be exposed to any radiolabeled host ligand of interest. The immobilized bacterial polypeptide bands that interact with and are bound by the labeled host ligand can then be identified by autoradiography. This type of receptor/ligand screen has been used successfully numerous times. For example, a S. pyogenes adhesin was identified using radiolabeled collagen as a ligand [118]; and in a more recent example, the Enterobacter cloacae mannosesensitive pilus adhesin for human cells was identified from purified pili subunit polypeptides after exposure to mannose-containing biotinylated albumin [92]. In addition to the basic receptor/ligand screen, variations of this technique have been used to study the binding of bacterial proteins to immobilized host ligands (e.g., [129]), and the binding of entire host cells to immobilized bacteria expressing an adhesin (e.g., [70]). Yet another variation of this method combines the expression of individual pathogen proteins on the surface of a surrogate host bacterium (that lacks the binding activity being studied) with affinity binding selection of surrogate host cells expressing the receptor to a ligand matrix. This methodology has been used to identify a Neisseria gonorrhoeae adhesin for the host cell ligand, gangliotetraosylceramide [93]. Affinity binding can often be used as a direct method for the identification of bacterial pathogenicity factors. For instance, both panning and affinity chroma-
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tography utilize immobilized host ligand as a means of purifying bacterial factors that interact with specific host substrates. A more recent innovation in these techniques has been developed, which is called Receptor activity-directed affinity Tagging (ReTagging) [55]. ReTagging is beneficial in that it utilizes a UV light-activatible crosslinker to covalently attach a biotin molecule onto a bacterial receptor when it comes into close contact with the host ligand (Fig. 1, see color plate). The attachment of the biotin molecule then allows direct identification and purification of the bacterial factor to which it is bound using streptavidin-coated magnetic beads (streptavidin possesses high binding affinity for biotinylated compounds). This method was recently utilized to identify the Helicobacter pylori receptor for the Lewis B histo-blood group antigen [55]. This simultaneous identification and purification of the bacterial receptor is a major advantage of the utilization of ReTagging. In addition, the affinity of the receptor-ligand pair enables identification and purification of low-abundance receptors.
E. Immunological Methods Many bacteria disrupt or circumvent the host immune system in order to produce a productive infection. Thus, it is fitting that researchers have developed strategies for discovery of pathogenicity factors that exploit the immunological system. Such strategies are usually based on the ability of antibodies to protect a host or surrogate host from disease. Before application of this strategy, a bank of B-lymphocyte hybridomas that produce monoclonal antibodies (MAbs) directed against antigens exposed on the bacterial surface must first be generated. Subsequently, each individual MAb can then be screened for the ability to protect the host from disease. The inability to cause disease represents a positive result and might indicate that the MAb is binding to and blocking the function of a pathogenicity factor residing on the surface of the pathogen. It should be noted, however, that mere binding of the MAb to the bacterial cell surface can be sufficient to protect the host from disease in that antibodies can act as opsonins for complement deposition and phagocytosis. For this reason, it is necessary to show protection using purified Fab fragments of the MAb, as Fab fragments lack opsonizing activity yet can block the function of the bacterial factor. Of course, studies in surrogate host systems that lack complement and/or phagocytic cells (e.g., cultured mammalian cells grown in medium supplemented with heat-inactivated serum) do not require the Fab control experiment. Once an MAb has been identified that provides protection of the host or surrogate host from disease, the MAb then becomes a powerful tool that can be used to identify the surface molecule to which it binds. Solubilized membrane protein extracts can be used in conjunction with the MAb of interest to direcdy purify the bacterial factor. This methodology has been used to identify many diverse bacterial factors (e.g., [78, 99]). Recently, this method was also used to
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look for factors involved in adherence of Mycobacterium tuberculosis to host cells. In this study an MAb that blocked adherence of M. tuberculosis to cultured mammalian nonprofessional phagocytic cells was identified from a bank of MAb generated against intact M. tuberculosis cells. This MAb was then used to purify and identify a phosphatidylinositol mannoside as the adhesin [52]. Since the production of hybridomas and subsequent screening of many individual MAbs can be very laborious, an often used variation on the immunological approach described above has been developed that uses the host immune system to select for immunogenic bacterial surface antigens during a sublethal infection. In this way, convalescence serum antibodies can then be gathered and used to identify bacterial surface proteins that were expressed during infection. In the most common implementation of this approach, bacterial outer membrane proteins are then electrophoretically separated or prepared as part of a bacteriophage X expression library. These polypeptide species can then be screened using the convalescence serum for the subset that were immunogenic during the course of the infection. Detection of protein species bound by serum antibodies requires binding of a secondary antibody conjugated to a colorimetric moiety. A further subset of these antigenic factors may indeed be pathogenicity factors, and their identification as such requires additional experimentation. This modified immunological approach has been beneficial as sera from lepromatous leprosy patients used to screen a X cosmid expression library of M. leprae genomic DNA [106], and resulted in the identification of a 15-kDa protein that is immunogenic to both B- and T-lymphocytes. The exact role this protein plays in pathogenicity remains as yet to be demonstrated. The application of immunologic strategies is advantageous in that it allows a broad approach to target for identification factors exposed on the bacterial surface. However, three limitations include the fact that these screens can often be quite laborious, many pathogenicity factors are either not exposed on the bacterial surface or are poorly immunogenic and thus would never be found (e.g., shiga toxin), and many highly immunogenic surface antigens are not pathogenicity factors (e.g., S. typhimurium flagella [76]).
F. Subtractive Hybridization The facts that related bacterial strains often have quite different pathogenic abilities and that gene expression patterns of pathogenic bacteria often change dramatically when going from an in vitro to an in vivo environment have led to the development of techniques that seek to identify differential expressed genes. One such technique is nucleic acid subtractive hybridization. In the first case, nucleic acids from two related bacterial strains, one pathogenic and the other nonpathogenic, can be physically compared to identify differentially expressed genes or differences in gene content that may be responsible for the
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pathogenicity difference. In the second situation, the nucleic acids from a single strain grown under a nonhost condition, and a host-like condition can be physically compared to identify differentially expressed genes, with the understanding that some of these may encode pathogenicity factors. The foundation of the subtractive hybridization approach relies on the fact that DNA sequences common to both (denatured) DNA preparations can be subtracted out as doublestranded DNA hybrid duplexes while unique single-stranded sequences remain. The "nonpathogenic" DNA is supplied in molar excess to ensure that complete hybridization of common sequences occur. In addition, the nonpathogenic DNA is typically modified by biotinylation so that subsequent to the annealing reaction the common DNA hybrids can be removed by utilization of a streptavidin matrix. Thus, only the single-stranded fragments of interest are left behind. It should be noted that subtractive hybridization can be performed using either cDNA or genomic DNA derived from the strains of interest. However, differences in gene expression in a single strain grown under in vitro and host-like conditions can only be detected by this method using cDNA. The traditional subtractive hybridization protocols rely simply on subtractive hybridization followed by either cloning of the end products or utilization of the products as probes to identify the genes from genomic libraries (e.g., [15, 95]). A more recent subtractive hybridization protocol has been designed to incorporate modifications that vastly improve the efficiency of the technique. This modified procedure has been called Representational Difference Analysis (RDA) [75]. RDA has been applied to discover bacterial pathogenicity factors. Some examples are the identification of genetic differences between the two closely related pathogens Neisseria meningitidis and A^. gonorrhoeae, which might be responsible for their different etiologies [114], and the recent identification of genetic differences between an emergent epidemic strain of V. cholerae, serogroup 0139, and its progenitor serogroup Ol strain [17]. The RDA method involves initial restriction enzyme digestion of DNA from the virulent strain (called the tester) followed by ligation of a PCR adapter/primer onto the ssDNA overhangs formed by digestion of the tester DNA. Next, the modified tester DNA is amplified by PCR. This amplified tester DNA can then be mixed with a molar excess of digested, but otherwise unmodified, DNA from the nonvirulent strain (the driver), and the mixture is denatured and allowed to anneal. During the course of the annealing reaction, most ssDNA tester fragments anneal with complementary driver ssDNA fragments. These dsDNA hybrids represent sequences common to both the tester and the driver and are thus not of interest. In contrast, though, ssDNA tester fragments that are unique cannot hybridize with driver ssDNA, but instead can only anneal back to the complementary tester ssDNA fragment. This restores the original adapter/primer arms on both ends of the resulting dsDNA fragment. These dsDNA tester fragments can then be reamplified by PCR. The resulting fragments represent unique sequences and can be further enriched by additional rounds of subtractive hybridization and
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amplification. As a final step, the amplified unique tester DNA fragments can be cloned and further analyzed. Despite improvements such as RDA, subtractive hybridization methods are still somewhat laborious and are fairly insensitive, that is, many genetic differences between tester and driver are not identified due to a variety of technical difficulties. A more recent technical improvement to the RDA protocol, however, increases both the efficiency and sensitivity of the method [3]. In the first implementation of this method, which is a slighdy modified version of the Suppressive Subtractive Hybridization (SSH) technique originally developed for use in eukaryotes [28], more than a dozen genomic sequences present in a monkey-isolate of H. pylori (tester) and not present in a human isolate (driver) were identified. Many of the identified sequences were smaller than the limit of detection of tradidonal subtractive hybridization screens, thus validating the improved sensitivity of this technique [3]. The method for SSH is similar to RDA but contains one major procedural difference. Instead of ligating a single adapter/primer onto the ends of the tester DNA restriction fragments, two different adapter/primers are used (Fig. 2, see color plate). First, the digested tester dsDNA is divided into two pools, 1 and 2. Adapter/primer 1 is then ligated onto fragments contained in pool 1, and adapter/primer 2 is ligated to fragments contained in pool 2. Each pool is then subtractively hybridized, separately, to a molar excess of unmodified driver DNA, as described above. After annealing, pools 1 and 2 are mixed together and another molar excess of denatured driver DNA is added. This final mixture is then allowed to undergo additional annealing. During this second annealing reacdon, any remaining common ssDNA from the tester DNA from pools 1 or 2 can anneal to the additional driver ssDNA added to the second annealing reacdon. Likewise, any unique tester ssDNA fragments originally derived from pool 1 sdll remaining after the first annealing reaction can hybridize with a complementary ssDNA tester fragment from pool 2. These latter dsDNA tester fragments will thus contain adapter/primer 1 at one end and adapter/primer 2 at the other. Unique tester dsDNA fragments that contain both adapter/primers 1 and 2 at their two respective ends can then be amplified exponendally in a single PCR reacdon using forward and reverse primers complementary to adapter/primers 1 and 2, respecdvely (Fig. 2, see color plate).
G. Differential Display Since most bacterial pathogens must express different subsets of genes on infecdon of a host, many different methods have been developed that attempt to direcdy analyze these differentially expressed gene products. One approach that is related to, yet distinct from, subtractive hybridizadon, is Differential Display (DD). The DD methodology was originally designed to study differendal gene
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expression in eukaryotes [71] but has been modified to allow for screening of differentially expressed genes between two populations of bacteria [127]. Because of this, DD can be used to identify pathogenicity factors by comparing gene expression either between a pathogenic and a nonpathogenic strain of one species or, between a strain grown in vitro and in vivo. DD allows for differences in gene expression to be visually determined by direct comparison of two sets of randomly primed PCR products, which are generated from the two cDNA pools to be compared. Pools of cDNA are first generated from total RNA purified from each bacterial population. These cDNA pools are made using an arbitrary primer of approximately 18 bases in length. Utilization of an arbitrary primer and low-stringency hybridization allows priming to occur at pseudo-random sites during the reverse transcription reaction. These cDNAs are then PCR amplified in a second reaction, wherein the first amplification cycle uses a low-stringency annealing step that results in products that have the primer sequence at both ends. These double-end primed products are then amplified exponentially in subsequent rounds of the PCR using the arbitrary primer, a stringent annealing temperature, and radioactive deoxynucleotides. After the final PCR amplification, PCR products are separated on an acrylamide gel and visualized by autoradiography to screen for band differences between products derived from the two cell populations. Unique bands represent genes that were differentially expressed between the two cell populations. Finally, these differentially expressed bands can be cut out of the gel and sequenced. DD has been successfully used to identify several bacterial pathogenicity factors. For example, S. typhimurium genes that are stress-induced by exposure to hydrogen peroxide were identified in the study that first reported the DD method modified for use in prokaryotes [127]. More recently, a slightly modified version of this protocol was used to identify a Legionella pneumophila gene that was induced during intracellular infection of macrophages [1]. In this report, cDNAs were made using random hexamer primers, and PCR amplification was done using pairs of arbitrary primers that were 17-23 bases in length. The strengths of DD depend a great deal on a number of experimental parameters. In particular, the length of the arbitrary primer(s) and stringency conditions used directly correspond to the number of bands that one generates per reaction. Generally between 1 and 50 bands are produced per reaction, but since many of these bands are artifactual—for example, corresponding to ribosomal RNA-derived products—it is necessary to screen products generated in several different reactions each using different arbitrary primers. This multipronged screening strategy greatly increases the likelihood of identifying bands that correspond to bona fide differentially expressed genes. Because of these technical limitations, DD is not a comprehensive method by any means and can be quite laborious. However, there are some procedural and theoretical advantages of DD. For example, very little total RNA is needed as starting material, so that DD can be used on relatively small numbers of bacteria recovered after exposure to a wide
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variety of in vitro and in vivo conditions. In addition, DD is reported to be a sensitive method, able to identify genes that are transcribed at very low levels. H. Reverse Genetics Many of the methods described above (II.A-II.E) result in identification of a polypeptide with the potential to play a role in pathogenicity. Although this is a reasonable endpoint for some studies such as those whose goal it is to identify and biochemically characterize a toxin, it is usually of further benefit to proceed with identification of the gene encoding the polypeptide. Not only does this open the door to many additional studies, it can allow for precise determination of the actual role in pathogenicity of the discovered factor. Specifically, after the gene responsible for a putative pathogenicity factor has been identified, it can be mutated and the resulting strain determined to lack the polypeptide of interest. The pathogenicity of the mutant strain can then be compared in side-by-side experiments with that of the isogenic parental strain in, for example, an animal model of disease to assess the relative importance of the putative pathogenicity factor (see III.B.3). In addition to this validation, the gene can then be manipulated to allow purification of large quantities of the polypeptide for additional biochemical, immunological, or structural studies. Although there are several methods by which the gene encoding a polypeptide can be identified, utilization of reverse genetics provides a much more efficient method than, say, random mutagenesis followed by screening for loss of production of the polypeptide. Reverse genetics proceeds by first determining the amino acid sequence of a portion (at least five contiguous residues) of the purified polypeptide by protein microsequencing [65]. Second, the codon usage preference of the bacterial species being studied is used to design and synthesize degenerate DNA oligonucleotides that are predicted to code for the determined amino acid sequence. Third, the degenerate oligonucleotides are used to identify the gene from the bacterial genome. This can be done directly by Southern blot analysis using the oligonucleotides as a probe, or indirectly by PCR amplification of an internal portion of the gene using degenerate oligonucleotides that correspond to two separate parts of the gene, followed by use of the PCR product as a Southern blot probe. This latter methodology requires that the amino acid sequence of two separate parts of the polypeptide be known. Reverse genetics has been used numerous times in both of these manners to identify bacterial genes encoding pathogenicity factors (e.g., [30, 53, 73]).
///. Genetic Screens Coincident with gaining the knowledge that DNA was the genetic material came the realization that DNA also encoded the biosynthetic information for pathogenicity factors [7]. The five and a half decades that have followed this seminal discovery have seen the development of many experimental techniques that have
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potentiated advances in understanding of bacterial pathogens and their ability to cause disease. Techniques such as sequence-specific DNA cleavage, ligation, and transformation, coupled with various mutagenesis protocols, allow scientists to manipulate DNA with amazing power. It is this ease of genetic manipulation that now allows scientists to identify and characterize pathogenicity factors from many organisms. It should be noted, however, that, even though genetic techniques have proven invaluable to culturable and transformable pathogens, many organisms are still unable to be grown in vitro and/or are untransformable. These organisms present a unique challenge to scientists wishing to study them and emphasize the importance of continued advances in molecular techniques. The remainder of this chapter attempts to describe many of the genetic strategies that have been employed throughout the last few decades of the twentieth century to facilitate identification of bacterial pathogenicity factors. Each technique, as described, can result in identification of a bacterial gene that is required for production of a putative pathogenicity factor. In practice, it is evident that the power of genetic techniques is only fully realized when used in combination with immunological or biochemical strategies. By implementation of combinations of strategies and techniques, scientists are able to gain a fuller understanding of the roles of pathogenicity factors in the disease process. Although a large number of genetic screens and selections have been designed to identify pathogenicity factors, only a sampling of these will be covered here for the sake of brevity. Emphasis will be placed on those strategies that have been particularly successful and/or those that introduce an interesting or novel concept.
A. In Vitro Screens 1.
LARGE-SCALE SCREENINGS
Most genetic strategies for identification of pathogenicity factors have in common the need to mutagenize the bacterial genome in a random fashion to generate a bank of mutant strains. Ideally, the mutagenesis is conducted in a manner such that each mutant strain contains a single, unique mutation, and the number of strains in the bank is comprehensive enough to ensure that most genes will have been mutated. The Poisson distribution equation, yV=[ln(l-P)]/[ln(l
-F)l
can be used to calculate the minimum number of strains (AO required to assure (with probability [P]) that any particular gene will be mutated, where F is equal to the average gene size divided by the genome size. For example, given an average gene size of 1 kb in a genome size of 4 Mb, the minimum number of strains in a mutant bank sufficient to assure that 95% of all genes were mutated would be 11,981. After construction of the mutant bank, a screen or a selection
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for strains with changes in pathogenicity is conducted. Intrinsically, a screen is more labor intensive than a selection, as it involves examination of individual strains to find those with the sought-after phenotype. In contrast, a selection is designed to enrich only those strains having the desired phenotype. In this way, the scientist is left with only the strains of interest at the completion of the enrichment. The most laborious yet comprehensive genetic screens are conducted on a large scale, whereby mutagenized strains are individually screened, one at a time, in some assay of pathogenicity chosen by the investigator. This method is powerful in that it allows the investigator the opportunity to assess each individual mutation's effect on the pathogenicity of the organism. In this manner, knowledge concerning each individual gene can, in theory, be gained. A classic example of a large-scale screening is the screening of 9,516 individual transposon insertion strains of 5'. typhimurium for loss of infectivity in microtiter plate wells containing monolayers of cultured murine peritoneal macrophages [36]. The infectivity of each strain was assessed by lysing each monolayer and titering viable bacteria on agar plates. From this screen, 83 strains exhibiting reduced pathogenicity were identified. Although laborious, large-scale screening is advantageous in that, if a large enough bank of mutant strains is screened, it is theoretically more comprehensive than any possible selection. The reason for this comprehensiveness is simple: in a large-scale screen each mutant strain is individually assayed for pathogenicity, whereas in a genetic selection strains that exhibit slightly attenuated pathogenicity are usually lost (or ignored). For example, if one were to search for gene mutations that adversely affected bacterial multiplication in the presence of hydrogen peroxide, a reactive oxygen species produced by host cell macrophages, a mutation that only slightly decreased (or increased) multiplication could be picked up in a screen. However, in a penicillin-based selection, where the penicillin selects for strains that are no longer able to replicate by killing all multiplying cells, a mutant strain that exhibited a slightly reduced multiplication rate would still be killed by the penicillin. Mutants that displayed an enhanced multiplication rate would also be killed in a selection of this type. All genetic selection strategies suffer this type of limitation to some degree. On the other hand, there are several major advantages to the selections discussed below. Because of the different strengths and weaknesses of different genetic strategies, investigators must carefully determine which strategy is most appropriate for isolation of the types of genes being sought after. When conducting genetic screens or selections, it is important that the researcher consider not only the type of screen or selection but also the method utilized for mutagenesis of the bacterial pathogen. As stated above, in most cases a bank of mutant strains containing highly random, single mutations is most desirable. Since the choice of mutagenesis strategy is an important consideration in all types of genetic screens and selections, a brief discussion of the advantages
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and disadvantages of several commonly used methods is included within this section. Physical and chemical methods for generating point mutations, such as UV light and A^-methyl-A^-nitro-A^-nitrosoguanidine, are probably the most frequently utilized types of mutagenesis due to the fact that they are highly random and can generate a tremendous number of different mutant alleles for any gene. The list of possible types of mutant alleles obtainable by utilization of these methods is about as complete as possible in that one can obtain null, partial function, dominant negative, dominant positive, suppressor, promoter-up, promoter-down, polar, or nonpolar mutations. However, a major disadvantage of these methods can be found in that multiple unlinked mutations are commonly introduced into individual cells. The presence of multiple mutations within a single strain gready complicates subsequent identification of the relevant mutation. This problem, however, can be overcome if an efficient means of genetic complementation exists for the pathogen being studied. Another commonly used strategy for mutagenesis involves utilization of transposon insertions. This method is advantageous in that it can be used to make stable, single insertions, and subsequent linkage of the transposon with the mutation greatly facilitates identification of the disrupted gene. Disadvantages of transposon mutagenesis include the fact that even the best of transposons insert only pseudo-randomly, which provides a much more limited spectrum of mutant alleles (relative to chemical mutagenesis), and finally, some pathogens, such as Campylobacter jejuni [64], are not currendy amenable to transposon mutagenesis. An in vitro transposidon protocol using a mariner transposon has been developed [65, 102] that improves generalized transposon mutagenesis in three ways. First, mariner can insert with a high degree of randomness in that it requires only an 5'-AT-3' dinucleotide target site. Second, mariner mini-transposons have been recendy engineered that produce either polar or nonpolar inserdon mutations (B. Akerley, E. Rubin, J. Mekalanos, and A. Camilli, unpublished data). Third, the products of in vitro transposition can be moved into transformable bacterial species followed by double-crossover insertion events, thus obviating the need for transposidon in vivo [2]. Suicide plasmid insertion-duplication mutagenesis is an alternative approach that theoretically can be more random than transposon mutagenesis, provides a linked marker for gene recovery, and can generate both polar or nonpolar mutations in most genes. In this method, random small fragments of the pathogen genome are ligated into a suicide plasmid vector, and the recombinant plasmids are then introduced into the pathogen by transformation, whereupon they integrate into the chromosome by homologous recombination. However, generadng a null allele in small genes can be difficult or impossible given that an internal portion of the gene coding sequence usually needs to be inserted into the suicide vector in order to ensure disrupdon of the gene, and given that the likelihood of proper inserdon (by homologous recombinadon) is limited direcdy by the size of the
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coding sequence contained within the plasmid. Despite the limitations of transposon and suicide plasmid insertion-duplication mutagenesis, both of these methods have the major advantages of: (1) establishing linkage between the observed phenotype and inactivation of a particular locus, and (2) providing a selectable marker tighdy linked to the mutation to allow the mutation to be moved to different genetic backgrounds. 2.
TARGETING EXPORTED PROTEINS
As was discussed in section II.B, many pathogenicity factors are surface-exposed or secreted proteins. A common requirement for all of these proteins is that they be exported across the bacterial cytoplasmic membrane. Several genetic strategies have been designed to target exported proteins for identification and mutagenesis. The most successful and widely used of these is TnphoA mutagenesis. This strategy combines transposon mutagenesis with a series of screens. First, a prescreen to identify strains harboring insertions into genes encoding exported proteins is conducted, and subsequently this subset of strains is screened to identify nonpathogenic mutant strains in a model of disease [113]. This approach relies on the use of TnphoA, which contains at the 5' end of the transposon a truncated/?/zoA gene that lacks a promoter and the DNA region encoding its signal sequence [84]. Because PhoA requires export to the periplasm for disulfide bond formation and subsequent enzymatic activity (as a phosphatase), only those TnphoA insertions occurring into genes encoding extracytoplasmic proteins will result in export of the protein fusion (if the fusion is in the correct reading frame). These exported PhoA translational fusion products are then enzymatically active and can be assayed for colorimetrically by the formation of blue colonies on media containing 5-bromo-4-chloro-3-indolyl-phosphate. This TnphoA mutagenesis strategy has been used to identify many pathogenicity factors such as the V cholerae toxin-coregulated pilus (TCP) pilus [113], Escherichia coli (EPEC) chromosomal and plasmid genes required for adherence to host cells [29], and S. choleraesuis genes required for transcytosis across epithelial cell layers [39]. In addition, other phoA fusion vectors have been constructed and used to identify secreted proteins from other bacterial species in which TnphoA does not function properly [59]. Despite the fact that the TnphoA strategy has been employed so successfully, there are of course limitations to the strategy that must be considered when deciding the appropriateness of the screen. By definition, the techniques described in this section preclude the identification of nonexported pathogenicity factors. Secondly, the expression of some fusion products can be toxic to the host bacterium. Aside from excluding some genes from ever being detected, that is, those whose expression when fused to phoA is deleterious to growth in vitro, this phenomenon poses a problem when assessing the effect of loss of an infectioninduced gene on pathogenicity. Specifically, an attenuated level of pathogenicity may result partially, or totally, from slower /// vivo growth of the bacteria due to
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toxic effects of the fusion product. Methods to alleviate this limitation have centered around the utilization of alternative reporter fusions that are perhaps not as deleterious to the cell. For instance, several other reporter systems have been developed that also identify exported proteins (e.g., p-lactamase [91, 101]), invasin [128], and nuclease [97]). However, as most of these alternative reporters allow for selection of strains expressing an exported fusion product, they will be discussed below under genetic selections. 3.
COORDINATE REGULATION SCREENS
Not only did the original V cholerae study using TnphoA take advantage of the knowledge that exported factors are often pathogenicity factors (III.A.2); it also took advantage of a pre-screen strategy to collect only strains harboring fusions that were coordinately regulated with a previously known pathogenicity factor [113]. Specifically, Tn/7/26>y4-mutagenized V. cholerae strains were grown under two separate in vitro conditions known to downregulate and upregulate, respectively, the expression of cholera toxin. Fusion strains expressing alkaline phosphatase activity only in the upregulatory growth condition could then be said to harbor TnphoA insertions in genes encoding exported polypeptides that are coordinately regulated with cholera toxin. The premise behind this strategy relies on the fact that many pathogenicity factors may often be coordinately regulated with a previously known pathogenicity factor. This premise has now been realized in several other pathogens aside from V cholerae. For instance, both the bvgAS regulon in Bordetella pertussis [40], and the phoPQ regulon in S. typhimurium have been shown to coordinately regulate expression of many pathogenicity factors [89, 110]. There are many other coordinate regulatory screens that have been designed that vary in the choice of reporter gene. Frequently used reporter genes include promoterless lacZ [126] and gfp [115] alleles. In contrast to the TnphoA translational fusion strategy, transcriptional fusions to cytosolic enzymes such as lacZ allow identification of virtually any gene that is coordinately regulated, that is, regardless of whether the gene encodes an exported or cytoplasmic protein. The two primary limitations of coordinate regulatory screens, though, are that, as discussed above, some gene fusions will be toxic to the bacteria, and the in vivo environmental conditions that induce expression of certain pathogenicity gene regulons remain unknown or are difficult to reproduce in the laboratory.
4.
HOST MIMICRY SCREENS
The requirement for bacterial pathogens to differentially regulate expression of genes depending on their environment continues to be a theme that can be exploited by the scientist to attempt to elucidate factors required for pathogenicity.
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In fact, this common theme runs through many of the strategies designed to elucidate pathogenicity factors that have been the most successful throughout the last few decades. Due to the facts that the environmental challenges that a pathogen encounters in its host are often different from one tissue to the next and that host environments are quite complex, screens designed to mimic certain host environmental parameters provide an attractive method for simplification of the process of pathogenicity factor identification. Although numerous mimicry screens have been designed, the most straightforward have proven to be those designed to look for gene fusions to a reporter such as lacZ, which are specifically induced during growth in a laboratory medium that mimics one particular host parameter. Examples of successfully employed screens include the identification of acid-inducible genes from various intestinal pathogens by exposing the bacteria to media having acidic pH values similar to those found in the human stomach or phagosomal microenvironment (e.g., [42]), and identification of high-temperature-inducible genes from facultative pathogens using an in vitro growth temperature equivalent to human body temperature (e.g., [5]). Host mimicry screens have not been limited to utilization of lacZ but have employed other reporter genes such as luciferase, gfp and phoA. In addition, a variety of host mimicking conditions have been employed. Due to the fact that many pathogenicity genes require more than a single signal to induce their expression, it is often advantageous for the investigator to be able to mimic several host signals at one time. It is for this reason that one commonly used in vitro model system is cultured mammalian cells. Cultured mammalian cells often provide many of the signals needed to simulate infection and have thus been used to identify pathogenicity factors produced by both adherent extracellular pathogens (e.g., [49, 94]) and intracellular pathogens (e.g., [61]). A recendy developed strategy combines the use of host macrophages as an inducing environment with Fluorescence-Activated Cell Sorting (FACS). This screen was utilized to search for S. typhimuriiim genes within the SPI-2 pathogenicity island locus, which are induced within the intracellular milieu of macrophages [24]. This study utilized gfp fusions to genes within the SPI-2 locus and resulted in identification of several genes that are transcriptionally induced within host cells and that are required for systemic spread in a mouse model of typhoid. One of the major advantages of host mimicry screens is the fact that there are coundess host-mimicking condidons that can be devised. In addition, most of these screens can be conducted with relative ease due to the fact that they take advantage of in vitro conditions to mimic the often more complex in vivo environment. In pardcular, the GFP/FACS screening strategy is attracdve due to the fact that it should allow near-saturating screens to be performed, as a thousand-fold enrichment of GFP-posidve bacterial cells can be achieved in a short period of dme [116]. However, it should be noted that host mimicry screens do suffer from the limitation that the host conditions required to trigger expression
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of some bacterial factors remain unknown or may be too complex to reproduce in the laboratory. This is less true for tissue culture models of infection than for other in vitro systems, but it applies nonetheless. Because of this limitation, several in vivo screens have been recently developed that utilize the intact host animal to provide a more complete inducing environment (see below).
B. In Vivo Screens 1.
RECOMBINATION-BASED IN VIVO EXPRESSION TECHNOLOGY
Because utilization of large numbers of animals as disease models can be quite costly and trying due to ethical and logistical reasons, scientists have spent a considerable amount of time trying to minimize the necessity to conduct large screens that require intact animals. It is for such reasons that large-scale screens (III.A.l) using animals as hosts can be quite limiting since it is required that each individual strain of interest be screened in a separate animal. Efforts to develop screens that allow multiple mutant strains to be tested per animal have recendy been inspired by the advent of In Vivo Expression Technology (I VET). I VET is a series of genetic selection methods devised to identify bacterial genes that are transcriptionally induced during infection of a host animal (see IV.D). These efforts have resulted in the development of two new screens: RecombinationBased In Vivo Expression Technology (RIVET), and Signature-Tagged Mutagenesis (STM). Each of these screens addresses fundamentally different questions, but both are advantageous in that they allow for utilization of fewer numbers of intact animals to screen for genes of interest. With RIVET, bacterial genes that are transcriptionally induced during infection are identified, while with STM (described in III.B.2) genes that play an essential role in pathogenicity are identified. There are two major obstacles that must be overcome to comprehensively screen for bacterial genes induced during animal infection. First, because traditional gene reporters such as lacZ and phoA encode labile products, a positive signal can be transient and may be lost on recovery of bacterial cells from infected tissues. Second, in order to assay these reporters, a large number of cells is needed. This of course is not possible when screening a library of different strains in an animal. The technique of rapidly screening for GFP-expressing bacterial cells by FACS (described in III.A.4) may largely overcome both of these obstacles for some host-pathogen systems. However, a limitation that applies to all previously used gene reporters including gfp, is that gene fusions that are transiently expressed during infection or that are expressed at very low levels are difficult or impossible to detect. The RIVET strategy overcomes all three of these limitations to some degree. With RIVET, transcriptional gene fusions are made to a promoterless gene encoding a site-specific DNA recombinase, such as the tnpR gene (encoding
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resolvase) of Tn 1000. The resolvase enzyme is able to mediate recombination between two directly repeated copies of a specific target DNA sequence, called resl, which have been inserted flanking a genetic marker within the chromosome of the pathogen of interest. If a particular gtnt-tnpR fusion is transcriptionally induced during infection, even transiently and/or at a low level, the resolvase that is produced will catalyze a heritable change in the bacterium by excising the marker from the chromosome. The resolved strain can then be screened or selected for after recovering the bacteria from host tissues (Fig. 3, see color plate). For example, in the original study describing RIVET, induced resolvase fusions catalyzed the excision of a tetracycline-resistance gene, resulting in conversion of the fusion strain to a tetracycline-sensitive phenotype [18]. The strains harboring induced fusions were then identified by replica-plating colonies onto an agar medium supplemented with tetracycline. In the first implementation of RIVET, over a dozen V cholerae genes were identified whose levels of transcription increased during infection of the small bowel of infant mice, and a few of these genes were shown to play a role in pathogenicity by mutational analysis [19]. In addition to its application to V. cholerae, RIVET has also been used to identify S. aureus pathogenicity genes in a murine renal abscess model of disease [77]. RIVET-based screens suffer from two limitations. First, because the site-specific DNA recombinase acts on only one substrate sequence, a low level of expression of the recombinase gene fusion is sufficient to catalyze the excision event. Although this exquisite sensitivity allows for detection of transient and/or low-level gene induction events, it unfortunately prohibits identification of pathogenicity genes that have high basal levels of transcription during in vitro growth. This is due to the fact that a high basal level of transcription results in a strain that is unable to be constructed in the unrecombined state. However, more recent modifications have largely overcome this limitation. Specifically, a series of tnpR alleles containing "down" mutations in the Shine-Dalgarno sequence have been constructed. These mutations reduce the efficiency of translation over a wide range of transcriptional levels. This modification allows tnpR to be fused to genes that have high basal levels of transcription in vitro, without excising the substrate. If, though, these gene fusions are further transcriptionally induced during infection, the concomitant increase in translation product will result in excision of the marker from the strain [109]. The second limitation of RIVETbased screens, which holds for most other genetic screens and selections, is that only a subset of the genes identified will encode factors whose loss results in attenuation of pathogenicity in models of disease.
2.
SIGNATURE-TAGGED MUTAGENESIS
Signature-Tagged Mutagenesis (STM) is a genetic screen whereby transposon insertion mutant strains that are attenuated for survival and/or growth within the
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host are identified [50]. Specifically, each host animal is infected with an input pool of limited complexity (usually 96 mutants), and the output of mutants recovered from host tissues is compared to the original input pool to identify strains that did not survive within the animal, that is, are missing from the output pool. In the most recent version of STM (Fig. 4, see color plate) [87], the strategy begins by marking each of 96 transposons with a small, unique DNA signature-tag which is inserted into a nonessential site within the transposons. Each of the resulting uniquely tagged transposons is then used to construct an insertion library in the bacterial pathogen. Strains from each of the 96 libraries are subsequently picked sequentially into microliter plate wells (see Fig. 4, see color plate), with each resulting microliter plate representing an input pool. A pooled sample of all 96 strains from each plate is then inoculated into an animal, and after a sufficient period of infection bacteria are recovered from one or more host tissues. The signature-tags are then PCR-amplified from both the input and output pools, and the representation of each strain in the input and output pools is measured by hybridizing the amplified tags to a master dot-blot filter that contains each of the original 96 DNA tags. Any strain present in the input pool, but is missing from the output pool, is attenuated for pathogenicity in the host animal. In the original implementation of STM, a large number of S. typhimurium pathogenicity factors were identified using a murine model of typhoid fever [50]. Since then, numerous STM screens have been successful in identifying pathogenicity factors from a variety of bacterial pathogens, such as V. cholerae [23] and S. pneumoniae [96]. In the first implementation of the modified STM protocol described above, several novel S. aureus pathogenicity factors were identified in a murine model of staphylococcal sepsis [87]. STM is a powerful genetic screen in that it allows many mutant strains to be screened per animal. In addition, the method is designed such that only genes that are essential for survival and pathogenicity in the animal are identified. Another advantage of STM, which is also shared with RIVET (III.B. 1), is that pathogenicity genes that are transiendy expressed, or expressed at low levels during infection, can in theory be identified. Perhaps the most notable limitation of STM, however, is that genes that play subtle roles in pathogenicity or have redundant functions are difficult or impossible to identify. Additional limitations of STM are that: (1) the presence of a colonization bottleneck in many disease models will necessarily reduce the allowable complexity of the input pool, and, relatedly, (2) a large inoculum, which can affect the natural course of the disease, is often needed to ensure adequate representation of all input strains. A significant property of both STM and large-scale screening that can make both methods powerful tools for analysis of potential pathogenicity factors, and which is not true for most other screening or selection strategies, is that a measure of the effect of a gene mutation on pathogenicity is built into the strategy. In contrast, the other strategies end with identification of bacterial factors whose expression levels are known to be increased during infection, or during in vitro
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growth in a host-mimicking condition. As these only lead to the possibility of a role in pathogenicity for such factors, a specific test of this possibility must follow. 3.
VERIFYING A ROLE IN PATHOGENICITY
As has been emphasized throughout this chapter, most genetic screens and selections result in identification of a gene whose role in pathogenicity is implicated, but not proven. Hence, it is usually desirable to fulfill what has been termed a molecular version of Koch's postulates in order to prove that the gene in question does play a role in pathogenicity [34]. The specific postulates are basically as follows: (1) The phenotype or property contributed by the gene should be associated with pathogenic strains. (2) A null mutation in the gene should attenuate pathogenicity in an appropriate model of disease. It should be noted at this point that the type of mutation that is least prone to artifacts is an in-frame deletion of virtually all of the gene coding sequence. For example, a deletion of only part of the gene could result in production of a truncated polypeptide that is toxic to the pathogen during infection. Alternatively, an out-of-frame deletion or insertional mutation might exert a polar effect on a downstream pathogenicity gene. (3) Pathogenicity should be fully restored after adding the gene back to the mutant strain. Such complementation is often best done by either inserting the gene back into the genome in a new location (e.g., [90]) or maintaining it on a low-copy plasmid. Insertion of the gene back into its original location, at the same time removing the mutant allele, does not rule out the possibility that a polar effect was responsible for the reduced level of pathogenicity originally observed. Optimally, the native promoter should be used to drive expression of the gene. However, this is often difficult and can be substituted for by engineering a heterologous promoter upstream of the gene. In practice, complementation of gene mutations usually ranges from easy to near impossible. For example, it is conceivable that some genes may require precisely regulated expression that can only be obtained in their native locations in the genome in order to mediate their effect on pathogenicity. A limitation of the second postulate is that mutations in some genes, which do indeed play roles in pathogenicity, may not result in a substantial decrease in pathogenicity when tested in the disease model. This might result from a limitation of the model of disease being used to measure pathogenicity, or it might result from the presence of another gene with redundant function that is also expressed by the pathogen. In the latter case, it would be ideal to identify and construct a null mutation in the redundant gene(s), and then attempt to fulfill Koch's molecular postulates for the first gene.
IV. Genetic Selections Since screens for pathogenicity factors can often be time consuming, requiring the isolation and analysis of many individual strains, it is of great benefit to the investigator if he/she can select (enrich) for the mutant strains that are being
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sought. Such selection strategies greatly decrease the number of strains that must be analyzed to find a pathogenicity gene. The basis of most genetic selections is that strains harboring mutations that attenuate pathogenicity are selectively amplified to facilitate their isolation and identification. Alternatively, some genetic selections are based on identification of the gene mutations that enhance pathogenicity. The latter type of selection has a built-in enrichment scheme to amplify the strains of interest in that it searches for enhanced survival and/or multiplication in the host. Although the latter type of selection is straightforward, the former is less so, particularly in instances where the only measure of attenuated pathogenicity involves death of the mutant bacterial strain in the host. In this section, several examples of both of these types of genetic selections, which have been successfully employed, will be discussed.
A. Direct Selections There are a number of methods that can be employed for selection purposes, but perhaps the most straightforward enrichment strategy for gene mutations that attenuate pathogenicity is to selectively kill virulent strains, thus enriching avirulent strains. This could be done, for example, by using convalescence serum containing antibodies against a pathogenicity factor(s) exposed on the bacterial cell surface to select strains from a library of mutagenized bacteria that no longer express the pathogenicity factor(s). These strains would be enriched as a direct result of pathogenic bacteria being selected against due to the opsonizing activity of the antibodies. This method has been used, for example, to identify capsular polysaccharide-defective mutants of Klebsiella pneumoniae [9]. It should be noted that this particular method is not generally applicable, as convalescence sera to some pathogens may contain bactericidal antibodies recognizing multiple surface molecules, the losses of which cannot be obtained by the result of a single gene mutation. A second type of direct selection is built on the fact that some pathogenicity factors that reside on the surface of bacterial cells serve as receptors for lytic bacteriophages. This knowledge provides the basis for selection of bacterial mutant strains that have lost expression of the pathogenicity factor and thus are resistant to bacteriophage infection. For example, a major pathogenicity factor of many Gram-negative bacteria, LPS, serves as a receptor for many bacteriophages (e.g., [46]). Therefore, a strain that is deficient in LPS biosynthesis would be resistant to infection and lysis by bacteriophages. In addition to LPS, it has been found that other bacterial surface molecules or structures that are pathogenicity factors can serve as bacteriophage receptors (e.g., V. cholerae TCP [121]). A third type of direct selection takes advantage of growth phenotypes that are associated with the presence or absence of some pathogenicity factors. For example, wild-type L. pneumophila are growth sensitive to high salt concentra-
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tions, but a variety of nonpathogenic derivatives have been noted to be salt resistant. This correlation, which may or may not be relevant to the pathogenesis of this organism, was used as the basis for a selection of transposon mutagenized strains that were salt resistant. This selection was highly successful and resulted in the isolation of multiple strains that were subsequendy shown to be nonpathogenic due to disrupdons in several previously unidentified pathogenicity genes [119]. A limitation of this strategy is, of course, that the presence or absence of most pathogenicity factors cannot be associated with a growth phenotype in vitro, and thus this method is not generally applicable. However, it is likely that some pathogenicity factors, in particular those metabolic factors that assist growth of the pathogen during infecdon, can be identified by similar types of genetic selections. To take a hypothetical example, proton-pump inhibitors that are bactericidal for //. pylori in vitro [109] might be used to select for mutations in the proton-pump encoding genes that in turn encode an essential pathogenicity factor necessary for surviving the low-pH environment of the human stomach.
B.
Complementation Approaches
Many bacterial pathogenicity factors have been identified by complementation selecdon strategies. The most common of these strategies selects for a small fragment of genomic DNA carried on a plasmid, which complements the nonpathogenic phenotype of a strain containing a spontaneous or chemically induced mutadon in an unknown gene. In most cases, the complemendng fragment of DNA contains a functional version of the mutated gene or operon, and thus serves to identify the gene of interest. Three examples of the many pathogenicity factors idendfied by complementation analysis include the inv locus of S. typhimuriwn [46a], the dot/icm genes of L. pneumophila [10], and the phase-variable tcpH gene of V cholerae [21]. It should be noted, however, that it is somedmes possible that the complementing fragment contains a gene or set of genes disdnct from the mutated gene, but which nevertheless can phenotypically complement (i.e., bypass) the mutation. Because complementadon is the most suitable approach to idendfy genes disrupted by chemical mutagenesis, and because chemical mutagenesis produces the widest range of gene mutadons and functions in almost any bacterial species (see III.A.l), this combination of approaches comprises a powerful strategy for idendfying pathogenicity factors. However, it should be noted that complementation strategies are limited to bacterial species for which efficient transformation protocols exist. A second type of complementation strategy expands on the above strategy by searching for a gain of function. This strategy works by selecdng for a small genomic fragment of a pathogen, which when transferred into a nonpathogenic
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Strain or heterologous species bestows some aspect of the pathogenic lifestyle. For example, the invasin protein of Y. pseudotuberculosis, which mediates invasion of nonprofessional phagocytic cells in the host, was identified by transforming a noninvasive E. coli strain with a library of Y. pseudotuberculosis genomic DNA, and then selecting for cells that had gained invasive ability [56]. The invasive strains of E. coli produced were selected for by the addition of an aminoglycoside antibiotic to the tissue culture medium to kill extracellular bacteria but not intracellular ones, as such antibiotics do not equilibrate with the interior of mammalian cells. A major attribute of this second type of complementation strategy is that an expressed genomic library of many pathogenic species, including some that are not genetically manipulatable, can be constructed in a nonpathogenic species that is much more easily manipulatable. However, this approach is limited by the need for relatively simple models of disease for which a single genetic locus can confer the sought-after phenotype on a heterologous bacterial species. As a result of this limitation, this type of approach has been used in the past usually to identify pathogenicity factors that either have enzymatic activities that the host bacterium lacks (e.g., hemolysins [31, 79]) or that complement a metabolic defect in the host bacterium (e.g., iron acquisition [8]). In addition, any factor identified by this method ultimately requires evaluation for a role, if any, in virulence in the original microorganism.
C. Selection for Nongrov^^ing Bacterial Mutants Penicillin selection, which was originally developed to facilitate isolation of auxotrophs, functions by killing growing cells and sparing nongrowing cells [68]. Penicillin selections have been used as the basis for a strategy to isolate nonpathogenic bacterial mutants that fail to grow in host tissues. For example, in the original application of this strategy, transposon insertion mutants of Listeria monocytogenes that failed to multiply within cultured macrophages were selected for after the addition of methicillin, a (3-lactam that is able to freely penetrate into the interior of mammalian cells [20]. Amongst the mutant strains isolated were some that harbored transposon insertions in the gene encoding listeriolysin O, an essential pathogenicity factor that facilitates access to the host cell cytoplasm where multiplication and cell-to-cell spread occur [98]. For a review on the role of listeriolysin O, please refer to the Chapter 16 in this book. An analogous strategy to penicillin selection, termed "thymineless death," was developed to isolate L. pneumophila mutant strains unable to multiply within cultured macrophages [10]. The method is based on the observation that thymine auxotrophs of many bacterial species, including L. pneumophila, die when
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multiplying in the absence of exogenous thymine whereas nonmultiplying cells survive. This allowed an enrichment of nonpathogenic L. pneumophila mutant strains that failed to multiply intracellularly in cultured macrophages. This study identified a multigene locus, called dot (also referred to as icm ([14]), which is required for intracellular multiplication. One of the most important attributes of both penicillin selection and thymineless death strategies that is also shared with STM is that nongrowing mutant strains can be identified direcdy from host tissues or cells. However, in contrast to STM, the former two selection strategies are probably limited to in vitro and tissue culture host model systems, as recovery of a nongrowing mutant strain from an intact animal could be exceedingly difficult. For example, a thymineless death strategy to select for L. monocytogenes mutants that are no longer able to multiply inside macrophages in an animal host would probably fail due to clearance of bacteria by the host immune system. Moreover, an in vivo penicillin selection would require the difficult task of maintaining sufficient antibiotic concentrations at the site of infection in order to select for nongrowing mutant strains. However, the latter technical difficulty would not apply to the thymineless death strategy if used in an intact animal host.
D. In Vivo Expression Technology In Vivo Expression Technology (IVET) is a strategy designed to identify genes that are transcriptionally active only during infection. The premise behind IVET is that some, perhaps many, genes that are transcriptionally active during infection encode pathogenicity factors. The system relies on the enrichment of bacterial strains harboring gene fusions to a marker whose expression is or can be selected for within the intact host animal [80]. In the first implementation of IVET, an S. typhimurium transcriptional gene fusion library iopurA was constructed in a strain background in which the native purA gene had been deleted. Because purine auxotrophs cannot survive during systemic infection of mice, only strains from the library that harbor transcriptional gene fusions to purA that were expressed during infection were complemented and therefore survived and multiplied (Fig. 5, see color plate). This method has been used to identify several genetic loci encoding pathogenicity factors in S. typhimurium [48, 81]. Additionally, IVET has been successfully used to identify pathogenicity factors in other pathogens, such as P. aeruginosa [122, 123]. The above IVET strategy requires prior knowledge of selectable genes, such as purA, whose expression is required for multiplication within the host. It is essential that the chosen selectable gene be required for growth in the same host compartment that the pathogenicity gene (to be identified) is expressed in. Thus, a limitation of IVET strategies that employ complementing genes like purA is that, if the selectable gene is required throughout the entire infectious process.
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then preferential enrichment will occur for strains harboring gene fusions that are constitutively expressed in the host animal. Thus, pathogenicity genes that are expressed at one stage of infection only, or which are transcribed at low levels, may not be identifiable via this approach. A second type of IVET selection is based on enrichment of bacterial strains harboring gene fusions to an antibiotic-resistance gene. In this method, a sufficient concentration of the corresponding antibiotic is maintained in the infected tissues to allow enrichment of strains containing active gene fusions. This method has been used to identify S. typhimurium pathogenicity factors [82]. Clearly, this method is more complicated than the auxotrophy complementation approach mentioned above since suitable antibiotics must be administered and proper concentrations maintained in infected host tissues. However, this property can provide some flexibility in designing the selection. For example, administering the antibiotic at only one stage of infection, or in one host organ, should facilitate identification of pathogenicity factors which may be expressed at only one stage of infection, or in only one host compartment, respectively. Recently, IVET has been combined with a direct selection scheme to identify pathogen genes that are transcriptionally induced during infection and which also encode exported proteins (J. J. Mekalanos and J. W. Tobias, unpublished data). The first component of this method proceeds by isolating pathogen DNA sequences that encode protein export signals, in a manner analogous to the TnphoA strategy (III.A.2). A plasmid library is constructed by ligating genomic fragments immediately upstream of a truncated p-lactamase gene that lacks the signal sequence coding portion required for protein export and subsequent enzymatic activity. The plasmids are moved into an E. coli host, and ampicillin is used to select for plasmids containing genomic fragments that encode protein export signals fused to the p-lactamase. Next, the selected pool of genomic fragments is excised from the plasmids and subcloned into pi VET 1 (see schematic diagram of pIVETl at the top of Fig. 5, see color plate). The resulting pool of pi VET 1 recombinants is then moved into the pathogen genome and subjected to the IVET selection to identify those strains containing infection-induced gene fusions, as described above (Fig. 5, see color plate). In the first implementation of this strategy, several S. typhimurium genes were identified. These included sti, a previously identified gene that encodes a glycosidase that interferes with IL-2 receptor function [85], and /v/-S2, a previously unknown gene whose putative product has a high degree of similarity to AmpD from E. coli, except that /v/-S2 encodes a putative signal sequence (the E. coli AmpD lacks a signal sequence). Roles for sti and ivi-Sl in pathogenicity have not been tested for, as yet. It is likely that this combination strategy will serve as a powerful method to identify additional infection-induced, exported factors from this and other pathogens. However, it should be noted that this combination strategy will be subject to the same limitations that apply to its two component methods.
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V. Genomic Approaches At the time of this writing, the complete genome sequences of 19 bacterial pathogens were publicly available, and another 40 or so were in progress (www.tigr.org). It is likely that within a decade the genomic sequences of almost all clinically important bacterial species will be known. This body of information, accompanied by the many computer algorithms already available for nucleotide and amino acid searching, have made possible a multitude of so-called, postgenomic strategies for identifying pathogenicity factors. In this chapter, several such strategies will be discussed, along with a few genomic strategies that do not require knowledge of a pathogen's genomic sequence. A.
Genome Walking
The past 30 years of molecular genetic research has shown conclusively that the genes encoding pathogenicity factors are more often than not physically linked on chromosomes or on extrachromosomal elements. Genetic linkage is particularly acute for genes that encode the factors necessary for production of complex factors such as pili (e.g., [83]) or type III secretion apparatuses (e.g., [25]). Although some genes encoding pathogenicity factors are not linked in this way (e.g., [47]), it nonetheless remains a fruitful strategy to target one's search for regions adjacent to previously identified pathogenicity genes. This is most easily accomplished by sequencing nearby genes, and analyzing the resulting nucleotide sequence for the presence of putative pathogenicity genes. Any such identified gene would then need to be mutated to assess its role, if any, in pathogenicity. One example of the numerous reports of genome walking is the recent completion of the Y. pestis Pmtl plasmid nucleotide sequence, which revealed the identity of at least seven genes encoding putative pathogenicity factors [54, 74]. A second genome walking strategy involves generating a series of mutations in specific DNA regions that surround a known pathogenicity gene. This strategy is usually accomplished by first cloning the region of interest into a plasmid, mutating the entire plasmid by transposon insertion mutagenesis or by site-directed means such as restriction enzyme-mediated frame-shift or deletion, and finally by moving the mutated plasmids back into the pathogen. The plasmids may then be tested for their ability to complement a complete deletion of the corresponding genomic locus or used to replace the genomic locus by allelic exchange. Strains that are isolated can then be tested for pathogenicity in a model of disease. This genome walking strategy has been used extensively. For example, a cluster of genes required for fimbrial expression in enteroaggregative E. coli, was identified by randomly inserting a transposon into a cloned DNA fragment known to contain genes essential for aggregation [103]. Genome walking has the advantage of discovering pathogenicity genes often with a minimal amount of labor, and it is also useful for identifying genes that function in concert to produce a complex virulence determinant. The only major
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limitation of this strategy is that additional studies need to be done on the putative pathogenicity genes identified to assess their roles in pathogenicity in a model of disease.
B. Genomic Analysis and Mapping by in Vitro Transposition Genomic Analysis and Mapping By In vitro Transposition (GAMBIT) was recently developed primarily as a method to identify genes of bacteria that are essential for in vitro growth [2]. However, GAMBIT can also be used to identify genes that are essential for growth during infection in a host. In this respect, GAMBIT is similar to STM (III.B.2). An essential step in the GAMBIT protocol is the initial PCR-amplification of large segments of the pathogen genome (-15 kb in size). This step, of course, requires knowledge of some, or all, of the genomic sequence of a pathogen in order to allow the design of sets of PCR primer pairs. If the entire genome sequence is known, it would technically be possible to use GAMBIT to identify all essential genes by contiguous amplification and analysis (see below) of 15 kb regions. As shown in Figure 6 (see color plate), when using GAMBIT, each PCR product is subjected to in vitro transposon mutagenesis, which results in random insertions along the length of the fragment. The mutated DNA fragments are then transformed into the pathogen, and strains in which a fragment has integrated by allelic exchange are selected for by the use of the antibiotic resistance marker that is carried on the transposon. Transformant colonies are pooled and subsequently used both to infect a host and to prepare total DNA (the input DNA). After an appropriate period of infection, bacteria are recovered from host tissues, and total bacterial DNA (the output DNA) is prepared. During the course of the infection, strains that harbor transposon insertions in essential pathogenicity genes are selected against. Both the input and output DNAs are used as templates in PCR reactions using one of the primers originally used to amplify the genomic fragment, and a primer that is complementary to a sequence within the inverted repeat present at each end of the transposon. The PCR products are separated on an agarose gel, and their estimated sizes are used in conjunction with the genomic sequence to identify the sites of transposon insertion along the genomic fragment. The patterns of bands derived from the input and output DNA form two "ladders." The two ladders are visually compared to identify gaps (missing bands in one or more regions) in the output-derived ladder that correspond to gene(s) that are essential for survival and/or multiplication in the host (Fig. 6, see color plate). GAMBIT is a new methodology and has not been used to date in a general screen for pathogenicity factors. Nevertheless, a discussion of its advantages and limitations is warranted. Like STM, GAMBIT has the unique advantage of directly identifying genes that are essential for infectivity. However, because GAMBIT is predicted to be more labor intensive than STM when screening the
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entire genome of a pathogen, the former will probably find greater use in analyzing individual loci for the presence of pathogenicity genes. As with most genetic methods, GAMBIT can only be used in pathogens that are readily transformable. Although GAMBIT was originally implemented in two naturally transformable bacterial species, Haemophilus influenzae and S. pneumoniae, it should also be useful for examining small genomic regions in less efficiently transformable species. Finally, GAMBIT has the advantage that near-saturating mutagenesis can be achieved for a locus, thus allowing the essentiality of small regions in any locus to be examined in great detail. For example, in the original GAMBIT study [2], it was found that, although transposon insertions into most of the secA gene of H. influenzae were lethal for growth in vitro, those in the carboxy-terminal coding region were well tolerated. This finding was in accordance with a previous report [100], and it thus serves to demonstrate the ability of GAMBIT to allow detailed analysis of loci of interest.
C. Computational Screens Perhaps the most efficient means of identifying pathogenicity factors using genomic sequence is to search for predicted polypeptides that have amino acid sequence similarity to previously known pathogenicity factors, and which therefore are predicted to function similarly. For example, a recent report identified the prepilin peptidase, PilD, of V. cholerae from genomic sequence analysis. After identification of this gene, the authors then confirmed the requirement of the pilD product for secretion of a number of pathogenicity factors including cholera toxin [45]. Numerous additional similarity search strategies exist. For example, one could search for putative polypeptides with amino acid similarity, and thus possible functional similarity, to host encoded proteins. Such pathogen encoded proteins may function as pathogenicity factors by interfering with or otherwise affecting host cellular functions. Aside from searches based on amino acid similarity, a number of nucleotide search strategies also exist that can be used to identify pathogenicity factors. For example, two characteristics of pathogenicity islands—a different GC content than the overall GC content of the species and the presence of flanking tRNA sequences—have been used to identify previously unknown pathogenicity islands (e.g., [11]). Another strategy could be to search for promoter or coding-sequence elements that are known to be conserved in other pathogenicity genes. For example, most of the pathogenicity genes previously identified in L. monocytogenes are transcriptionally activated by the PrfA protein, which binds to a conserved 14-bp palindrome within their promoters [43]. Thus, after the L. monocytogenes genome sequence (currently in progress; www.pasteur.fr) is completed, it might be fruitful to identify and analyze the subset of genes that contain this palindrome in their promoter regions. Conserved virulence gene
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promoter elements have been defined in a number of other pathogenic species as well, for example, B. pertussis [60, 104]), and thus this approach might be widely applicable. A third potential strategy could be to search for genes that contain small sequence repeats that might undergo slipped-strand mispairing during replication in order to phase-vary the expression of a bacterial factor [51]. For more information on phase variation, please refer to Chapter 14 in this book. Genes that phase-vary are likely to encode proteins that are seen by the host immune system, or that are involved in the biosynthesis or export of such factors, and thus are more likely than not to play a role in pathogenicity (e.g., [58]). As more pathogen genomes are sequenced, pathogenicity gene discovery by computer algorithm searching will become commonplace. Although computational screens are rapid and efficient means of identifying putative pathogenicity genes, it must be understood that these screens are only a starting point, as genes of interest must still be studied by traditional means, such as mutagenesis and complementation to demonstrate a role in pathogenicity.
D.
Transcriptional Profiling and the Use of Microarrays
The advent of extensive microbial and eukaryotic DNA sequence databases has prompted the development of DNA chip and microarray technologies in several commercial and academic laboratories [13, 26, 105, 107, 124]. These methods provide a powerful new way to measure the expression of virtually every gene in a genome by simultaneously measuring the concentration of individual messenger RNAs in an experimental sample. This methodology is referred to as "transcriptional profiling" and has applications in virulence gene identification. In brief, DNA samples that correspond to each individual gene of an organism are spotted by a high-resolution robot (the microarrayer) on the surface of, for example, an appropriately treated glass slide. Each gene has a known location on the microarray, and literally thousands of genes can be arrayed per glass slide. These DNA spots then serve as hybridization targets that will bind complementary nucleic acid labeled with appropriate fluorescent tags. In practice, mRNA is extracted from cell preparations, converted to fluorescently labeled cDNA, and hybridized to the microarray. The resulting hybridization intensities are measured by simply inserting the slide into a special fluorescence confocal microscope (the scanner) that can measure and record the individual fluorescent intensities of each spot in the microarray in a matter of minutes. There are clearly numerous applications for microarray technology. The conceptually easiest applications to envision involve comparisons of various sorts—for example, mutant strain versus parental strain, or bacteria grown in laboratory medium versus those grown in host-derived sample or host cells. In each of these experimental formats, bacteria are grown under a given condition or are allowed to interact with host cells for a defined period of time. Next, RNA
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is extracted and converted to fluorescently labeled DNA probes by reverse transcription. In each case, the comparison of RNA-derived probes from two different sources is made by differentially labeling (e.g., with red and green tags) and then hybridizing the two different probe preparations to the same DNA microarray. Comparison of DNA sequence distribution between different strains or even different culture preparations of a given strain provides an additional application for microarray technology that will fmd use in pathogenicity gene identification. In theory, hybridization to genomic arrays can be used to identify DNA segments that undergo amplification or rearrangement in response to the selective pressure of growth in a mammalian host. Because genetic elements encoding or controlling the expression of pathogenicity genes can undergo amplification [87], inversion [62, 130], or deletion [86], the mapping of any genomic segment that is rearranged in vivo provides a potentially powerful way of identifying genes involved in host-microbe interactions. Variations in sequence content between different clinical isolates might alert investigators to the presence of new pathogenicity-encoding plasmids, phages, and transposons, and pathogenicity islands that have moved horizontally into strains or have been recently lost. Thus, microarray analysis is expected to augment other established methods such as subtractive hybridization and differential display (discussed in II.F and II.G), which have been effectively used to identify genetic differences between closely related clinical isolates. The use of microarrays to define infection-induced genes, or genes coordinately regulated with other pathogenicity genes, will become more common as this technology is explored and improved. Other applications of microarrays will be developed. For example, it is easy to imagine that one could make a custom microarray carrying all oligonucleotide "tags" used to label individual transposons in the STM protocol (III.B.2). Such an array could be hybridized with PCR-derived probe pools derived from transposon mutants before and after animal passage, thus allowing quantification of the relative ratios of various mutants in a mixture without the need to grid mutants out spatially. Thus, like other IVET and STM techniques, microarray technology will eventually provide a new approach to defining which bacterial genes are induced during infection. The primary limitation of microarray technology, currently, is the high cost of manufacturing the microarrays. However, as the utilization of microarrays becomes more commonplace, and as the technology continues to develop, it is likely that the extreme costs associated with this technology will become more affordable.
VL Concluding Remarks As we settle into the postgenomic era, microbiologists will continue to use a combination of biochemical, genetic, and genomic techniques to identify patho-
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genicity factors, although the latter will become a more frequent starting point. In particular, the biggest advances in pathogenicity gene discovery will likely come from large-scale genomic analyses, using either computational methods or DNA microarray technologies. Specifically, these two methods should facilitate identification of sets of pathogenicity genes with related function and/or that are coordinately regulated, many of which may be recalcitrant to discovery by current methods. For example, assessing the roles in pathogenicity of factors that have redundant function is very difficult, because knocking out the expression of one factor may not attenuate pathogenicity in the model of disease being used. However, knowing the identity of the redundant genes would allow a multiple gene knockout strategy to be used to assess their roles. In addition, many pathogenicity genes that are small in size may have eluded identification because they are difficult to mutate by transposon or insertion-duplication mutagenesis. Thus, knowing their locations would allow a site-directed mutagenesis strategy to be used to construct null mutations in them, in order to assess their collective roles in pathogenicity. It is likely that we are at the dawn of new discoveries of pathogenicity factors. With the technical advances of the last few years, in particular the ability to sequence large genomes, it is likely that many of the genes that are involved in pathogenicity will soon be discovered. This jackpot of discovery will more than likely lead to a general slowing of the pace of discovery of new pathogenicity genes within the more well-studied pathogens that are genetically manipulatable. In contrast to this slowing down of gene discovery, though, the scientific community will continue to be faced with a frontier of discovery that is perhaps even more challenging: a true understanding of the actual functions of bacterial pathogenicity factors during infection of the human host.
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113. Taylor, R. K., Miller, V. L., Furlong, D. B., and Mekalanos, J. J. (1987). Use of p hoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. Natl Acad. Sci. U.S.A. 84(9), 2833-2837. 114. Tinsley, C. R., and Nassif, X. (1996). Analysis of the genetic differences between Neisseria meningitidis and Neisseria gonorrhoeae: Two closely related bacteria expressing two different pathogenicities. Proc. Natl. Acad. Sci. U.S.A. 93(20), 11109-11114. 115. Valdivia, R. H., and Falkow, S. (1996). Bacterial genetics by flow cytometry: Rapid isolation of Sahnonella typhimuriiim acid-inducible promoters by differential fluorescence induction. Mol. Microbiol. 22(2), 367-378. 116. Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L., and Falkow, S. (1996). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. Gene 173(1), 47-52. 117. van den Berg, B. M., Beekhuizen, H., Willems, R. J., Mooi, F. R., and van Furth, R. (1999). Role of Bordetella pertussis virulence factors in adherence to epithelial cell lines derived from the human respiratory tract. Infect. Immiin. 67(3), 1056-1062. 118. Visai, L., Bozzini, S., Raucci, G., Toniolo, A., and Speziale, R (1995). Isolation and characterization of a novel collagen-binding protein from Streptococcus pyogenes strain 6414. J. Biol. Chem. 270(1), 347-353. 119. Vogel, J. P., Roy, C, and Isberg, R. R. (1996). Use of salt to isolate Legionella pneumophila mutants unable to replicate in macrophages. Ann. N.Y. Acad. Sci. 191, 271-272. 120. Waldor, M. K., Colwell, R., and Mekalanos, J. J. (1994). The Vibrio cholerae 0139 serogroup antigen includes an 0-antigen capsule and lipopolysaccharide virulence determinants. Proc. Natl. Acad. Sci. U.S.A. 91(24), 11388-11392. 121. Waldor, M. K., and Mekalanos, J. J. (1996). Lysogenic conversion by a filamentous phage encoding cholera toxin [see comments]. Science 272(5270), 1910-1914. 122. Wang, J., Lory, S., Ramphal, R., and Jin, S. (1996). Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients. Mol. Microbiol. 22(5), 1005-1012. 123. Wang, J., Mushegian, A., Lory, S., and Jin, S. (1996). Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection. Proc. Natl. Acad. Sci. U.S.A. 93(19), 10434-10439. 124. Wang, K., Gan, L., Jeffery, E., Gayle, M., Gown, A. M., Skelly, M., Nelson, P S., Ng, W. V., Schummer, M. (1999). Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray. Gene 229{\-2), 101-108. 125. Weintraub, A., Widmalm, G., Jansson, P E., Jansson, M., Hultenby, K., and Albert, M. J. (1994). Vibrio cholerae 0139 Bengal possesses a capsular polysaccharide which may confer increased virulence. Microb. Pathogen. 16(3), 235-241. 126. Weiss, A. A., Melton, A. R., Walker, K. E., Andraos, S. C., and Meidl, J. J. (1989). Use of the promoter fusion transposon Tn5 lac to identify mutations in Bordetella pertussis y/r-regulated genes. Infect. Immun. 57(2674), 2674-2682. 127. Wong, K. K., and McClelland, M. (1994). Stress-inducible gene of Salmonella typhimurium identified by arbitrarily primed PCR of RNA. Proc. Natl. Acad. Sci. U.S.A. 91(2), 639-643. 128. Worley, M. J., Stojiljkovic, I., and Heffron, F. (1998). The identification of exported proteins with gene fusions to invasin. Mol. Microbiol. 29(6), 1471-1480. 129. Yu, J. L., Mansson, R., Flock, J. I., and Ljungh, A. (1997). Fibronectin binding by Propionibacterium acnes. FEMS Immunol. Med. Microbiol. 19(3), 247-253. 130. Zhao, H., Li, X., Johnson, D. E., Blomfield, I., and Mobley, H. L. (1997). In vivo phase variation of MR/P fimbrial gene expression in Proteus mirabilis infecting the urinary tract. Mol. Microbiol. 23(5), 1009-1019.
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CHAPTER 5
Mechanisms of Bacterial Pathogenesis in Plants: Familiar Foes in a Foreign Kingdom
JAMES R. ALFANO ALAN COLLMER
I. Introduction II. An Overview of Bacterial Plant Pathogens and Plant Diseases A. Three Types of Gram-Negative Bacterial Pathogens: Tumorigenic, Stealth Necrogenic, and Brute-Force Necrogenic B. The Fate of Nonpathogenic Bacteria in Plants C. The Overriding Importance of Protein Secretion Systems in Pathogenesis III. Tumorigenic Agrobacterium tumefaciens: Using the Type IV Secretion System to Transform the Host into a Factory for Bacterial Nutrients IV. Necrogenic, Stealth Pathogens: Parasites Strongly Dependent on the Hrp (Type III) Protein Secretion System A. The Hypersensitive Response (HR) Plant Defense Syndrome B. Gene-for-Gene (avr-R) Interactions and the Antiparasite Surveillance System of Plants C. hrp and hrc Genes and the Type III Protein Secretion System of Plant Pathogenic Bacteria D. Harpins and Pilins: Proteins Secreted in Culture in Abundance by the Hrp System E. Avr Proteins as Injected, Interchangeable Effectors of Parasitism V. Necrogenic, Brute-Force Pathogens: Soft-Rotters Dependent on Type II Secretion of Pectic Enzymes VI. Other Virulence Factors of Gram-Negative Plant Pathogens Compared with Those of Animal Pathogens A. Attachment Factors: Important in Agrobacterium but Role Unclear in Necrogenic Pathogens B. EPS: A Special Role in Wilt Diseases of Plants
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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VII. VIII. IX. X.
C. Toxins: Some Similarities, but Differently Defined D. Iron Uptake Mechanisms: Common Strategies for Overcoming a General Eukaryote Defense E. Resistance to Host Antimicrobial Peptides: Related Mechanisms Underlying Bacterial Virulence in Mouse and Potato F. Regulation of Virulence: A Potpourri of Common Components, Strategies, and Signals Host Innate Immune Systems: Common Components in Pathogen Recognition and Defense Signaling The R Gene Surveillance System: An Innate Immune System with Elaborate Recognition Specificity Pseudomonas aeruginosa: Dual-Kingdom Pathogenesis Conclusions References
202 203 204 205 206 207 209 210 211
/. Introduction With vast areas of their surface bearing perforations large enough to admit most bacteria, and without an adaptive immune system, plants would seem easy targets for hungry prokaryotes. But, surprisingly, plants are relatively resistant to bacterial attack, and most of the bacterial diseases that do afflict them are caused by strains in a handful of species. When the interactions between these successful pathogens and plants are viewed from the perspective of animal pathogenic microbiology, we find a mix of the familiar and the alien. The plant pathogens use familiar, conserved regulatory components and secretion systems to deploy key virulence proteins, and the attacked plant cells often respond defensively with familiar factors like oxidative bursts, nitric oxide synthesis, and programmed cell death. However, the virulence proteins themselves and the antiparasite surveillance systems that trigger these defenses are significantly different. Both the familiar and alien features are becoming more relevant to students of animal pathogenesis. Aspects of conserved virulence systems are sometimes more experimentally accessible in the plant pathogens, and the prevalence of horizontally transferred pathogenicity islands highlights the universal nature and potential interchangeability of these systems. Food safety concerns encourage more attention to the association of many animal pathogenic bacteria with fruits and vegetables and the possible interactions of these bacteria with plant pathogens. And the development of a model system involving a Pseudomonas aeruginosa strain pathogenic to both plants and animals suggests that novel genes involved in animal pathogenesis may be efficiendy identified using a plant system. The purpose of this chapter is to provide an introduction to the world of bacterial plant pathogenesis that is accessible to students of animal pathogens. We will delineate the conserved and
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unique mechanisms and emphasize those features that will contribute most to a broader understanding of mechanisms of bacterial pathogenesis.
//. An Overview of Bacterial Plant Pattiogens and Plant Diseases The most important prokaryotic pathogens of plants are Gram-negative bacteria in the genera Agrobacterium, Envinia, Pseudomonas, Xanthomonas, Ralstonia, and Xylella, Gram-positive bacteria in the genera Clavibacter and Streptomyces, and Mollicutes in the genus Spiroplasma and the Candidatus genus Phytoplasma. The Mollicutes and Xylella are fastidious parasites that are typically delivered by insects into the vascular system of plants. The other bacterial pathogens are generally facultative parasites that colonize the intercellular spaces between plant cells (or the nonliving xylem cells that conduct water up from the roots). Thus, most bacterial plant pathogens colonize a microniche in the host that is separated from the host cytoplasm not only by a plasma membrane, but also by a cell wall that is ca. 200 nm thick and comprised of a matrix of polysaccharide and protein thought to exclude macromolecules with a Stokes radius >4.6 nm [1].
A.
Three Types of Gram-Negative Bacterial Pathogens: Tumorigenic, Stealth Necrogenic, and Brute-Force Necrogenic
Most of what we know about pathogenic mechanisms is based on work with the Gram-negative bacteria, and in subsequent sections we will distinguish three types of pathogens within this group (Table I). Agrobacterium tumefaciens represents the first type, which is distinguished by its tumorigenicity. The pathogen transforms plant cells with bacterial DNA, producing tumors, but generally not necrosis, in the host (Fig. 1). In contrast, bacteria in the genera Erwinia, Pseudomonas, Xanthomonas, and Ralstonia are necrogenic. That is, they have a characteristic ability to elicit plant cell death. Pseudomonas syringae is representative of a subgroup of the necrogenic pathogens and of the second pathogen type we will discuss in that it is host specific and triggers host cell death only after prolonged "stealthy" parasitic multiplication. Eminia carotovora, on the other hand, is representative of another subgroup of the necrogenic pathogens and of the third pathogen type we will discuss in that it is relatively host promiscuous and kills host tissues rapidly during *'brute-force" pathogenesis. We will use the descriptive terms "stealth" and "brute-force" here instead of the formal terms
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JAMES R. ALFANO AND ALAN COLLMER
Model Gram-Negative Plant Pathogens
Pathogentype/ representative species
Model hosts
Typical disease symptoms
Major virulence factor secretion pathway"
Key virulence factors secreted
tobacco, carrot. and other dicots
crown gall tumors
Type IV
T-DNA
Pseudomonas syringae pvs.
tomato, Arabidopsis, legumes
necrotic lesions. often with chlorotic halos on leaves and fruit
Type III
Avr proteins^
Xanthomonas campestris pvs.
pepper, tomato. brassicas, rice
necrotic lesions on leaves and fruit
Type III
Avr proteins^
Erwinia amyiovora
apple and pear
fire blight
Type III
harpins, DspE (and other Avrlike proteins?)
Ralstonia solanacearum
tomato, tobacco. banana
wilt and necrosis of whole plant
Type III
Avr proteins?
potato, Saintpaulia, and many other plants with fleshy tissues
maceration "softrof' of fleshy plant organs
Type II
pectic enzymes
Tumorigenic Agrobacterium tumefaciens Necrogenic: host-specific ("stealth")
Necrogenic: host-promiscuous ("brute-force") Erwinia carotovora and E. chrysanthemi
^Note that plant cell-wall-degrading enzymes secreted by the type II pathway also contribute to the virulence of X. campestris and R. solanacearum and that the type III pathway contributes to the ability of E. chrysanthemi to initiate infections at low levels of inoculum [27, 77]. As discussed in the text, the known Avr proteins are thought to represent a larger class of effector proteins injected into host cells by the type III pathway.
biotroph and necrotroph, respectively, which denote the feeding relationship of the parasite with host cells [2]. The necrogenic pathogens produce a wide array of symptoms in plants (Fig. 1). For example, P. syringae typically causes small necrotic lesions, often surrounded by a toxin-induced yellow halo, on leaves. Ralstonia solanacearum colonizes the xylem cells in the water-supplying vascular system and causes plants to wilt. Erwinia amyiovora attacks blossoms and then vascular tissues in
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Fig. 1 Representative disease symptoms and plant responses caused by Gram-negative plant pathogens. (A) Crown gall of cherry caused by A. tumefaciens; note the massive gall exposed after removing soil. Reprinted through the courtesy of Wayne A. Sinclair. (B) Brown spot of bean caused by P. syringae pv. syringae; note the necrotic lesion (arrow) that has developed after several days of symptomless bacterial multiplication. Reprinted through the courtesy of Susan S. Hirano [269]. (C) Southern bacterial wilt of tomato caused by R. solanacearum; note the wilted plant on the right, which was grown in infested soil. Reprinted through the courtesy of H. David Thurston. (D) Fire blight of pear; note the blackened, collapsed stem and white strands of extruded bacterial ooze. Reprinted through the courtesy of Steven V. Beer. (E) Bacterial soft rot of potato caused by E. carotovora; note the sharply demarcated zone of macerated tissue in this close-up of a sectioned, infected potato tuber. (F) The hypersensitive response (HR) elicited in tobacco following infiltration with P. syringae pv. tomato at a concentration of 1 x 10^ cells/ml; note the infiltrated panel (arrow) has collapsed 24 hr after infiltration.
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the shoots of apple trees, causing aptly named "fire blight" symptoms. And E. carotovora macerates fleshy plant tissues, producing "soft rots" in organs such as potato tubers. Beyond these major differences, there is another level of more subtle variation in plant diseases. For example, a field of tobacco attacked by P. syringae may have a dynamic mixture of strains that differ only in their ability to produce a toxin: those producing the toxin will cause "wildfire" symptoms with massive yellow halos around lesions, whereas the others will produce more limited "angular leaf spot" symptoms [3]. As will be discussed below, pathogenesis by the necrogenic pathogens is a highly multifactorial process involving much apparent redundancy and subtle variation in virulence factors. Because of this and the diversity of susceptible plants, a small handful of pathogen species using highly conserved virulence factor delivery systems can cause hundreds of different diseases. Host specificity is a key part of this pathogen variability and is well defined at the pathovar and race level. That is, P. syringae is divided into over 40 pathovars based primarily on specificity for different plant species. For example, many strains off! syringae pv. syringae are virulent on bean but "avirulent" on tobacco, whereas strains of P. syringae pv. tabaci have the opposite specificity. Over 100 pathovars similarly have been defined for Xanthomonas campestris (although taxonomic studies support the redefinition of some pathovar groups as new species [4]). Races can be distinguished within many pathovars off! syringae and X. campestris based on host range among cultivars of the host species. For example, P. syringae pv. glycinea is pathogenic to soybean, but race 6 is avirulent on Harosoy and a few other cultivars of soybean, where it triggers a strong defense syndrome known as the hypersensitive response (HR). The search for the basis of this specificity and the strong defenses elicited in resistant cultivars has driven major advances in our understanding of how these bacteria attack plants.
B. The Fate of Nonpathogenic Bacteria in Plants When nonpathogenic bacteria, such as Escherichia coli and Pseudomonas fluorescens, or disarmed pathogen mutants are introduced into the intercellular spaces of a plant leaf, they neither grow nor trigger the strong defense reactions associated with avirulent pathogens [5, 6]. However, they do trigger several weak, transient, localized defense responses, including an oxidative burst, plant cell wall papilla deposition, and increased expression of the phenylpropanoid pathway (which leads to phenolics involved in wall fortification, defense signaling, and pathogen inhibition) [7-9]. These responses may also be triggered nonspecifically by two common bacterial surface features, LPS and flagella. LPS isolated from a variety of bacteria can elicit weak defenses [10-12], and plants also respond to flagellins or a 20-amino-acid domain that is conserved in the N terminus of flagellins from bacteria as diverse as Escherichia and BaciUus spp. [13]. Virulent
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necrogenic stealth pathogens, such as P. syringae, suppress these defense responses [8]. What actually accounts for the stasis of nonpathogenic bacteria in plant leaves (or what enables pathogens to grow) remains a puzzle. It is likely that pathogens, in addition to suppressing defenses, also possess active mechanisms to trigger the release of nutrients to the surface of plant cells. This has been established for tumorigenic A. tumefaciens and for necrogenic brute-force pathogens (which lyse host cells) [14, 15]. However, the relative importance of defense suppression and nutrient acquisition in the success of the necrogenic stealth pathogens is unknown. Interestingly, mixed inoculations of these bacteria and nonpathogens result in growth of the nonpathogens [16], which indicates that pathogen-induced changes in host metabolism render plants broadly susceptible to bacteria. This issue has direct implications for human health given the contamination of many horticultural products with fecal coliforms and the finding that Salmonella grows better in vegetable tissues that are diseased [17]. In this regard, it is also noteworthy that in some situations (e.g., in the commercial production of alfalfa sprouts) human pathogens like Salmonella can grow significantly on the surface of plant tissues [18].
C. The Overriding Importance of Protein Secretion Systems in Pathogenesis The primary determinants of pathogenicity in the Gram-negative plant pathogens are T-DNA for tumorigenic Agrobacterium and secreted proteins for the necrogenic pathogens in Pseudomonas, Xanthomonas, Envinia, and Ralstonia. Other factors, such as peptide toxins and extracellular polysaccharides, appear to have a secondary role and are discussed later. Typically, necrogenic pathogen mutants altered in the production of a single virulence protein are only partially reduced in virulence. Redundancy among such proteins seems to be the rule. In contrast, mutants deficient in protein secretion pathways are generally nonpathogenic. This is true for the type IV pathway in Agrobacterium, the type III pathway in stealth necrogenic pathogens, and the type II pathway in brute-force necrogenic pathogens [15, 19]. Indeed, screens for random mutants that have lost pathogenicity generally yield deficiencies in the respective secretion systems [19]. The pathogenic personality of the three types of pathogens is largely determined by the unique capabilities of the dominant secretion pathway, which are to deliver a nucleoprotein complex into plant cells (type IV), to inject effector proteins into plant cells (type III), or to efficiently secrete massive quantities of degradative enzymes to the bacterial milieu and the surface of plant cells (type II). The type IV secretion system is also described as "conjugation-like" and is used by Bordetella pertussis to secrete pertussis toxin, as discussed below. The type III secretion system is also referred to as the "Hrp system" in plant pathogens and
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the "contact-dependent" secretion system in animal pathogenic Yersinia, Salmonella, and Shigella because of its stimulation by contact with host cells [20]. And, the type II secretion system is also known as the "main terminal branch of the general secretory pathway" [21]. Aspects of the operation of these pathways that are particularly relevant to pathogenesis will be treated in subsequent sections, and an earlier chapter in this book is devoted to protein secretion systems (see Chapter 2 by Harper and Silhavy).
///. Tumorigenic Agrobacterium tumefaciens; Using the Type IV Secretion System to Transform ttie Host into a Factory for Bacteriai Nutrients Central to the pathogenesis of A. tumefaciens is the Ti (tumor inducing) plasmid and the transfer of a DNA segment (T-DNA) from the plasmid to the cytoplasm of the plant cell, where it is imported into the nucleus and integrated into the plant genome. The T-DNA encodes enzymes that synthesize plant growth hormones, thereby producing neoplastic growths, "crown galls," which develop at the crown of the plant just below the soil line. T-DNA also encodes opines and amino acid or sugar derivatives that provide a specialized food source for Ti-plasmid-bearing A. tumefaciens in the surrounding soil. This interkingdom DNA transfer system has aspects of both conjugation and protein secretion systems [22, 23]. For example, T-DNA borders are similar to the origin of transfer region oriT sites; in some plasmids, T-DNA is transferred in a single-stranded form, and components of the translocation apparatus are similar [22]. However, both T-DNA transfer and bacterial conjugation systems transfer both DNA and proteins, and, as discussed below, the VirE2 protein is probably transferred independently of T-DNA [23,24]. Thus, these conjugal transport systems can also be thought of as protein secretion systems. Further supporting this concept is the finding that these systems (encoded by virB genes in Agrobacterium and trb genes in conjugation systems) share extensive similarity with the Ptl secretion system present in the animal pathogen B. pertussis, which is dedicated to the secretion of the pertussis protein toxin [23, 25, 26]. These systems are presently being referred to as type IV protein secretion systems [27]. Agrobacterium pathogenesis and the associated type IV secretion system are summarized below and illustrated in Figure 2. A more detailed description can be found in several recent reviews that focus on different aspects of this phenomenon [15, 28, 29]. Agrobacteria in the soil are attracted to plant roots by a variety of signals, including monocyclic phenolics and monosaccharides associated with plant cell wall repair [15]. (The subsequent attachment to plant cells is discussed below.) The signals are thought to be released from plant wounds at the root-shoot
5.
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BACTERIAL PLANT PATHOGENS
Hormone prodyction
Fig. 2 Overview of the type IV secretion system of Agrobacterium and key events in the development of crown gall disease. Signals from wounded plant cells (white hexagons) induce the expression of vir genes via the VirA/VirG two-component regulatory system. The type IV secretion apparatus (encoded by the virB operon) delivers single-stranded (ss) T-DNA (squiggled line) across both the inner membrane (IM) and the outer membrane (OM) of the bacterial cell, and the cell wall (CW) and plasma membrane (PM) of the plant cell. T-DNA bound covalently to VirD2 (white trapezoids) at the 5'-phosphoryl end of the T-DNA may be transferred separately from the ssDNA-binding protein, VirE2 (hatched circles). Inside the plant cell, the T-DNA becomes coated with VirE2 and is targeted by the nuclear localization signals contained in both VirD2 and VirE2 to enter the nucleus through a nuclear pore (NP), whence it integrates into the plant genome. The transformed plant cell then produces tumor-promoting growth hormones and also opines, which support bacterial growth and induce tra genes directing conjugational spread of the Ti plasmid.
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interface and are perceived by a two-component regulatory system comprised of the VirA and VirG proteins, which in turn activates virulence (vir) gene expression [30]. The vir genes are carried on the Ti plasmid within a 35-kb region that is made up of at least 6 operons involved in different aspects of tumorigenesis. With the exception of 11 chromosomal genes, some of which are known to be involved in bacterial attachment or sugar perception, the Ti plasmid carries all of the genetic information necessary to transfer T-DNA complexes from the bacterial cell to the plant nucleus. The T-DNA carries the information for tumor formation and opine production in the plant, whereas opine utilization by the bacterium is directed by genes carried on the Ti plasmid outside of the T-DNA region [28]. Since the ability to utilize these compounds is rare in nature, this trait gives plasmid-bearing strains a competitive advantage over other neighboring soil bacteria. Furthermore, opines stimulate a conjugational transfer system encoded by the Ti plasmid, resulting in transfer of the plasmid to Agwbacteriiim strains lacking it. Hence, the prime beneficiary of this curious disease is the ca. 200-kb Ti plasmid. The T-DNA complex translocation apparatus is encoded primarily by the virB operon. A current focus of research involves localization of different VirB proteins and determining specific protein-protein interactions occurring among components of the translocation apparatus [29]. Since most of these proteins are localized to the inner and outer bacterial membranes, the VirB proteins likely assemble to form a pore through both bacterial membranes, permitting one-step secretion of T-DNA complexes from the bacterial cell. It is important to note that the type IV (Ptl) system in B. pertussis secretes pertussis toxin subunits to the periplasmic space by using an export system similar to the E. coli Sec system. The subunits are assembled into the mature toxin in the periplasm and secreted to the extracellular milieu via the type IV secretion system. Thus, it appears that in different bacteria type IV secretion systems can secrete dissimilar macromolecules and initiate the secretion of these molecules from either the bacterial cytoplasm or the periplasmic space [23]. The substrate that travels the type IV pathway in Agrobacterium, the T-DNA complex, is a single-stranded DNA molecule containing the VirD2 protein attached at the 5'-end. The T-DNA molecule itself apparently provides no secretion signals targeting it for the type IV pathway; therefore, all of the information that enables DNA to be delivered into the plant cell apparently resides in the protein component of the complex. VirD2 (along with VirDl) nicks the T-DNA and attaches to the 5' end of the released single-stranded DNA [31, 32]. The binding of VirE2 to the T-DNA is not needed in the bacterium but is essential in the plant, and VirE2 is likely to be secreted independendy of the T-DNA. That is, the T-DNA complex can be translocated into the plant cell without VirE2 as long as VirE2 is supplied by another Agrobacterium strain (producing VirE2 but not T-DNA) or by expression of VirE2 in transgenic plants [33, 34]. VirD2 and VirE2 contain functional nuclear localization signals, implicating both proteins in transporting the T-DNA to the nucleus [28, 34, 35]. The T-DNA is randomly and
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Stably inserted into the plant genome. Plant molecular biologists have exploited the Agrobacterium system by replacing T-DNA genes with desired genes and a selectable marker, and then infecting germinal tissues or somatic cells capable of regenerating whole plants to construct transgenic plants expressing foreign genes. The recent discovery of type IV secretion systems in the animal pathogens Legionella pneumophila and Helicobacter pylori indicates that type IV secretion systems may be widely important in bacterial pathogenicity [36-38]. Because of the well-studied potential of the Agrobacterium type IV system to transfer both protein and DNA into host cells, it will be interesting to learn what macromolecules are transferred by these novel systems.
IV. Necrogenic, Stealth Pattiogens: Parasites Strongly Dependent on ttie Hrp (Type III) Protein Secretion System The stealth pathogens can trigger strong plant defenses in nonhost plants, or even in host plants in the latter stages of pathogenesis (e.g., in the common leaf spot diseases where lesions typically are limited). The capacity to trigger these defenses is intimately linked with the ability of the bacteria to be pathogenic, and the defense reactions (specifically the HR) have traditionally provided more convenient assays for this basic bacterial ability [39]. Hence, we will start with a general overview of plant defenses and a description of the antiparasite surveillance system of plants before moving on to the all-important bacterial type III (Hrp) protein secretion system and its parasite-promoting protein traffic.
A. The Hypersensitive Response (HR) Plant Defense Syndrome At the heart of plant resistance against most necrogenic, stealth pathogens is the HR, a defense-associated, rapid (usually within 24 hr), programmed death of plant cells in contact with an "incompatible" pathogen (i.e., an avirulent bacterium that is virulent on some other plant) [39, 39a]. Single bacterial cells can trigger the HR death of single plant cells in a one-to-one manner [40], and if bacteria are infiltrated into leaf intercellular spaces at a level higher than 5x10^ cells/ml, then usually enough plant cells die to produce a readily assayed macroscopic tissue collapse (Fig. 1). The HR is elicited in nature only by plant pathogens (including bacteria, fungi, viruses, and nematodes), and the gene-for-gene interactions and signals underlying this phenomenon are discussed in the next section. Paradoxically, plant cell death itself does not appear necessary for effective resistance
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JAMES R. ALFANO AND ALAN COLLMER
against P. syringae [41]. However, the HR syndrome includes a panoply of antimicrobial responses, and exploring these has revealed much about plant defense systems. The ability to respond with the HR is not restricted to any specialized cell types but is a general property of plant cells that enables defense anywhere in the plant to be immediate and localized. Many of the defense responses are further localized to the plant cell wall at the site of contact with the pathogen. These are early responses occurring within the first few hours of pathogen contact and include an oxidative burst [42, 43], dityrosine crosslinking of tyrosine-rich proteins in the wall [44], increased levels of phenolic compounds associated with wall fortification [45], callose synthesis and deposition of a callose-rich papilla between the cell wall and the plasma membrane [46], and redistribution of phospholipase D in the plasma membrane to the site of contact [47]. The affected plant cell and its neighbors then produce phytoalexins (low-molecular-weight antimicrobial compounds produced in response to microbes) [48], and a variety of "pathogenesisrelated" (PR) proteins that are secreted into the cell wall and intercellular fluids or into the vacuole (from which they are released if the cell dies) [49]. If enough plant cells in the region detect pathogen, then a signal for systemic acquired resistance (SAR) is sent throughout the plant, leading to induction of a subset of PR proteins, known as SAR proteins, in distal tissues. SAR development takes several days, and it confers increased (but often not complete) resistance against a broad array of pathogens for several weeks [49]. Salicylic acid is involved in SAR signaling, although it may not be the long-distance signal [50, 51]. (Serendipitously, because of the defense-inducing effects of salicylates, the standard medical prescription for two aspirins and a phone call in the morning is not entirely off the mark with plants.) There is litde evidence that these HR-associated defense responses are tailored to the potential invader. Many of them are also triggered (but more weakly and without host cell death) by nonpathogenic bacteria, and an SAR that is triggered by a bacterium, a fungus, or a virus is equally (usually partially) effective against members of all three groups of pathogens [49].
B.
Gene-for-Gene (avr-R) Interactions and the Antiparasite Surveillance System of Plants
We now know that the HR is triggered when a bacterial avirulence (avr) gene product interacts with the product of a cognate plant resistance (R) gene in a "gene-for-gene" manner [52]. Figure 3 depicts the dynamic nature of gene-forgene interactions. It is such interactions that control race-cultivar specificity within various pathovars off! syringae andX. campestris as well as similar highly host-specific interactions of plants with many fungi, viruses, and nematodes. It is also likely that avr-R gene interactions are important in host-range determination
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at the level of pathogen pathovar and host species. For example, R syringae pv. tomato carries several avr genes that interact with R genes in soybean, thereby contributing to the avirulence of this tomato pathogen on soybean [53]. Thus, the R gene products form the antennae of a surveillance system that is constitutively arrayed against a broad array of stealth parasites. We will further discuss the R gene system below in the section on innate immunity. Interestingly, most (but not all) of the known R genes recognizing bacteria encode cytoplasmic plant proteins [54]. Thus, the recognition event that betrays unsuccessful stealth parasites appears to occur most commonly inside of plant cells.
C. hrp and hrc Genes and the Type III Protein Secretion System of Plant Pathogenic Bacteria The hrp genes were discovered in P. syringae and so named because mutants lose their ability to elicit the HR in nonhosts or to be pathogenic in hosts [6, 55]. hrp gene clusters have now been at least partially characterized in several other Gram-negative plant pathogens, including X. campestris pv. campestris (black rot of crucifer), X. campestris pv. vesicatoria (bacterial spot of pepper), R. solanacearum (Southern bacterial wilt of tomato), E. amylovora (fire blight of apple), Pantoea (Erwinia) stewartii (Stewart's wilt of com), E. chrysanthemi (soft rot), E. carotovora (soft rot), and Erwinia herbicola pv. gypsophilae (gypsophila gall) [56]. Thus, they appear to be universal among the necrogenic pathogens, although they are not essential for the virulence of the brute-force pathogens. They have not been reported for A. tumefaciens. hrp genes are always clustered and appear to be flanked by genes encoding Avr proteins, harpins, and other potential virulence factors in apparent pathogenicity islands [57-60]. In P. syringae and E. amylovora, the Hrp pathogenicity islands have integrase and tRNA sequences at one border [60a] (J. F. Kim and S. V. Beer, unpublished results), which is typical of pathogenicity islands [61, 62]. hrp genes encode the type III protein secretion pathway, and nine of the hrp genes have been renamed ''hrc'' (HR and conserved) to indicate that they encode conserved components that are also present in the type III secretion systems of Yersinia, Shigella, and Salmonella [63]. The hrc genes were given the last letter designation of their Yersinia ysc homolog. With the availability of more DNA sequences for comparison, it now appears that there are hrp homologs of yscD and yscL, and these can be conceptually included among the hrc genes [56], thus bringing the total to 11. Ten of these widely conserved genes have homologs involved in flagellum biogenesis and flagellar-specific secretion, whereas hrcC encodes a "secretin," an outer membrane protein with homologs involved in type II and filamentous phage secretion [64]. Secretins are thought to multimerize and form a channel through the outer membrane [65]. Thus, the hrc genes appear to
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Round
Wild relatives
Host
J
Cuitivar 0
"i
"2
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"3"' Resistance
Race 0
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Fig. 3 Gene-for-gene interactions of plants and stealth parasites. Panel A depicts the gene-for-gene concept portrayed in terms of a "game" played between plant breeders and the parasite. Round 1 depicts a highly inbred cuitivar of a crop species being attacked by a stealth parasite (e.g.. P. syringae, X. campestris, a rust fungus, etc.) that presumably has many adaptations for parasitism, but the only relevant features depicted are two molecules, encoded by the avrl and avr2 genes, that have the potential to be recognized by the host. In round 1, however, the plant lacks the factor(s) needed to detect race 0, and disease ensues. In round 2. recognition of the parasite is achieved by introgressing into the crop a major resistance gene {R\) derived from a wild relative. The interaction between the products of/?i and avr\ betrays the parasite, and strong. HR-associated defenses are triggered. Thus, the introduction of a single gene into the plant produces a new. resistant cuitivar. In round 3, the wide planting of cuitivar 1 has put strong selection pressure on the emergence of a new race of the parasite that lacks a functional avr\. There may be a fitness cost to this avr loss, but it is often slight or undetectable. The R\ gene in cuitivar 1 has now been ""defeated." and the crop is susceptible to this new race. In round 4, the plant breeder introgresses a second R gene, which is effective against both races 1 and 0. The resultant cuitivar 2 is widely planted, which leads to round 5. when a new race arises that lacks avn_ function. The game can go on for many more rounds because stealth parasites appear to carry a great number oi avr genes and wild relatives of the crop (typically in the geographic origin of the crop where complex host and parasite populations are in equilibrium) have a similarly large supply of/? genes.
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B.
HR Test cultivar
Avirulent donor race
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Virulent recipient race Cosmid library
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Tomato {Pto)
m
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Fig. 3 (cont'd) Panel B depicts the universal strategy established by Staskawicz and Keen and their coworkers [53, 255, 270] for cloning bacterial ovr genes. The process requires a test cultivar that is resistant to the donor race and susceptible to the recipient race. A cosmid library of donor DNA is constructed in E. coli and then conjugated into the recipient race. Random transconjugants are inoculated into the test cultivar, and those that have acquired the avr gene are identified on the basis of their avirulence phenotype. That is, they elicit the HR instead of multiplying to high levels and causing disease. Note that the donor can also be another pathovar that is virulent in some other plant species and avirulent on all cultivars of the test species. Panel C clarifies the location of Avr-R protein interactions, which was schematically depicted at the spatial interface between the host and parasite in panels A and B, but for several bacterial Avr proteins appears to occur inside the plant cell. For example, as discussed in the text, there is convincing evidence that recognition of AvrPto and AvrBs3 occurs in the plant cytoplasm and nucleus, respectively.
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have been recruited from two different protein secretion systems and to direct the translocation of proteins across the bacterial envelope. The Hrp systems of plant pathogens have been exploited to advance our understanding of some universal type III secretion system functions. For example, the Yersinia pestis LcrD and Bacillus subtilis FlhA proteins, which are homologs of HrcV, were initially thought to be regulatory proteins [66, 67]. However, the unambiguous secretion phenotype of an E. amylovora hrcV mutant provided evidence for the function of this protein family in secretion [68]. Similarly, the HrcC protein of Z. campestris pv. vesicatoria was the first type III secretin shown to induce the phage shock protein operon in E. coli, thus providing evidence that it forms a multimeric channel in the outer membrane [69]. Also, the observation that P. syringae pv. syringae hrcC mutants accumulate HrpZ (a type Ill-secreted harpin protein discussed below) in the periplasm, whereas mutants deficient in hrcU or several other hrc genes accumulated HrpZ only in the cytoplasm, provided the first direct evidence that the flagellar homologs specifically directed secretion across the inner membrane in a ^^c-independent manner [70]. The primary function of the type III protein secretion system is to deliver effector proteins into host cells (as will be discussed below). Thus, there are likely to be additional components of Hrp systems that function outside of the bacterial cell to enable effector proteins to cross the plant cell wall and plasma membrane. These components may be quite different between plant and animal pathogens because of the fundamental differences in the surfaces of the host cells. The hrp gene clusters of various plant pathogens can be divided into two groups based on protein similarities, gene arrangements, and regulatory components (discussed below) [71]. The hrp clusters oiR syringae and Erwinia spp. are in group I, those of X. campestris and R. solanacearum are in group II. Interestingly, the 11 /zrc-class genes are the only genes that are obviously conserved between the two groups. Although this may reflect the fact that extracellular components of the type III secretion system are more variable than those contained within the bacterial envelope [72], it is also possible that there are significant differences in the way these two groups of pathogens translocate proteins across the host cell wall.
D.
Harpins and Pilins: Proteins Secreted in Culture in Abundance by the Hrp System
It is important to note at the outset that there are conceptual discrepancies between the biological activities of isolated harpins and the phenotypes of harpin mutants, and this leaves the function of these proteins somewhat enigmatic. Harpins are defined as glycine-rich proteins that lack cysteine, are secreted by the Hrp system, and possess heat-stable HR elicitor activity when infiltrated into the intercellular spaces of leaves of tobacco and several other plants [19]. The first harpin was
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discovered in culture supematants of an E. coli transformant strongly expressing a functional E. amylovora hrp cluster [73]. Mutations in the encoding hrpN gene in E. amylovora essentially abolished the ability of the bacterium to elicit the HR in nonhost tobacco or to be pathogenic in susceptible immature pear fruit [73]. That is, hrpN mutants had a strong Hrp phenotype. These observations suggested that the HrpN harpin protein was the long sought bacterial elicitor of the HR. Genes encoding proteins with similar properties were subsequently found in R syringae [74, 75], R. solanacearum [76], E. chrysanthemi [77], and other plant pathogens, including the highly virulent strain CFBP1430 of £". amylovora [78]. No significant sequence similarity was found between the harpin genes in different bacterial genera, although the encoded proteins all had the properties that defined them as harpins (the PopA protein of R. solanacearum is unique in selectively triggering the HR in plants in which the bacterium is avirulent). Unexpectedly, with the exception of E. chrysanthemi, harpin gene mutations had little or no effect on the ability of the bacteria to elicit the HR. These observations suggested that harpins are not generally responsible for bacterial HR elicitation or that each bacterium has redundant harpin genes. Observations with the HrpZ harpin for P. syringae pv. syringae highlight the puzzling nature of harpins. The hrpZ gene is conserved in divergent P. syringae pathovars [75], and the isolated protein elicits an apparent programmed cell death in plants that is indistinguishable from the HR elicited by living bacteria [74]. Furthermore, a functional cluster of cloned R syringae pv. syringae hrp genes is greatly reduced in its ability to direct HR elicitation in E. coli when hrpZ is deleted [79]. However, mutation of hrmA [80, 81], which is in a variable region flanking the conserved hrp cluster in the cloned genes, has the paradoxical effect of abolishing the ability of this heterologous system to direct HR elicitation in tobacco without diminishing HrpZ synthesis or secretion [79]. Thus, isolated HrpZ is sufficient to elicit an HR in tobacco leaves, but HrpZ produced by bacteria in plants is not. hrmA has several properties of an avr gene (explained in Fig. 3 and below) that interacts with an unknown R gene in tobacco [82], and these observations suggest that the HrmA protein is the actual elicitor of the HR in this particular system and that HrpZ has a secondary or supporting activity in HR elicitation. Genes encoding HrpW, a second harpin, have recently been found near the hrp gene clusters of E. amylovora and P. syringae [59, 60]. In both bacteria, the N-terminal half of HrpW has general characteristics of harpins, whereas the C-terminal half is homologous to a newly defined class of pectate lyases found in fungal and bacterial plant pathogens. The harpin domain has elicitor activity, but neither the whole protein nor the pectate lyase domain possesses detectable pectolytic activity. As will be discussed in a later section, pectate lyases are major virulence factors in the brute-force pathogenesis of soft-rot Erwinia spp. because they efficiendy destroy plant cell wall structure. Furthermore, the a-l,4-linked galacturonide (pectic) polymers they attack are thought to control the porosity of plant cell walls [1]. Thus, it is intriguing that the R syringae pv. syringae HrpW pectate lyase domain binds specifically to calcium pectate [60]. The R syringae
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HrpZ harpin binds to plant cell walls and has biological activity only with walled cells [83]. Thus, the current evidence points to plant cell walls as the site of harpin activity, but the function of these proteins remains a puzzle. One of the most abundant proteins secreted by the P. syringae Hrp system in culture is the HrpA pilin [84, 85]. HrpA is the subunit of a pilus that is formed on bacteria in an Hrp-dependent manner and is required for pathogenicity and elicitation of the HR. The Hrp pilus is 6-8 nm in diameter, and thus similar to the pilus required by A. tumefaciens for T-DNA transfer [85, 86]. E. Avr Proteins as Injected, Interchangeable Effectors of Parasitism Avr proteins are so named because of the avirulence phenotype they can confer to pathogenic bacteria, as depicted in Figure 3A. Over 30 bacterial avr genes have been cloned on the basis of this phenotype, as described in Figure 3B [87, 88]. Unlike harpins, no bacterial Avr protein has shown HR elicitor activity when infiltrated into the intercellular spaces of test plant leaves. There are several lines of evidence that typical Avr proteins act inside plant cells following injection by the Hrp system. These lines of evidence also highlight several experimental tools that are unique to plant pathosystems: (1) Cloned hrp gene clusters functioning heterologously in nonpathogenic bacteria, like E. coli, can direct elicitation of an <3vr-/?-dependent HR if the appropriate avr gene is expressed in the bacterium and the set ofhrplhrc genes is intact [89, 90]. That is, the type III secretion system is both sufficient and necessary for bacterial delivery of Avr signals, even though the Avr proteins typically do not appear to be secreted to the bacterial milieu, and the proteins do not have elicitor activity when exogenously delivered to the surface of plant cells (which argues by default for targets in the plant cell interior). (2) Similarly, avr genes can trigger an R gene-dependent HR if expressed inside plant cells following delivery by biolistics or A. tumefaciens-mtdxdiitd transformation or transient expression systems [90-94]. (3) The R gene products expected to interact with known bacterial Avr proteins are predicted to reside in the plant cytoplasm [54]. (4) The bacterial AvrPto and the plant Pto proteins interact in the yeast 2-hybrid system, and mutations that affect this physical interaction also affect biological activity [91, 92]. (5) The AvrBs3 protein and homologs carry nuclear localization signals, AvrBs3 is localized to the plant nucleus in transient expression assays, and these targeting signals (or heterologous replacements) are required for HR elicitation [94, 95]. However, transfer of an Avr protein from a bacterium to a plant cell has not been observed directly, and it is likely that relatively small amounts of Avr proteins are actually transferred into host cells. These observations suggest that the central event in the pathogenesis of the stealth necrogenic bacteria is the Hrp-mediated delivery of Avr proteins into plant cells, as depicted in Figure 4. That is, the primary function of Avr proteins is in virulence, even though their avirulence activity is epistatic in plants carrying a cognate R gene. There are two main lines of evidence that Avr proteins (meaning injected effector proteins; an alternative term, ''Hop," for Hrp-dependent outer protein, has also been proposed [71]) are important to parasitism. First, mutations
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Hypervarlabia rtgion wttfn B¥rgenes Conserved hrp/hrc cluster Conserve<J reglop with avr and harpin genes
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PARASITISM Avr proteins collectively promote parasitism by: ^suppressing defenses? -promoting nutrient release?
HR and DEFENSE Any Avr protein can betray pattiogen if appropriate R protein is present, thereby triggering strong defense
Fig. 4 Model of stealth, necrogenic pathogenesis based on Hrp (type III secretion)-mediated delivery of Avr-like proteins into plant cells, avr genes are shown in an Hrp pathogenicity island and on a plasmid, and are likely exchanged among plant pathogens via various mobile genetic elements. The machinery translocating Avr proteins into the host cytoplasm is shown in three parts, which direct translocation across the bacterial inner membrane (IM), outer membrane (OM), plant cell wall (CW), and plasma membrane (PM), although the process is likely continuous. Whether the HrpA pilus penetrates the plant cell wall and is a conduit for protein transfer is uncertain. The HrpZ and HrpW harpins are proposed to interact with the plant cell wall, possibly to foster Avr transfer, but this also is uncertain. Inside plant cells, the Avr proteins may collectively promote parasitism via interaction with unknown susceptibility targets. However, if any one of them is recognized by the /?-gene surveillance system, the HR is triggered and a panoply of defenses are mounted.
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in the Hrp secretion genes abolish the parasitic ability of pathogens like P. syringae, suggesting that injected proteins are collectively required for this ability. It is also possible that harpins and possibly other proteins secreted by the Hrp system operate outside of plant cells to direcdy promote parasidsm. Unfortunately, no mutant has been reported in a plant pathogen that allows secretion of harpins but not injection of Avr proteins. Such a mutant would permit testing of the relative contribution to virulence of "inside" and hypothetical "outside" (of the host cell) effector proteins. yopD mutants represent such a class in Yersinia [96]. Perhaps in plant pathogens the extracellular components are more integral to secretion across the bacterial envelope than they are in Yersinia. Regardless, our working hypothesis is that injected proteins are the primary effectors of parasitism, and all other proteins are secondary or support the delivery of these Avr-like proteins. The second line of evidence for the role of Avr proteins in parasitism is that approximately one-third of the avr mutants studied to date are quantitatively reduced in their ability to grow in planta or to produce full symptoms in susceptible plants [87]. For example, AvrBs2 homologs are widespread in the X campestris group, and avrBs2 mutants are significantiy reduced in their ability to grow in host tissues [97]. AvrBs3 homologs appear to be similarly widespread, and in X. campestris pv. malvacearum (angular leaf spot of cotton) multiple AvrBs3 homologs contribute quantitatively to the water-soaked lesions associated with the disease [98]. Experiments involving heterologous expression of avr genes inside plant cells suggest that Avr proteins can be deleterious even in the absence of a known cognate R gene if expressed too strongly [90, 99]. Whether this results from excessive interaction with susceptibility targets in the host is unknown. What Avr proteins do inside host cells to promote the growth of bacteria on the surface of the cell remains a puzzle. The known Avr proteins represent a menagerie with respect to sequence and physical properties, and comparisons with sequences in the databases generally have not been informative. Only one Avr protein has an observed biochemical activity: AvrD directs the synthesis of syringolides (low-molecular-weight C-glycosides) that elicit the HR in soybean in an /?pg4-dependent manner [102, 103]. One Avr protein has a sequence suggesting a biochemical activity: AvrBs2 shows similarity with agrocinopine synthase (opine production in tumors) of A. tumefaciens, which suggests a direct role in nutrition of the pathogen [101]. And one Avr protein, AvrRxv from X. campestris pv. vesicatoria, has a homolog among the effector proteins of animal pathogens, namely, YopJ/P of Yersinia and AvrA of Salmonella (discussed further below) [100]. Finally, members of the AvrBs3 family are unique in possessing 13.5-17.5 direct 34 amino-acid repeats that control Avr specificity, and, as discussed above, these proteins appear to function inside plant nuclei [87, 104, 105]. Studies of the localization of Avr proteins in P. syringae and X. campestris have failed to detect secretion out of the bacterial cytoplasm either in culture or in
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planta [106-108]. However, a cluster of hrplhrc genes from E. chrysanthemi, functioning heterologously in E. coli, can secrete the P. syringae AvrB and AvrPto proteins in culture and trigger an avr-dependent HR in appropriate test plants [109]. One interpretation is that Avr protein secretion is dependent on contact with host cells in P. syringae and X. campestris, but this regulation is less stringent in the Hrp system of the brute-force pathogen E. chrysanthemi. This heterologous secretion system opens new avenues for research. Genes encoding effector proteins traveling the Hrp pathway can now be screened on the basis of a protein secretion phenotype in culture without the need for test plants that happen to carry cognate R genes, and targeting signals in Avr proteins and putative plant signals triggering Avr secretion can now be explored more efficiently. The discovery of this system also suggests that a stage in brute-force pathogenesis involves the injection of Avr proteins into host cells and that avr genes can be swapped among diverse plant pathogenic bacteria without loss of the ability of their products to be substrates for the Hrp system. This observation is consistent with the previously reported functional conservation of the translocation machinery for virulence proteins of Yersinia, Salmonella, and Shigella [110]. One question raised by the broad specificity of type III secretion systems is whether animal and plant pathogens can reciprocally deliver heterologous effector proteins into their respective host cells. The E. amylovora dspE and P. syringae pv. tomato avrE genes provide a case study for the functional interchangeability of Avr-like genes between different genera of plant pathogenic bacteria [111]. dspE (disease specific; also known as dspA in strain CFBP1430) is required for E. amylovora to cause disease in pear and apple hosts but not to elicit the HR in nonhost tobacco (presumably because other avr products trigger defenses in tobacco) [111,112]. avrE, on the other hand, was found through its ability to confer avirulence to P. syringae pv. glycinea by the method depicted in Figure 3B [53]. avrE mutation partially reduces the parasitic fitness of one strain of P. syringae pv. tomato [113]. DspE and AvrE are homologs sharing 30% identity, and they appear to be functionally similar: expression of dspE in P. syringae pv. glycinea causes it to become avirulent on soybean, and avrE can restore at least partial virulence to an E. amylovora dspE mutant [111]. Whether the site of DspE and AvrE action is inside of plant cells is unknown. However, DspE does travel the type III pathway, as it is secreted in an Hrp-dependent manner by E. amylovora in culture [112, 114]. The targeting signals in Avr proteins appear to be recognized by the Hrp systems of diverse bacteria, which suggests that avr genes may be readily swapped among these bacteria. Indeed, there is growing evidence that avr genes are quite mobile. Many are carried on plasmids. Most of the known P. syringae avr genes are associated with mobile genetic elements or are linked to the hrp gene cluster in loci possessing different avr genes in different strains [58, 60a, 115]. Also, avr genes may be inactivated at a high frequency by insertion elements, thus permitting the pathogen to evade recognition by the host [87, 116].
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V, Necrogenic, Brute-Force Pathogens: Soft-Rotters Dependent on Type II Secretion of Pectic Enzymes These bacteria attack a wide range of plants, particularly those with fleshy tissues. Bacterial soft rot of potato tuber is a representative disease commonly encountered when a potato that looks healthy on the outside is squeezed and found to be liquefied, or macerated, inside. The soft-rot erwinias, E. carotovora and E. chrysanthemi, also cause systemic, persistent infections and vascular necroses in vegetatively propagated ornamental plants, such as African violet (Saintpaulia) and chrysanthemum [117]. The many diseases caused by these pathogens differ in three fundamental ways from those caused by the stealth pathogens in that pathogenesis is: (1) usually dependent on bruising, wetness, hypoxia, or other environmental conditions that compromise the host; (2) not determined by gene-for-gene interactions but rather by the quantitative balance of multiple pathogen and host factors; and (3) driven by pectic enzymes secreted by the pathogen (via the type II secretion pathway). Pectic enzymes are produced by many bacteria, including some human pathogens, like Yersinia pseudotuberculosis [118], but their involvement in virulence appears unique to plant pathogens [14]. Pectic enzymes cleave a-1,4galacturonosyl linkages in plant cell wall polymers by p-elimination (pectate or pectin lyases) or by hydrolysis (polygalacturonases) [14]. The primary (unlignified) walls of plants contain a variety of structural polysaccharides, including pectic polymers, hemicelluloses, and cellulose [119]. In dicots and some monocots, the pectic polymers are particularly important because they are both structurally critical and enzymatically accessible in both the cell wall and the middle lamella (which glues the walls together to form a tissue) [120, 121]. Application of a purified E. chrysanthemi pectate lyase to potato tuber tissue causes the middle lamellae to disintegrate and the cell walls to fail to retain turgid protoplasts [122]. That is, a single pectic enzyme can produce the major symptoms—tissue maceration and cell killing—that are characteristic of the disease. E. chrysanthemi secretes an arsenal of pectic enzymes that modify (two pectin methylesterases and a pectin acetyl esterase), nibble (exo-poly-a-D-galacturonosidase and exo-polygalacturonate lyase), and chop (one pectin lyase and at least eight pectate lyase isozymes) pectic polymers [14, 123-126]. Mutagenesis has revealed that none of these enzymes is essential for pathogenesis, although the pectate lyases with very alkaline isoelectric points are particularly active in maceration [14]. Also, mutations in individual pectate lyase pel genes have different effects on virulence in different hosts, which suggests that isozyme multiplicity contributes to the wide host range of these bacteria [127]. And, of course, mutations in the type II secretion pathway through which all of these enzymes pass (except for the SOS-inducible pectin lyase) virtually abolish virulence [14].
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Pectic enzymes and their products can also elicit plant defenses. Specifically, oligogalacturonates that have between 10 and 15 galacturonosyl residues induce genes encoding the phenylpropanoid pathway and the synthesis of phytoalexins and PR proteins (but not the HR) [ 128]. These defense responses are also triggered by pectolytic culture fluids of E. carotovora, and are diminished by combinations of pectic enzymes that prevent the accumulation of elicitor-active oligomers [129-131]. E. chrysanthemi pectate lyase isozyme Pell is processed by E. chrysanthemi extracellular proteases to a smaller form that is highly active in eliciting necrosis and phytoalexin production [132]. Whether this defense elicitation is acting through enzyme products is uncertain. If tobacco seedlings are pretreated with low levels of pectic enzymes from soft-rot erwinias, they become resistant to subsequent attack by a variety of bacterial pathogens, so it appears that the soft-rotter's main weapon also has the potential to foil pathogenesis [129, 130, 133]. As will be discussed below, E. carotovora has a regulatory mechanism for apparently delaying pectic enzyme production until the bacterium has a "quorum" population that is able to overwhelm these host defenses.
W. other Virulence Factors of Gram-Negative Plant Pathiogens Compared witli Ttiose of Animal Pathiogens A. Attachment Factors: Important in Agrobacterium but Role Unclear in Necrogenic Pathogens A stage in bacterial pathogenesis of both plant and animals that would seem intuitively important is attachment to host cells, and the essential role of attachment factors in many animal pathogens has been extensively studied [134]. Factors involved in attaching to plant surfaces have also been investigated; they are considered essential for the virulence of Agrobacterium but not for the necrogenic pathogens [135]. In Agrobacterium, attachment to plant cells occurs in two steps. Initially, agrobacteria in the soil loosely bind to plant cells [136]. Afterward, cellulose filaments are produced, resulting in a tight association with the plant cell [137]. The binding to the plant cell appears to involve specific receptors located on the plant cell surface because it is possible to saturate the number of bacteria that can bind. In animals, one component of the cellular matrix is vitronectin, and there are several examples of animal pathogens attaching to vitronectin or other components of the cellular matrix [138, 139]. Vitronectin-like proteins have been isolated from a number of plant species, and these proteins may act as receptors iov Agrobacterium [140]. Attachment of Agrobacterium to carrot cells is reduced in the presence of anti-vitronectin antibodies [141]. Several Agrobacterium mutants have been isolated that are reduced in their ability to attach to plant cells and are defective in their ability to synthesize or secrete cellulose, p-l,2-glucans.
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or acidic polysaccharides that are associated with the bacterial envelope or surface [142-145]. Another class of attachment-defective mutants involves proteins with homology to ABC transport systems [146]. Carrot cell binding is restored to these mutants by a conditioned medium in which wild-type bacteria and plant cells had been grown, suggesting that the defect is in secretion of a bacterial factor promoting attachment or in uptake of a plant signal [146]. Many plant pathogenic bacteria produce pili and fimbriae, but these appendages generally are not required for pathogenicity [135], and instead they appear to aid the attachment of bacterial cells to microniches on the epidermis during epiphytic growth. Pili are also produced by the type IV and type III secretion systems of plant pathogenic bacteria, and they have been shown to be essential for the virulence of A. tumefaciens and P. syringae, respectively, as discussed above [85, 86]. Since both of these secretion systems are capable of transferring macromolecules through the plant cell wall and plasma membrane, it is possible that these dedicated pili have a primary role as conduits rather than as attachment factors. B. EPS: A Special Role in Wilt Diseases of Plants Many bacterial species, including pathogens of animals and plants, can produce copious amounts of exopolysaccharides (EPSs) as secreted capsular material or as a loose slime. In animal pathogens, capsular material is thought to help the pathogen evade the inflammatory response of the host [147], and EPSs may help the pathogen colonize certain niches in the animal. For example, alginate made by P. aeruginosa has been implicated in the ability of the bacterium to persist in the lungs of patients suffering from cystic fibrosis [148]. In plant pathogens, EPS acts as a virulence factor in those pathogens that produce wilt and extensive water-soaking symptoms, although it is generally not absolutely required for pathogenesis [149]. The regulation and biosynthesis of EPS has been most extensively studied in the plant pathogens R. solanacearum [150, 151], £". amylovora [152, 153], P. stewartii [154], and X. campestris [155, 156]. EPS is thought to act in pathogenesis by restricting water flow in the xylem (causing the plant to wilt), producing a hydrophilic gel in leaf intercellular spaces (promoting water-soaking), or by altering the accessibility of antimicrobial compounds and defense-related signals [149]. Since EPS is also made by nonpathogens, it is important to note that these compounds may have a role in protection against general environmental stresses (e.g., desiccation) away from the host. C. Toxins: Some Similarities, but Differently Defined Unlike bacterial exotoxins of animal pathogens, which often appear central to pathogenicity, toxins of plant pathogenic bacteria (phytotoxins) generally function as supporting virulence factors [134, 157]. Most of the known phytotoxins from Gram-negative plant pathogens have been discovered in different P syringae
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pathovars. These are secondary metabolites that show no host specificity, typically do not contribute to bacterial multiplication, and are highly diffusible in plant tissue [157]. Bacterial phytotoxins can be broadly classified into two groups based on the type of symptoms they cause: necrosis-inducing toxins that kill plant cells and produce necrotic lesions, and chlorosis-inducing toxins, which cause yellowing of plant tissue, usually due to the destruction of chloroplasts. The actual role of phytotoxins in pathogenesis is somewhat unclear. Toxins are produced in nonpathogenic strains [158], and R syringae pathogenic strains that have spontaneously deleted tabtoxin biosynthetic genes retain pathogenicity [3]. Furthermore, many toxins have antimicrobial activity, which suggests that their primary targets may be other plant-associated microorganisms [157]. However, several phytotoxins appear to affect signaling processes within plant cells, which suggests that their targets are in the host. For example, the related necrosis-inducing toxins syringomycin and syringopeptin have pore-forming activity in membrane bilayers and cause rapid ion fluxes across membranes [159-161]. It is postulated that by permitting Ca^"*^ influx into plant cells these toxins activate various cellular signahng pathways [161], and, indeed, syringomycin induces kinase-mediated phosphorylation of plant proteins [162]. In their pore-forming activity, the syringomycin-like phytotoxins are functionally similar to a large group of exotoxins from animal pathogens that range from iturins, which are small peptides, to the RTX toxins, which are larger proteins [163-166]. The syringomycin-like phytotoxins appear to cause similar host cell responses in plant cells [160, 161]. Moreover, syringomycin, like many RTX toxins, is secreted via a type I secretion pathway [167]. Whereas all phytotoxins are secondary metabolites (mostly small peptides), many animal pathogen exotoxins are proteins, which may be grouped as A-B enzyme toxins, pore-forming toxins that disrupt membranes, proteolytic toxins that block the vertebrate central nervous system, or a number of other toxins that do not fit in any of these groups [134]. Some of the proteins that are delivered to animal cells via type III systems are considered A-B exotoxins [168, 169], and it will be interesting to see if a more complete inventory of proteins traveling the type III pathway in plant pathogens includes any homologs of these proteins.
D.
Iron Uptake Mechanisms: Common Strategies for Overcoming a General Eukaryote Defense
Iron presents two potential threats to pathogens of both plants and animals. On the one hand, the levels of iron not sequestered by the host are usually too low to support the essential iron needs of the invading bacterium. On the other hand, free iron can catalyze the production of highly reactive hydroxide radicals that may enhance localized, oxidative defenses mounted by the host. Microbial iron uptake systems may ameliorate both problems, and siderophore systems are prevalent in
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pathogens of both plants and animals [ 170, 171]. The role of siderophores in plant pathogenesis has been extensively explored in E. chrysanthemi 3937, particularly in the context of the ability of the bacterium to systemically invade African violet plants by moving through the vascular system [170]. E. chrysanthemi produces two siderophores, the catechol-type chrysobactin and achromobactin (which lacks both catechol and hydroxamate). The former is expressed in the plant and is important for systemic invasion but not localized maceration; the latter is expressed at high basal levels in the presence of mineral iron [172-174]. Plants do not produce transferrin-like proteins, but polyphenols possessing multiple 6>-dihydroxyphenyl groups may contribute to the nutritionally restrictive levels of free iron observed in plant fluids [172, 175]. Interestingly, chrysobactin is produced in such high levels in infected plants that it appears to reduce the amount of iron available to host cells [172].
E.
Resistance to Host Antimicrobial Peptides: Related Mechanisms Underlying Bacterial Virulence in Mouse and Potato
Antimicrobial peptides are effectors of innate immunity in vertebrates and insects [176]. Plants also produce several classes of antimicrobial peptides, including thionins, defensins, and lipid transfer proteins, all of which contain 4, 6, or 8 cysteines [177]. Plant antimicrobial peptides are constitutively abundant in many storage organs and are induced in other tissues upon pathogen attack. At the subcellular level, they accumulate in either the vacuole or the intercellular spaces between plant cells [177]. Transgenic overexpression in tobacco of barley genes encoding either an a-thionin or a lipid transfer protein resulted in plants with increased resistance to P. synngae pv. tabaci (wild-fire of tobacco), which suggests that these two antimicrobial peptides may have a role in plant defense against bacteria [178, 179]. This notion is further supported by the observation that R. solanaceamm mutants with altered LPS structures lose resistance to these two antimicrobial peptides and virulence to the same degree [180]. There is growing evidence that mechanisms enabling pathogens to resist antimicrobial peptides are widely important for bacterial virulence [181]. For example, in S. typhimurium, the sapA-F (sensitivity to antimicrobial peptides) operon confers resistance to the antimicrobial peptides protamine and melittin, and sap mutants lose this resistance and virulence in a mouse model [182, 183]. Because of the similarities of SapA to solute-binding proteins and other Sap proteins to ABC transporters, the Sap system was proposed to act by transporting antimicrobial peptides away from membrane targets and into the bacterial cell, possibly for degradation [183]. E. chrysanthemi also carries the sapA-F operon, which confers resistance to snakin-1 and a-thionin, two antimicrobial peptides found in potato, and a sap mutant has reduced virulence in potato tuber tissue [184]. The sap operons in the two bacteria are colinear in arrangement, and their
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products share between 69 and 78% sequence identity [184]. Thus, although adapted for the antimicrobial peptides of their respective hosts, homologous Sap systems are required for the virulence of bacteria attacking different kingdoms.
F.
Regulation of Virulence: A Potpourri of Common Components, Strategies, and Signals
The regulation of virulence factors in plant pathogens is mediated by proteins that belong to many of the same families that regulate virulence and other bacterial functions in animal pathogens [185, 186]. For example, HrpL is a member of the ECF (extracytoplasmic factor) family of sigma factors and activates the transcription of hrp genes in the group I Hrp systems of P. syringae and E. amylovora [187-189]. The expression of hrpL is dependent on two regulatory proteins, HrpR and HrpS, that have similarity with known a^'^-dependent promoter-enhancerbinding proteins [187, 190, 191]. In addition, the Hrp system of R syringae is negatively regulated by HrpV, which has no known homologs, and appears to act upstream of HrpR/S in the regulatory hierarchy [192]. In contrast to the group I Hrp systems, a homolog of the AraC family of positive activators is required to activate the group II Hrp systems of X. campestris and R. solanaceamm [193195]. Two upstream activators have been identified for these systems: HrpG in X. campestris is homologous to two-component response regulators [196], and PrhA shows homology to several TonB-dependent outer membrane siderophore receptors (although it is not regulated by iron levels), and is further noteworthy in being specifically required for induction of hrp genes in the presence of plant cells [197]. Virulence in plant pathogens, as in animal pathogens, is controlled by complex regulatory networks. For example, the battery of pectic enzymes produced by E. chrysanthemi is controlled by a regulatory system that involves at least three regulatory proteins (KdgR, PecS, and PecT) responding to a plethora of environmental cues (reviewed in [123]). In E. carotovora, both pectic enzyme and hrp gene transcripts are regulated at the level of mRNA stability by the RsmA/B system [198]. In R. solanacearum, the production of EPS and several extracellular virulence proteins is controlled by a hierarchy of regulatory proteins that involves at least three two-component regulatory systems, an LysR-type transcriptional regulator, and a volatile factor that may function analogously to an autoinducer [199-201]. The expression of virulence in X. campestris pv. campestris is also dependent on a diffusible signal molecule, but this factor is distinct from the R. solanacearum volatile factor and the E. carotovora acyl-homoserine lactone autoinducer, which is discussed below [202]. Plant and animal pathogens also share several global regulators of virulence. For example, in P. syringae, the LemA/GacA two-component regulatory system controls the production of syringomycin, tabtoxin, protease activity, and, in
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certain pathovars, lesion formation. The GacA protein is also required for P. aeruginosa virulence in mouse [203-205]. The sigma factor RpoS, which is involved in regulation of many genes during stationary phase, also is required for expression of virulence genes in both Salmonella spp. and E. carotovora [206208]. Cyclic-AMP receptor protein (CRP) homologs are required for the virulence of X. campestris, E. chrysanthemi, and P. aeruginosa [209-211]. And, finally, many different bacteria monitor their population density with a quorum-sensing regulation (LuxR-LuxI type) system based on production of a diffusible acyl-homoserine lactone autoinducer [212, 213]. Long thought to be limited to Photobacterium (Vibrio) fischeri, this system was subsequendy found in the pathogens E. carotovora and P aeruginosa, and then in a variety of other bacteria [214-217]. It is postulated that the production of pectic enzymes by E. carotovora occurs only when the bacterial population in the infection site is high enough to permit the destructive effects of the enzymes to overpower their side-effects of eliciting host defenses [216, 217]. Although regulatory systems based on diffusible signals appear important to the virulence of many plant pathogens, the signals and circuitry vary. For example, the LuxR homolog (EsaR) in P stewartii is unusual in acting as a negative regulator that is inactivated by the autoinducer [218], and, although E. chrysanthemi has homologs of Luxl and LuxR, neither is required for pectic enzyme production or virulence [219]. Environmental signals such as temperature, iron availability, nitrogen, and pH are also important signals for pathogenicity in both plant and animal pathogens [220-223].
VIL Host Innate Immune Systems: Common Components in Pattiogen Recognition and Defense Signaling The receptor-based plant surveillance system, consisting in part of resistance (R) gene products, represents an ancient defense mechanism that appears related to innate immune systems in vertebrates and insects [54, 224, 225]. For example, the tobacco A^ gene, an R gene conferring resistance to tobacco mosaic virus, encodes a protein with a domain that is similar to the Drosophila Toll protein and the mammalian interleukin-1 (IL-1) receptor [226, 227]. The TIR (Joll-IL-l region) domain is likely involved in downstream defense-signaling events. In tobacco plants resistant to TM V, the N protein recognizes a TM V Avr determinant, the TMV replicase protein [228], thereby triggering the HR [225]. In adult Drosophila, the pathway initiated by Toll results in production of the antifungal peptide drosomycin [229]. In mammals, the IL-1 receptor is activated by IL-1 and initiates a signaling cascade that results in the Rel-related transcriptional activator NF-KB being transported to the nucleus, where it activates transcription of many genes encoding acute phase and proinflammatory proteins [230]. Several additional human receptors with Toll homology have been identified [231], and one
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has been shown to activate production of antimicrobial peptides and cytokines, thereby also implicating it in stimulation of acquired immunity [232]. Among the limited set of R proteins characterized so far, those containing the TIR domain confer resistance against fungi (and TMV) but not bacteria [54]. Also, there are at least three genetically distinguishable signaling pathways downstream of different R genes [233-234]. However, there does not appear to be a set of R proteins or defense responses dedicated to any class of pathogens. The cloned R genes that recognize bacterial pathogens fall into three classes. The tomato Pto gene encodes a serine/threonine kinase [235]. The RPS2 and RPMl genes from Arabidopsis encode intracellular proteins that, like the N protein (and many R proteins), possess an internal nucleotide-binding site (NBS) and a leucine-rich repeat (LRR) domain; but, unlike the N protein, they have a leucine zipper instead of a TIR domain near the N terminus [236, 237]. The rice Xa-21 gene encodes a putative membrane-spanning protein with an extracellular LRR domain and a cytoplasmic kinase domain [238]. Interacting avr genes have been identified for all of these R genes except Xa-21. There are several additional indications of the similarity of the innate immune systems of plants, insects, and mammals. (1) The tomato Pto kinase and the rice Xa21 kinase have similarity to kinases involved in innate immunity pathways in insects {Drosophila pelle kinase) and mammals (human IRAK kinase) [235, 238-240]. (2) The NPRl/NIMl protein, which acts downstream of at least some R genes in Arabidopsis, contains ankyrin repeats and other regions similar to those in the IKB and Cactus proteins from mammals and Drosophila, respectively [241, 242]. These proteins inhibit the Rel-related transcription factors NF-KB and Dorsal present in their respective systems. The similarities between NPRl/NIMl, IKB, and Cactus suggest that NPRl/NIMl may be acting in a similar way in plants. (3) In plants, reactive oxygen species function in defense signaling, wall fortification, and possibly in pathogen killing, and they are generated by an NADPH oxidase similar to that in neutrophils [42]: both NADPH oxidases are inhibited by diphenylene iodonium [243], antibodies to the neutrophil p47 and p67 proteins crossreact with plant proteins of the same size [244], and plant homologs of the gp91P*^°^ subunit gene have been identified [245, 246]. (4) Similarly, as in animals [247], nitric oxide is involved in redox-mediated defense gene induction, with cyclic GMP and cyclic ADP-ribose apparently functioning as second messengers [248, 249].
VIIL The R Gene Surveillance System: An Innate Immune System with Elaborate Recognition Specificity The ability of the plant R gene surveillance system to specifically recognize a vast array of subspecific groups (e.g., pathovars and races) among potential pathogens distinguishes it from the innate immune system of animals. That is, in addition to the system that recognizes general features of bacteria and their activities in the
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cell wall (e.g., LPS and pectic fragments), plants have evolved a more elaborate germline-encoded system that recognizes specific virulence proteins of pathogens. This second recognition system bears resemblance to the adaptive immune system in vertebrates in its specificity and strong responsiveness. However, there appear to be three fundamental differences in the development of pathogen recognition by the R gene surveillance and adaptive immune systems: (1) In plants, the reservoir for recognitional capacity resides in the germlines of different individuals in complex populations, not in somatically varying cells (lymphocytes) within a given individual. (2) In vertebrates, recognition of a pathogen by the adaptive immune system appears to be dependent on costimulatory instruction by the innate immune system [224, 250], whereas the selective advantage of defensive recognition of individual parasite proteins appears sufficient to drive the evolution of R genes. (3) In vertebrates, recognitional diversity is generated by somatic gene rearrangement, whereas in plants it is generated typically by meiotic recombination involving R genes in complex loci. Although some R genes are found in simple loci or in allelic series, most exist in complex loci containing tandemly repeated members of R gene families, with plant genomes containing many of these loci [54]. This arrangement of R genes fosters duplications, deletions, and sequence exchanges, with polymorphism in intergenic regions suppressing sequence homogenization among members [251, 252]. Furthermore, comparisons of the sequences of tomato Cf genes and homologs, which are R genes conferring resistance to the fungus Cladosporium fulvum, reveal that solvent-exposed residues in LRR domains are hypervariable, suggesting a role in recognition specificity [251 ]. The finding of more nonsynonymous than synonymous nucleotide substitutions in the LRR domains of rice Xa21 homologs further supports the concept that these domains are subject to adaptive selection for pathogen recognition [253]. The possibility that animals also have an R gene surveillance system, such that naive individuals can specifically recognize a parasite protein and trigger defenses, has been raised by Galan in a provocative commentary addressing the homology between the X. campestris pv. vesicatoria AvrRxv, Yersinia spp. YopJ/P, and Salmonella enterica AvrA proteins [254]. The R gene that corresponds to avrRxv has not yet been isolated; however, AvrRxv induces an HR on resistant plants that is likely due to an R gene interaction, as indicated by cultivar-differential effects [255]. YopJ/P induces apoptosis in macrophage cell lines [256, 257], which raises the possibility that this is mediated by an R protein analog in mammalian cells. There are additional similarities between these proteins: (1) they are dispensable for virulence; (2) the sporadic distribution oiavrA resembles that of many avr genes in plant pathogens [100]; and (3) AvrA, YopJ/P, and probably AvrRxv are secreted via the type III system [100, 256, 257]. IpaB, which is secreted via the type III system of Shigella flexneri, also induces apoptosis in macrophages [258, 259]. Unlike the HR in plants, which is clearly a defense response, the function of programmed cell death of macrophages in response to these proteins is uncertain. In one scenario, apoptotic macrophages release IL-1,
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which induces a proinflammatory response as part of a defense. Alternatively, the removal of bactericidal cells, which results in inflammation-induced tissue damage, could promote further bacterial invasion [258]. Comparative investigations of the innate immune systems of plants and animals will help resolve these questions.
IX. Pseudomonas aeruginosa; Dual-Kingdom Pathogenesis The numerous similarities between plant and animal pathogens described in the preceding sections invite the question of whether some bacteria can attack both plants and animals. The potential hazard of several plant-associated bacteria to immunodeficient or otherwise compromised human patients has long been known [260]. Burkholderia {Pseudomonas) cepacia and P. aeruginosa present two important examples of dual-kingdom pathogens, and each yields different lessons. B. cepacia causes sour skin of onion, a rot of the outer, fleshy scales of the bulb [261], and it is also a prevalent pathogen of the human respiratory tract, particularly in patients with cystic fibrosis [148]. Bacteria classified as B. cepacia are remarkably diverse in their genome size and metabolic abilities, and some strains proliferate in the rhizosphere around plant roots and suppress fungal diseases. These strains are being exploited as biological control antagonists (see, e.g., [262]). However, because of the apparently extreme plasticity of the B. cepacia genome and the uncertain taxonomic distinction between "environmental" and "clinical" isolates, the wisdom of such agricultural use of ^. cepacia is now being debated [263, 264]. In contrast, P. aeruginosa UCBPP-PA14 provides an example of a single bacterial strain that has been established to cause disease in both plant (Arabidopsis and lettuce) and animal (mouse) models, and it is being used to find novel virulence factors [205, 265]. P. aeruginosa has been described as a "quasi-pathogen" of plants [266], and as the "quintessential opportunist" pathogen of humans [147]. Nevertheless, strain UCBPP-PA14 causes soft-rotting in leaves of the model plant Arabidopsis in an ecotype-specific manner (a characteristic of many true pathogens), and it causes a high level of mortality in a mouse full-thickness thermal burn assay [205]. Furthermore, experiments with strain UCBPP-PA14 are yielding important insights into P aeruginosa virulence mechanisms in both hosts. Directed mutations in the toxA, plcS, and gacA genes, which were previously implicated in bacterial virulence in one of the hosts, revealed these factors to be essential for virulence in both hosts [205]. Subsequently, a collection of mutants (ca. 25% genome saturation) carrying random TnphoA insertions was screened in the plant (lettuce) assay system for loss of virulence, thereby yielding
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dsbA and seven novel genes that are also needed for virulence in the animal model [265]. Thus, both individual effectors (exotoxin A and phospholipase C) and global regulatory or putative deployment factors (GacA two-component responseregulator and DsbA disulfide bond oxidoreductase) are important in both hosts, and the available (but still incomplete) genetic evidence suggests that the mechanisms for virulence in the two host kingdoms are congruent. Finally, the basic validity of this system is supported by the finding that homologs of both GacA and DsbA are required for the virulence of highly virulent, dedicated pathogens of each host kingdom [265].
X. Conclusions Gram-negative bacteria have evolved at least three distinct strategies for parasitizing plants. The Agrobacterium strategy of transforming the host has no known counterpart among animal pathogenic bacteria. In contrast, similarities are extensive between plant and animal "stealth and interdiction" [267] pathogens like P. syringae and K enterocolitica, which inject major virulence proteins into their hosts via the type III pathway, and they are similarly extensive for brute-force pathogens like E. carotovora and P. aeruginosa, which secrete tissue-degrading enzymes via the type II pathway on achieving quorum populations for attacking their hosts. Similarly, plant defense and innate immunity systems in animals have a growing list of homologous components. These similarities are not surprising. For example, a system that injects virulence proteins into host cells and is encoded by horizontally transferred pathogenicity islands would be expected to support the evolution, "by quantum leaps" [61], of virulence in a wide range of bacterium-eukaryote pathosystems. Similarly, evolving multicellular eukaryotes would be expected to retain at least some ancient signaling components and defense factors like oxidative bursts and antimicrobial peptides. However, there are three unique features of plant pathosystems and the experimental approaches they afford that warrant highlighting. The first unique feature is the plant cell wall, which provides the common meeting ground for all plant-bacterium interactions. Wall fortification is the function of several of the most rapidly deployed plant defenses, and wall destruction is the main virulence strategy of the brute-force pathogens. The second unique feature is the /?-gene surveillance system, whose evolutionary elaboration appears to have driven an arms race involving avr-gene proliferation and exchange among the stealth pathogens. The third unique feature is the experimental advantages afforded by Arabidopsis and other plant genetic systems, the Agrobacterium transformation system, and a suite of tools that permits rapid and rigorous evaluation of disease factors in plant-bacterium pathosystems. For example, tens of thousands of Arabidopsis plants can easily be screened for mutants with altered susceptibility, and Agrobacterium-mtdisLied transient-expression and transformation systems allow facile screening for the effects of pathogen proteins inside plant cells [91,
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92, 99, 268]. Similarly, plant pathogen-derived type III secretion cassettes, functioning in nonpathogens like E. coli, facilitate the study of naturally delivered, individual effector proteins [89, 90, 109]. If some of the many plant pathogen effector proteins are indeed suppressors of plant defense signaling pathways, they may provide novel tools for exploring innate immunity in general. The availability of well-developed experimental systems on both sides of the interaction makes plant-bacterium pathosystems ideal for exploring common defenses of multicellular eukaryotes and the virulence mechanisms bacteria use to overcome them.
Acknowledgments We thank Kent E. Loeffler for photography and Terrence P. Delaney and Jihyun F. Kim for critical review of portions of this manuscript before submission in December 1998. Work in the authors' laboratories was supported by grants MCB-9631530 (AC) from the National Science Foundation and 97-35303-4488 (AC) and 98-35303-6464 (JRA) from the National Research Initiative Competitive Grants Program, U.S. Department of Agriculture.
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259. Chen, Y., Smith, M. R., Thirumalai, K., and ZychUnsky, A. (1996). A bacterial invasin induces macrophage apoptosis by binding directly to ICE. EMBO J. 15, 3853-3860. 260. Starr, M. R (1981). Prokaryotes as plant pathogens. In "The Prokaryotes" (M. P Starr, H. Stolp, H. G. Truper, A. Balows, and H. G. Schlegel, eds.), pp. 123-134. Springer-Verlag, New York. 261. Burkholder, W. H. (1950). Sour skin, a bacterial rot of onion bulbs. Phytopathology 40, 115-117. 262. Mao, W., Lumsden, R. D., Lewis, J. A., and Hebbar, P K. (1998). Seed treatment using pre-infiltration and biocontrol agents to reduce damping-off of corn caused by species of Pythium and Fusarium. Plant Dis. 82, 294-299. 263. Govan, J. R. W., and Vandamme, P. (1998). Agricultural and medical microbiology: A time for bridging the gaps. Microbiology 144, 2373-2375. 264. Holmes, A., Govan, J., and Goldstein, R. (1998). Agricultural use of Burkholderia (Pseudomonas) cepacia: A threat to human health? Emerg. Infect. Dis. 4, 221-227. 265. Rahme, L. G., Tan, M. W., Le, L., Wong, S. M., Tompkins, R. G., Calderwood, S. B., and Ausubel, F. M. (1997). Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors. Proc. Natl. Acad. Sci. U.S.A. 94, 13245-13250. 266. Cho, J. J., Schroth, M. N., Kominos, S. D., and Green, S. K. (1975). Ornamental plants as carriers of Pseudomonas aeruginosa. Phytopathology 65, 425^31. 267. Bliska, J. B., Galan, J. E., and Falkow, S. (1993). Signal transduction in the mammalian cell during bacterial attachment and entry. Cell 73, 903-920. 268. Glazebrook, J., Rogers, E. E., and Ausubel, F. M. (1996). Isolation of Arabidopsis mutants with enhanced disease susceptibility by direct screening. Genetics 143, 973-982. 269. Hirano, S. S., Rouse, D. I., Clayton, M. K., and Upper, C. D. (1995). Pseudomonas syringae pv. syringae and bacterial brown spot of snap bean: A study of epiphytic phytopathogenic bacteria and associated disease. Plant Dis. 79, 1085-1093. 270. Staskawicz, B. J., Dahlbeck, D., and Keen, N. T (1984). Cloned avirulence gene oi Pseudomonas syringae pv. glycinea determines race specific incompatibility on Glycine max (L.) Merr. Proc. Natl. Acad. Sci. U.S.A. 81. 6024-6028.
CHAPTER 6
Yersinia AoiFE P. BOYD GUY R . CORNELIS
I. Introduction A. Course of Yersinia Infection B. Avoidance of Phagocytosis C. Control of Cytokine Production and Release D. Virulence Factors II. The Adhesive Factors A. Invasin B. Ail C. Myf/pH 6 Antigen D. YadA E. Conclusion III. Iron Acquisition IV. Pathogenicity Islands V. Yst Enterotoxin VI. The Yersinia Virulence Plasmid A. Role of the Virulence Plasmid B. Ysc Secretion System C. Yops D. Regulation of Transcription of the Virulon Genes E. Syc Cytosolic Chaperones F Translocation of Effector Yops across the Eukaryotic Cell Plasma Membrane G. The Yop Effectors and Their Targets VII. Conclusion References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of" reproduction in any form reserved. ISBN 0-12-304220-8
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/. INTRODUCTION A.
Course of Yersinia Infection
Yersinia spp. are Gram-negative bacilli belonging to the Enterobacteriaceae family. Based on differentiating biochemical characteristics, 11 species have been identified and further divided into biogroups. Serotypes have been established based on the reaction of antibodies to different lipopolysaccharide structures. The serotypes are linked to geographical distribution, severity of human disease, and animal reservoir. Of the 11 species in the Yersinia genus, only three are human pathogens: Y. pestis, Y. pseudotuberculosis, and K enterocolitica. Y pestis is the agent of bubonic plague, which is best known as the black death of the Middle Ages that killed millions of people. More recently, there has been an increase in cases of Y pestis seen in the United States, and isolated outbreaks have also occurred in India [1]. K enterocolitica, which is the most prevalent in humans, and Y pseudotuberculosis cause a broad range of gastrointestinal syndromes, ranging from acute gastroenteritis to mesenteric lymphadenitis and, on rare occasions, they can even provoke systemic infections such as septicaemia and meningitis [2]. Humans generally become infected with Y pestis via a bite of a flea that has acquired K pestis from a blood meal on an infected animal, such as a rat (Fig. 1). In the flea, infection is limited to the alimentary tract, which becomes so blocked with bacteria that Y. pestis is regurgitated into the next animal the flea feeds on. In humans and other animals, K pestis spreads from the site of the flea bite into the regional lymph nodes and the characteristic buboes (swollen lymph nodes) of bubonic plague form. The bacteria may then spread into the bloodstream to cause septicaemia, and also to the spleen, liver, and lungs. Pneumonic plague is transmitted by respiratory droplets from the lungs of an infected individual and causes an overwhelming pneumonia. K pestis is an obligate parasite, and this is in contrast to K enterocolitica and K pseudotuberculosis, which can survive outside animal hosts and are foodbome pathogens. Y enterocolitica infection occurs mainly by ingestion of contaminated food or water, and pork meat is a frequent source of this pathogen [3]. Because of its ability to grow at low temperatures, K enterocolitica can multiply in refrigerated stored blood bags, which can lead to severe posttransfusional septic shock [4]. Once ingested, K pseudotuberculosis and K enterocolitica attach to the intestinal mucus and intestinal cells, and Y. enterocolitica produces an enterotoxin that induces diarrhoea (Fig. 1). Y enterocolitica and Y pseudotuberculosis are able to cross the gut epithelium and proliferate locally in the underlying tissue. The bacteria selectively enter via the M (microfold) cells, and reach the intestinal lymphoid aggregates known as Peyer's patches [5-7]. M cells are specialized epithelial cells that carry out transcytosis; they take up small quantities of the intestinal contents and release them at their basolateral side into the Peyer's patches. Entry into the Peyer's patches leads to an enormous recruitment of phagocytic polymorphonuclear leukocytes (PMNs), formation of microabcesses comprising extracellular Yersinia, appearance of dead apoptotic cells and, even-
6.
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K pestis 1
Respiratory droplets
Y. pestis
Y, enterocolitica Y. pseudotuberculosis
Fig. 1 Infection routes of the three pathogenic Yersinia species. K pestis enters the body from a flea bite and moves to the lymphatic nodes, where it forms buboes. From the lymphatic system it can reach the bloodstream and eventually the lungs, to cause pneumonic plague, which can be transmitted by respiratory droplets. Y. enterocolitica and Y. pseudotuberculosis are food-borne pathogens that enter the body via the intestinal tract and pass through the M cells of the Peyer's patches to reach the lymph system. Y. pseudotuberculosis can also pass into the bloodstream.
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tually, complete destruction of the cytoarchitecture of the Peyer's patches [5, 7]. Monocytes infiltrate the Peyer's patches and mature into inflammatory macrophages to produce cytokines such as interleukin-12 (IL-12), gamma interferon (IFNy), and tumor necrosis factor alpha (TNF-a), which aid in development of the immune response [8-10]. Professional phagocytes severely restrict the rate at which Yersinia multiplies in the host tissues, thereby allowing the host to develop a specific protective immunity. However, Yersinia can fight back by impairing phagocytosis, inhibiting phagocytic killing, triggering apoptosis, and suppressing the normal release of TNF-a and other cytokines. Once established in the Peyer's patches, the bacteria can disseminate to the mesenteric lymph nodes and eventually to the liver and spleen [7, 11].
B. Avoidance of Phagocytosis The alterations in tissue morphology that occur after Yersinia infection result from both the virulence properties of the bacteria and the defense features of the immune system of the host and, hence, reflect the fragile equilibrium between these two factors. In spite of their different infection routes, all three Yersinia spp. share a common tropism for lymphoid tissues and a common capacity to resist the nonspecific immune response. All the histological and microscopic studies confirm the affinity of Yersinia for lymphoid tissue and the reticuloendothelial system, and have also provided evidence for a predominantly extracellular multiplication of Yersinia in the lymphatic organs [5, 12]. Once the bacteria have passed through the M cells to the Peyer's patches, it seems that the majority remain extracellular. However, one cannot exclude an intracellular state of Yersinia at later stages of the infection, especially when the bacteria transmigrate from the Peyer's patches to deeper tissues. In accordance with these in vivo observations. Yersinia spp. are resistant to phagocytosis by macrophages [13, 14] and PMNs [15-17] in vitro. Thus, Yersinia spp. are predominantly extracellular pathogens, and their survival strategy strongly relies on their ability to escape phagocytosis.
C. Control of Cytokine Production and Release Cytokines play a major role in limiting and eliminating bacterial infection. One of their most important effects is to cause cells of the immune system to become activated, proliferate and differentiate, and so drive the development of the immune and inflammatory responses against the infection. The major cytokines released in response to bacterial infection are interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 6 (IL-6), tumor necrosis factor (TNF), and prostaglandins. TNF-a, which is mainly released by macrophages, plays an essential role in the
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local host defense mechanism against Yersinia in intestinal tissues, possibly by activating phagocytes [9]. Epithelial cells also participate in modulation of the immune response against infection by Yersinia via the release of proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-a, monocyte chemotactic protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and melanoma growth-stimulating activity (MGSA) [18-20]. These cytokines play a role in the initiation or amplification of the inflammatory response: IL-8 and MCP-1 act as potent chemoattractants and activators of neutrophils and monocytes, respectively; TNF-a activates neutrophils and mononuclear phagocytes, while GM-CSF prolongs the survival of neutrophils and monocytes and increases the response of those cells to other proinflammatory stimuli, which can further amplify the inflammatory response. Epithelial cells thus appear to be programmed to provide a set of chemotactic and activating signals to adjacent and underlying immune and inflammatory cells in the earliest phases after microbial infection. However, as we will discuss later in this chapter, cytokine release is inhibited by pathogenic Yersinia spp., potentially providing a powerful survival mechanism. D. Virulence Factors A number of virulence factors are encoded in the chromosome of Yersinia spp., including the invasin InvA, the adhesive factors Myf/pH 6 antigen and Ail, the enterotoxin Yst, and iron acquisition proteins (Table I). In addition, all three pathogenic Yersinia spp. harbor a common 70-kb virulence plasmid that encodes an array of tightly regulated and sophisticated antihost factors.
//. The Adhesive Factors A pathogen must be able to cope with and subvert the host immune responses. The strategies used by Yersinia to establish an infection are to resist phagocytosis by macrophages and PMNs, to inhibit cytokine production, and to induce apoptosis [21]. To achieve these effects. Yersinia adheres to receptors on the surface of host cells and injects toxic proteins inside the host cell cytosol, thereby inducing dysregulation of signal transduction pathways (see below). Yersinia spp. possess a number of adhesion factors that mediate bacterial attachment to eukaryotic cells, including Inv, YadA, Ail, and Myf/pH 6 antigen. A.
Invasin
Invasin (Inv) is a chromosomally encoded outer membrane protein that is expressed in both Y enterocolitica and Y pseudotuberculosis [22-25]. Nonpathogenic strains of these two species often lack a functional Inv and are missing other virulence determinants [26]. Surprisingly, the Y pestis inv gene is inactivated by an insertion sequence [27]. Inv promotes both attachment to and invasion into eukaryotic cells, including nonphagocytic cells, by Yersinia spp. [22-24, 28].
232 Table I
AoiFE P. BOYD AND GUY R. CORNELIS
Adhesion Molecules and Chromosomal Virulence Factors Required for virulence
Protein
Function
Inv
Adherence and invasion
K pseudotuberculosis and pathogenic K enterocolitica strains
Not absolutely; enhances Peyer's patches colonization
Ail
Adherence and invasion
K pseudotuberculosis and pathogenic K enterocolitica strains
No for Y enterocolitica; unknown for Y pseudotuberculosis
YadA
Adherence and invasion
Y. pseudotuberculosis and pathogenic K enterocolitica strains
Yes for Y enterocolitica; no for Y pseudotuberculosis
Myf/pH 6 antigen
Adherence
Y. pestis, Y. pseudotuberculosis, and pathogenic K enterocolitica strains
Yes for Y pestis; others unknown
Yst
Enterotoxin
Pathogenic K enterocolitica strains
Required for diarrhea
FyuA/ Psn-Irp
Iron acquisition
K pestis, Y. pseudotuberculosis and highly pathogenic Y enterocolitica strains
Yes for Y pestis and Y enterocolitica; unknown for Y pseudotuberculosis
Present in
Introduction of the inv gene into other bacterial species, such as Escherichia coli, confers the capacity to invade eukaryotic cells upon these bacteria [24, 25]. Inv is not required for virulence in Y. pseudotuberculosis or Y. enterocolitica, but it does favor efficient translocation of the bacteria from the intestinal tract into the Peyer's patches and so to the underlying tissues [29-31]. As Inv is important at this initial stage of infection, it could be advantageous for the ingested Yersinia to have the adhesin already present on its surface to direct the bacterium to the M cells, and indeed Inv is maximally produced at 23°C [32]. However, the addition of sodium and a reduction in the pH to 5.5 allows production of Inv at 37°C at levels that are equal to those observed at 23°C [33]. It thus seems that Inv expression is possible inside the host animal at 37°C with the appropriate environmental conditions, and, in agreement with these results, Inv can be detected on Yersinia in Peyer's patches 2 days after infection [33]. Inv expression is maximal when bacteria enter stationary phase, and SspA (a stationary phase regulator) is required for optimal Inv expression [34]. The inv gene is located in the middle of a gene cluster encoding the flagella proteins [35]. Interestingly, the expression of Inv is inversely related to that of flagella, (i.e., the
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environmental conditions and regulatory proteins that enhance Inv expression decrease expression of flagellin [34]). It makes sense that these two factors would be regulated in this way, as motile bacteria would not be able to move freely if they were attached to a surface and attachment would not be favored by bacteria trying to swim away all the time! The Inv receptor on eukaryotic cells is (31 integrin [36]. Integrins are able to couple extracellular adhesion events to numerous signaling pathways within the mammalian cell, some of which allow association of the pi chain to cytoskeletal-associated proteins. Once attached, multiple points of contact are established between the cell and bacterium, and the bacterium is taken up into an endocytic vacuole by what is called a zipper mechanism [37]. Uptake is accompanied by cytoskeletal rearrangements with accumulation of actin, filamin, talin, and (31 integrins around the incoming bacteria. Invasion can be inhibited by cytochalasin D, which disorganizes actin filaments, and this property has been used to study the effects on host cells of externally located bacteria [38, 39]. It has also been shown that tyrosine protein kinases are involved in the invasion event [40]. The (31 integrin-binding domain is located in the 192 C-terminal amino acids of the 986-aa Inv protein and incorporates a 76-aa disulfide loop [41,42]. An RGD motif is important for the binding of fibronectin to (31 integrins, and attachment of Yersinia to cells via the Inv protein is inhibited by RGD-containing peptides. However, Inv does not contain an RGD sequence. Instead, amino acids around residue D911 are critical for binding, and residues around D811 are important for high-affinity binding [43, 44].
B. Ail Ail is the second chromosomally encoded, membrane-associated protein that promotes attachment to and subsequent invasion of eukaryotic cells by Yersinia spp. [23]. Like Inv, Ail is expressed in virulent strains of Y pseudotuberculosis and Y enterocolitica but is lacking in Y. pestis due to an insertion sequence. Also, Hke Inv, Ail confers invasion properties on E. coli [23, 45, 46]. Ail is a less powerful adhesin than Inv [46], but it does have the additional ability of rendering Yersinia spp. resistant to killing by serum complement [47, 48]. Ail is expressed by Yersinia during infection; but, nevertheless. Ail is not required for virulence in Y enterocolitica—neither to establish infection nor to cause systemic infection [49, 50]. Unlike Inv, Ail is preferentially expressed at 37°C under aerobic conditions in the stationary phase [48]. In an as-yet-uncharacterized manner, ClpP protease seems to play a role in negative transcriptional regulation of Ail at lower temperatures [51]. A number of Ail homologs have been identified in other pathogenic bacteria. Structural models for these proteins predict the presence of eight membrane-span-
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AoiFE P. BOYD AND GUY R. CORNELIS
ning domains alternating with hydrophilic inner and outer loops. The Rck protein of Salmonella typhimurium promotes resistance to complement killing and cellular invasion [52]; PagC, also in S. typhimurium, is essential for full virulence in mice and survival within macrophages [53]; Lom of bacteriophage lambda is expressed in the outer membranes of E. coli lysogens [54]; and OmpX of E. coli is required for a^ activity [55]. Rck and Ail promote serum resistance and eukaryotic cell invasion, properties not shared by PagC, E. coli OmpX, and Lom [54]. However, the Ail homolog Tia of enterotoxigenic E. coli (ETEC) does promote adhesion and invasion [56], as do OmpX of Enterobacter cloacae [57] and lalB of Bartonella bacilliformis, the agent of human Oroya fever [58].
C. Myf/pH 6 Antigen Myf (mucoid Yersinia factor) in K enterocolitica and its homolog pH 6 antigen in Y. pestis and Y, pseudotuberculosis are chromosomally encoded proteins that form a fibrillar structure of strands, bundles, and aggregates of 15-kDa subunits surrounding the bacteria [59-61]. Like Inv and Ail, expression of these proteins is restricted to pathogenic strains [61]. They enhance thermoinducible binding of Yersinia spp. to eukaryotic cells and can bind intestinal luminal mucus [31, 62]. pH 6 antigen binds specifically to (31-linked galactosyl residues in glycosphingolipids [63]. In addition, synthesis of this antigen has been found to be induced inside macrophages, suggesting it may also play a role in intracellular survival [60]. Most interestingly, pH 6 antigen is required for full virulence of Y pestis [59], and this distinguishes it from Inv and Ail, which are genetically inactivated in Y pestis. Myf/pH 6 antigen is produced from a locus of five genes, one of which, myfA/psaA, encodes the main fibrillar subunit [59-61, 64]. myfBlpsaB and myfClpsaC are required for transport of the subunit across the bacterial membrane and assembly of the subunit into fibrillae [59, 61]. MyfB/PsaB is a member of the PapD family of periplasmic chaperones and could act as a chaperone for MyfA/PsaA, while MyfC/PsaC could act as an usher in the outer membrane. The two inner membrane proteins, MyfE/PsaE and MyfF/PsaF, are required for transcription of myfA/psaA [59, 64, 65]. The C termini of MyfE/PsaE and MyfF/PsaF are periplasmic, while the cytoplasmic N terminus of MyfE/PsaE has similarity to the DNA-binding domain of transcription regulators of two component regulatory systems. This suggests that together these two proteins can sense environmental signals and transduce these signals to the interior of the bacterium. myfE/psaE and myfFlpsaE are transcribed constitutively while transcription of the myfA/psaA operon occurs at 37°C in an acidic medium—hence, the name pH 6 antigen in Y pestis.
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D. YadA The virulence plasmids of Y. enterocolitica and K pseudotuberculosis encode an outer membrane protein called YadA [66-68]. The yadA gene of Y. pestis has a single basepair deletion that results in a reading frame shift and an mRNA with a reduced half-life, so that the YadA protein is not produced in this species [69]. YadA can bind a variety of eukaryotic extracellular and cell surface molecules, including collagens, fibronectin, and laminins [70-73], and can also mediate internalization of the bacteria into eukaryotic cells, a process in which interaction with pl-integrins plays a role [74, 75]. In addition, YadA contributes to the protection of Y. enterocolitica against killing by the antimicrobial polypeptides from the cytoplasmic granules of PMNs that are normally released into the phagolysosome containing the phagocytosed Yersinia [76]. YadA can also protect against killing by human serum [66, 77] due to binding of factor H by YadA, which leads to inactivation of C3b and a subsequent decrease in the deposition of membrane attack complexes on the bacterial surface [78, 79]. This reduction of complement-mediated opsonization could be involved in the resistance of Yersinia to phagocytosis [80]. There is a major difference between K enterocolitica and K pseudotuberculosis regarding the role of the yadA gene in virulence in mice. A Y. enterocolitica yadA mutant is attenuated for virulence [49, 81-83], as YadA is required for survival and multiplication in the Peyer's patches, and perhaps for dissemination of the bacteria from the Peyer's patches to other sites in the body [49, 81]. In contrast, a Y. pseudotuberculosis yadA mutant is just as virulent as the wild type and can colonize the Peyer's patches just as efficiently [30, 68, 84]. An inv yadA double mutant strain maintained the same virulence as the parental strain, demonstrating that neither Inv nor YadA plays an important role during Y. pseudotuberculosis infection [30]. The differences between the Yersinia species with regard to the role of YadA in virulence probably results from the interplay of multiple adhesion factors of varying importance. Structure-function studies have revealed a number of functional domains in the YadA protein. The N terminus of YadA comprises a typical secretion signal sequence [85] and is followed by amino acids 29-81, which are required for adherence to PMNs [82]. Residues 83-101 compose one of the hydrophobic domains of YadA and are required for autoagglutination, and binding to collagen and laminin [83, 86]. Histidines 156 and 159, which are also important for collagen and laminin binding, are needed for binding to fibronectin and to epithelial cells [81]. Finally, the hydrophobic C-terminal amino acids of YadA are involved in surface exposure of the protein and polymer formation [83]. E.
Conclusion
To summarize, four Yersinia spp. adhesins have been identified—Inv, Ail, YadA, and Myf/pH 6 antigen. Expression of each of these adhesins can be induced at 37°C in the appropriate conditions, but expression is restricted to virulent strains
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only. K pestis does not express Inv, Ail, or YadA. These adhesive factors may not be necessary in Y, pestis, as it enters the body by a different route than Y. pseudotuberculosis and Y. entewcolitica. Alternatively, Y. pestis could possess additional adhesion factors, such as the Fraction 1 capsule or the Pla protease (plasminogen activator) which can bind weakly to collagen [1]. YadA is required for virulence in K entewcolitica, and pH 6 antigen for virulence in Y. pestis, but none of the four adhesins has been shown to be absolutely necessary for virulence in all three Yersinia species. This suggests that there is probably a combination of bacterial adhesins and eukaryotic cell receptors that mediate pathogenicity and that there is a certain amount of redundancy in their functions, and/or that these adhesins play a second role in Yersinia lifestyle other than attachment to eukaryotic cells (e.g., YadA and Ail are both involved in resistance to killing by serum).
///. Iron Acquisition Iron is an essential nutrient for Yersinia spp., but, in the host, iron is chelated by mammalian proteins, making it less available to the bacteria. To overcome this problem. Yersinia spp. possess a number of iron acquisition systems. In Y enterocolitica the presence of iron acquisition systems makes a significant difference between low- and high-pathogenicity strains [87, 88]. The importance of iron during infection is demonstrated by Y. enterocolitica bacteremia, which occurs under conditions that make iron more available to the bacteria (e.g., in patients that are overloaded with iron and/or are treated with desferrioxamine, an iron-chelator that Yersinia spp. can readily utilize). The highly pathogenic strains Y pestis, Y pseudotuberculosis 0:01, and Y. enterocolitica biotype 1B share a common iron uptake system called FyuA/PsnIrp [89-91]. This system uses yersiniabactin, a siderophore that can remove iron from a number of mammalian proteins, due to its extremely high affinity for ferric iron [92]. Functional yersiniabactin production and uptake are required for growth at 37°C in iron-depleted media and for virulence [89, 93-95]. Seven genes— called irpl, irp2, irpS, irp4, irp5,fyuA/psn and ybtA—have been identified at this locus so far [94, 96]. irpl encodes HMWPl (high molecular weight protein 1), which is a cytosolic 385-kD protein required for yersiniabactin synthesis. irp2 encodes HMWP2—a 228-kD protein also required for yersiniabactin synthesis. The N terminus of HMWPl resembles polyketide synthases and the C terminus resembles HMWP2. irpS encodes a 41-kD protein of unknown function and irp4 encodes a 30-kD protein (YbtT) that resembles thioesterases. The 56-kD YbtE encoded by irp5 resembles dihydroxybenzoic acid activators and is also required for yersiniabactin synthesis. The proteins encoded by these five genes are presumably the yersiniabactin synthesizing enzymes. The sixth gene in this locus encodes the highly specific yersiniabactin receptor called FyuA in K enterocolitica and Psn is Y pestis. This 74-kD outer membrane protein also serves as a receptor for pesticin—a bacteriocin produced by Y. pestis.
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237
Energy-dependent transport of inorganic iron is induced by iron-deficient conditions in all three pathogenic species of Yersinia. Under iron-replete conditions, iron transport is repressed and excess iron is stored inside the bacteria. Over 30 Y. pestis proteins have been shown to be regulated by iron [97]. This regulation is mediated to a large extent by Fur [98, 99], a cytosolic protein that on binding ferrous iron changes conformation and binds DNA in front of Fur-regulated genes at a specific site called a Fur box, thus preventing transcription. In the presence of iron. Fur downregulates transcription of fyuA/psn, irp2 and most, but not all, other iron-regulated proteins [89]. Under these conditions, yersiniabactin production and uptake is repressed due to transcriptional repression of irp2 andfyuA/psn by Fur. In the absence of iron, Fur cannot bind DNA and transcription proceeds, and Ybt and its receptor are produced. Ybt chelates iron from the environment, then attaches it to Psn/FyuA, and the iron is transported inside the bacteria. Other factors are also involved in iron regulation: YbtA is an AraC-like regulator that is required for transcription of fyuA/psn and irp2, and it is encoded by a gene located upstream of irp2 [100]. Ybt A also acts to control the system as it downregulates its own transcription. In addition, yersiniabactin is itself required for upregulation of genes involved in its own production and uptake, including irp2 andfyuA/psn [94, 96]. Thus, there is autoregulation of genes involved in the synthesis and uptake of yersiniabactin. A number of other iron acquisition systems and iron-regulated proteins have been identified. For instance, in K enterocolitica the exogenous siderophore desferrioxamine B is bound and taken up by the 77-kD outer membrane protein Fox A [101]. This explains why septicaemia with low-virulence Y. enterocolitica is sometimes observed in patients who receive desferrioxamine B to treat iron overload caused by multiple blood transfusions. Still to be investigated is the conservation of these systems among the Yersinia species, the specific role of each system, the function of the components within each system, and the importance of each system in virulence.
IV. Pathogenicity islands The fyuAlpsn-irp region has many characteristics of pathogenicity islands: it carries genes necessary for a high-pathogenicity phenotype, it is present only in pathogenic strains, it is greater than 35 kb long (45 kb total), it is bordered by a tRNA gene at one side {asn tRNA), it contains insertion sequences, it has a GC content different from that of the rest of the chromosome (60% versus 47-50%), and it is not found in other bacterial species {E. coli) [102-104]. As this region determines the level of pathogenicity in Y. enterocolitica between strains of high virulence (e.g., serotypes 0:8 and 0:21) and those of low virulence (e.g., serotypes 0:9 and 0:3), it has been called an HPI (high-pathogenicity island). In Y. pestis, the fyuAlpsn-irp region is linked to a haemin storage (hms"^) and pigmentation (pgm"^) locus to form a complete 102-kb region [104]. The haemin storage locus allows Y pestis to use haem and haem-protein complexes—for example, myoglobin or haemoglobin—as a source of iron. When grown in the
238
AoiFE P. BOYD AND GUY R. CORNELIS
presence of haemin or Congo red, bacteria harboring this locus give rise to pigmented colonies, and so these are given the phenotype pgm"^. This large region is conserved among different strains, but in the lab it is easily lost, most probably due to recombination between insertion sequences located at the extremities of the 102 kb [105]. It is maintained under natural conditions, as it is essential for in vivo survival (iron acquisition) and transmission to new hosts via flea vectors (haemin storage system) [106].
V. Yst Enterotoxin Pathogenic strains of Y. enterocolitica produce a heat-stable enterotoxin called Yst, which is responsible for the diarrhoea associated with Yersinia infection [107, 108]. Yst is homologous to the STI toxin of E. coli and to rat guanylin, an endogenous activator of the intestinal guanylate cyclase [109]. Like STI, Yst stimulates cGMP synthesis in the intestinal brush border, leading to an overall effect of fluid loss and lack of fluid absorption [110-112]. The STI toxin is very tissue specific, and receptors for it are apparently present only in the intestine. Yst is synthesized as a 71-aa pre-pro-Yst nascent protein, of which the 18 N-terminal amino acids comprise a signal sequence that is cleaved off during secretion [107, 113]. A further 23 central amino acids are removed during or after secretion, and the mature active peptide thus contains just 30 amino acids. Yst is produced during the stationary phase at 30°C, but increased osmolarity and pH at 37°C also induce high expression of this protein [114]. Accordingly, yst transcription is controlled by the stationary phase sigma factor RpoS and by two proteins that affect DNA supercoiling—the YmoA histone-like protein and Yrp, which is homologous to E. coli host factor 1 [114-116].
VL The Yersinia Virulence Plosmid A. Role of the Virulence Plasmid The virulence plasmid, referred to as the pYV plasmid in K enterocolitica, encodes the Yop virulon—an integrated virulence apparatus (Fig. 2) [117a-c]. Experimental infection of mice with laboratory-constructed derivatives lacking the virulence plasmid showed that numerous bacteria were located intracellularly in vacuoles of PMNs and mononuclear cells but were unable to cause infection. In contrast, virulence plasmid-carrying bacteria were not phagocytosed and
239
6. YERSINIA
repBA
oriR
yopO
arsCBR/^'^
yadA
yopl\/l
pYVeO:9
yscM2
yopD ^ yopB Hi sycD g j^^^
intracellular delivery
69.5 kb
IcrG spyAB
plasmid partition
secretion secretion and its control
effectors and chaperones
secretion
secretion yscM
regulation
s > yscuj'x^
yopH
virC
p
o/T
/ f ,^,R
//
^ ^ yscV(lcrD) _ tyeA yopN
virB
virF y^*^^ ^"^ (virG)
Fig. 2 Map of the pYV plasmid of K enterocolitica. The genes encoded by the pYV plasmid and the functions of their protein product. Adapted with permission from Iriarte and Cornelis (1998) [175].
remained extracellular, even though they were surrounded by inflammatory cells [118]. Thus, virulence plasmid-encoded proteins are involved in the resistance of Yersinia to phagocytosis by PMNs and macrophages. These proteins are also involved in inhibition of the PMN oxidative burst [119], in induction of programmed cell death in macrophages [120-122], and in inhibition of the cytokine release that is normally induced by Yersinia infection [19, 20, 123], so limiting the host's inflammatory response to the infection. The cell types that are the actual targets of these Yersinia proteins in vivo are presently not known, and, although macrophages and PMNs are obvious in vivo targets, epithelial cells of the gastrointestinal tract and endothelial cells may also be relevant targets. Epithelial cells not only constitute a barrier against bacterial invasion, but they also synthesize and secrete a number of cytokines, while endothelial cells have an important role in development of the immune and inflammatory responses by recruiting PMNs through the increased surface expression of adhesion molecules.
240
AoiFE p. BOYD AND GUY R. CORNELIS
The Yop virulon includes a set of secreted proteins (the Yop proteins), the Yop-specific chaperones (the Syc proteins), a Yop-dedicated secretion system (the Ysc system), and a regulatory network. Some of the secreted Yop proteins are required for the translocation across the eukaryotic cell membrane of other Yops that interfere with normal cellular processes in the cytosol of the eukaryotic cell.
B. Ysc Secretion System The virulence plasmid encodes a set of proteins that are involved in establishing and building a Yop-specific secretion apparatus through the bacterial membranes of Yersinia spp. Homologous secretion systems are also present in a number of other bacteria, all of which interact with eukaryotic cells. These include animal pathogens—Salmonella spp.. Shigella flexneri, enteropathogenic and enterohaemorraghic E. coli (EPEC and EHEC), Pseudomonas aeruginosa. Chlamydia psittaci, and Bordetella spp.—and plant pathogens that elicit the hypersensitive response and pathogenesis—Erxvinia amylovora, Pseudomonas syringae, Xanthomonas campestris, and Ralstonia solanacearum [21, 124-126]. This system is also involved in the nodulation of legumes by the symbiotic nitrogen-fixing Rhizobium sp. NGR234 [127]. The Ysc system belongs to the family of type III secretion systems, which are called contact-dependent because intimate contact between the eukaryotic cell and bacteria triggers secretion and allows delivery of bacterial proteins inside eukaryotic cells. In Yersinia, 29 ysc secretion genes have been identified within four contiguous loci, and, although a mutation in almost any one of these genes causes a lack of secretion, little is known about the exact function of the various encoded components. Four proteins—LcrD, YscD, YscR, and YscU—span the inner membrane (Fig. 3) [128-132], while YscC is an outer membrane protein that belongs to the family of secretins, a group of outer membrane proteins involved in the transport of various macromolecules and filamentous phages across the outer membrane [130, 133, 134]. The VirG lipoprotein is required for efficient targeting of this YscC complex to the outer membrane [133], where it forms a ring-shaped pore structure with an external diameter of about 200 A and an apparent central pore of 50 A. YscN contains ATP-binding motifs (Walker boxes A and B) resembling the p catalytic subunit of FQEI proton translocase and related ATPases, and presumably energizes the secretion of Yops [135]. Four }^5c-encoded proteins are secreted: YopR, encoded by yscH, YscO, YscP and YscM/LcrQ [136-139]. YopR is not required for secretion of the other Yops, but it is involved in pathogenesis since the LD50 of the yscH mutant is 10-fold higher than that of the wild-type strain [138]. YscM/LcrQ is involved in the feedback control of Yop synthesis (see §VI.D.2) [138-141]. The type III Ysc secretion proteins are thought to form a structure similar to that of the apparatus involved in flagellar assembly [142, 143].
6.
241
YERSINIA
f^ Aimm 1
fiPfliiniiniiiifioM
mm
Mumu
MUM
ii IM O5 GOO
folding or association
Yop
y
'g
Fig. 3 Model of the Ysc secretion apparatus. The Syc chaperones bind to their newly synthesized partner Yops to prevent Yop misfolding, inappropriate protein-protein interactions and/or degradation. The Yops associate with the secretion apparatus, pass through, and are then released from the bacterium. The Ysc machinery comprises the YscN ATPase, the inner membrane proteins YscR, YscU, and YscD, the lipoproteins VirG and YscJ, the proteins YscS, LcrD, and YscT, and, in the outer membrane, the secretin YscC. Reprinted with permission from Cornells et al. (1998) [21].
242
AoiFE P. BOYD AND GUY R. CORNELIS
C. Yops The Ysc system is required for the specific secretion of the virulence plasmid-encoded Yop proteins [144, 145]. 14 Yops have been identified so far, and they seem to be very well conserved among the Yersinia species (Table II, Fig. 4). Most of the Yop proteins are essential for virulence [146-151]. The information necessary for Yop secretion is contained in the N terminus [39, 144], and the shortest region Table II
Yops Secreted by Y. entewcolitica W22703
Yop
Size: predicted and apparent (kDa)
Secretion signal (N-terminal amino acids)
Translocation signal (N-terminal amino acids)
Required for virulence
YopO/ YpkA
82, 84
77
77
Yes
YscP YopH
50, 66 51, 51
ND" 17
N/A^ 71
ND Yes
YopM YopB LcrV YopD
42, 42, 37, 33,
48 44 41 37
40 ND ND ND
100 N/A ND ND
Yes Yes Yes Yes
YopT
36, 36
124
124
No
YopN YopP/YopJ
33, 35 33, 30
15 43
N/A 99
ND No
YopE
23, 25
11
50
Yes
YopQ/YopK
21, 20
ND
N/A
Yes
YopR YscMl
18, 17 12, 12
ND ND
N/A N/A
Yes ND
"ND = not determined.
N/A = not applicable.
Function Effector—serine/ threonine kinase Unknown Effector—tyrosine phosphatase of FAK and pHO'^^'; prevents phagocytosis and phagocytic killing Effector? Translocator Translocator effector? Translocator; negative regulator of secretion; effector? Effector—depolymerizes actin; causes cytotoxicity Controls Yop secretion Effector—inhibits NFkB activation; IkB is possible target Induces apoptosis; prevents cytokine induction Effector—depolymerizes actin; causes cytotoxicity; prevents phagocytosis Controls size of translocator pore Unknown Negative regulator of secretion
6.
243
YERSINIA
-Ca"" +Ca2*
YopO
S '
YSCP
Y o p M •"wW'"*™*
67
43
30
YopE
YopQ
igi 20
YopR
14
Fig. 4 The secreted proteins of K entewcolitica. Acrylamide gel separation of the proteins secreted by K entewcolitica grown in calcium deficient media (-Ca^"^) and in media containing calcium (+Ca +). The names of the Yops are shown on the left-hand side of the figure and the molecular weight standards in kD on the right-hand side.
244
AoiFE P. BOYD AND GUY R. CORNELIS
shown to be sufficient for secretion of a Yop was 15 amino acids for YopE [152, 153]. However, unlike proteins secreted by the type II system, there is no cleavage of this secretion signal during export from the bacteria [145, 154, 155]. There is no similarity between the secretion domains of the Yops with respect to amino-acid sequence, hydrophobicity profile, distribution of charged residues, or prediction of secondary structure, which suggests that a conformational structure is important for secretion. It has also been suggested that there may be a signal in the yopE mRNA that directs the nascent polypeptide to the secretion apparatus [156]. A second, and weaker, secretion signal corresponding to the chaperonebinding site (see further) has also been described for YopE [157]. D.
Regulation of Transcription of the Virulon Genes
In vitro, Yop secretion only occurs at 37°C in the absence of Ca^"^. This secretion correlates with growth arrest, a phenomenon known for a long time as "Ca^"^ dependency." There seem to be two different regulatory networks. The first permits full expression of all the virulence plasmid-encoded virulence functions when the environment is ideal and the temperature reaches 37°C, while the second only prevents Yop production in the presence of 2.5 mM Ca^"^ ions. 1.
EFFECT OF TEMPERATURE ON YOP VIRULON TRANSCRIPTION
Transcription of many virulence plasmid genes—including all the yop genes, sycE, yadA and the virC operon—is dependent on VirE/LcrF, a member of the AraC family of regulators [134, 158-160]. On the other hand, VirF/LcrF seems to be dispensable or less important for transcription of the virA and virB operons encoding the Ysc secretion apparatus [161], and of several other genes like sycH [162]. VirF binds to a 40-bp region localized immediately upstream from the RNA polymerase-binding site. These VirF-binding sequences are located in an AT-rich region, appear either isolated or repeated in opposite orientation and contain the 13-bp consensus sequence TTTTaGYcTtTat (in which nucleotides conserved in >60% of the sequences are in uppercase letters and Y indicates C or T) [163]. As VirF/LcrF expression is regulated by temperature at both transcriptional and posttranscriptional levels, this explains in part the temperature regulation of these genes. YmoA, a histone-like protein, is involved in inhibiting transcription at lower temperatures [164]. It has been hypothesized that temperature could dislodge the YmoA transcriptional inhibitor from the promoter regions of VirF/LcrF-sensitive genes, so VirF/LcrF could bind and transcription could proceed [165]. 2.
EFFECT OF CA^^ ON YOP VIRULON TRANSCRIPTION
While Ca^"^ has little effect on transcription of the ysc machinery loci, it has a very clear inhibitory effect on yop expression [166, 167]. Thus, the presence of Ca^"^ ions blocks not only the secretion of Yops but also their synthesis. This
6.
YERSINIA
245
secretion blockage by Ca^"^ probably occurs at the bacterial surface, such that when secretion is compromised a feedback inhibition mechanism blocks transcription of yop genes. This hypothesis is reinforced by the fact that mutations in v/M, virB and virC loci, which encode the Ysc secretion apparatus, severely downregulate expression of iht yop genes [128-131, 135, 158]. Three proteins are involved in the control of Yop release: YopN [154, 168], LcrG [169, 170] and TyeA [168]. yopN, IcrG, and tyeA mutants express and secrete Yops in the presence and absence of Ca^"^ (see later) [154, 169, 170]. The fact that these mutants are derepressed for Yop expression even in repressive conditions (presence of Ca^"^ ions) leads to the idea that feedback regulation operates via a negative regulator. By analogy with the secreted antisigma factor involved in the regulation of flagellum synthesis, Rimpilainen et al [140] suggested that feedback inhibition could be mediated by a negative regulator called LcrQ/YscM that is normally expelled via the Yop secretion machinery. Overproduction of LcrQ abolishes Yop production, and, in the absence of secretion (presence of Ca^+ or mutation in the genes coding for the secretion machinery), an IcrQ mutant indeed synthesizes more Yops than the wild type. In the presence of Ca^"*", this mutant secretes YopD and LcrV. LcrQ is rapidly secreted when bacteria are shifted from a medium containing 2.5 mM Cd?^ (nonpermissive conditions for Yop secretion) to a medium containing a Ca^+ chelator (permissive for Yop secretion), which fits quite well with the "secreted negative regulator" hypothesis [141]. In K enterocolitica, two different YscM proteins—YscMl and YscM2—fulfill the role of LcrQ in Y. pseudotuberculosis [139]. LcrQ/YscM is probably not a transcriptional repressor and one or more other virulence plasmidencoded proteins, including YopD, are required to act with this protein in the feedback inhibition mechanism [171, 172]. E.
Syc Cytosolic Chaperones
Syc chaperones (specific Yop chaperones) are present in Yersinia to aid in secretion of specific Yops. The genes encoding these chaperones are generally situated beside the genes encoding the Yops they aid. Several Syc chaperones have been described, including SycE/YerA for YopE, SycH for YopH, SycN for YopN, SycT for YopT, and SycD/LcrH for both YopB and YopD [162, 163, 173-177]. The Sycs are required for efficient secretion of their corresponding Yops but not for their synthesis. In addition, they bind specifically to their Yop. YopE and YopH have discrete domains (residues 15-50 for YopE and residues 20-70 for YopH) that bind their cognate chaperones, while SycD binds to several domains on YopB [177, 178]. Surprisingly, the chaperone-binding domain of YopE and YopH is not the secretion domain, but rather the translocation domain (see§VI.Rl). Although the Syc chaperones seem to have a common role in protein secretion, they are only weakly related in terms of primary amino acid sequence. However, they do share some common features: (1) an acidic pi; (2) a size in the range of 15 to 19 kD, and (3) a C-terminal amphiphilic a-helix. SycE, SycH, and SycT
246
AoiFE P.
BOYD AND GUY R. CORNELIS
possess a conserved "leucine-repeat" motif (LLWXRXPLXXXXXXXLXXXLEXLVXXAEXL) in this a-helix, where most of the hydrophobic residues, essentially leucines, are present on the same side of the helix [179]. Sycs strongly enhance, but are not required for, secretion of the Yops, as residual secretion of YopE or YopH was still observed in the absence of SycE or SycH, respectively [162, 163]. It was also shown that Yops deleted of their chaperone-binding domains are normally secreted in the absence of their chaperones, in contrast to wild-type Yops, which are much less secreted in the absence of the chaperone [178]. As the amount of a Yop is generally decreased inside Yersinia in the absence of the cognate Syc as compared to in its presence [162, 177], Sycs could have a protective role for their respective Yops. In support of this idea, SycD protects E. coli from cell lysis due to overexpression of YopB [177]. Thus, there are three possibilities (which are not necessarily mutually exclusive) for the role of the Syc chaperones and the chaperone-binding domains: (1) the chaperone-binding domain binds another protein that interferes with secretion, and binding by Syc inhibits this interaction; (2) the chaperone-binding domain inhibits interaction with the Ysc secretion machinery, perhaps as a result of misfolding, and binding by Syc overcomes this inhibition; and (3) the chaperone-binding domain is a site for a protease, and binding by Syc protects against degradation of the Yop.
F.
Translocation of Effector Yops across the Eukaryotic Cell Plasma Membrane 1.
DISCOVERY OF TRANSLOCATION
The secreted Yops include translocators and effectors. The translocators (YopB, YopD and LcrV) presumably form a pore in the eukaryotic cell membrane through which the effector Yops (YopE, YopH, YopP, YopO, YopT, YopM) enter the cytosol of the host cell (Fig. 5). A domain important for translocation of the Yop effectors has been shown to reside in their N-terminal domain, immediately following the secretion domain [152, 153]. This translocation domain is also the chaperone-binding domain, raising the possibility that the Syc chaperones play a role in the translocation of Yops, as well as in their secretion [178]. The translocation event is carried out by extracellular bacteria adhering to the eukaryotic cell surface [39, 180]. Contact between the bacteria and the cell is necessary for translocation of the Yops inside the eukaryotic cell, as neither Yops produced by nonadherent bacteria nor purified secreted Yops have the ability to enter eukaryotic cells [39, 180, 181]. The adhesins primarily involved in enhancing attachment of K pseudotuberculosis and Y. enterocolitica to allow Yop translocation are YadA and Inv [39, 180-182]. For Y. pestis, which lacks Inv and YadA, other adherence factors must fulfill this role (e.g., pH 6 antigen).
6.
247
YERSINIA
EUKARYOTIC CELL depolymerization of actin
phosphorylation
apoptosis & ^ inhibition of cytokine release
Ca2+ dephosphorylation ^^^J of signal transduction ^ > ^ ^ proteins _^€^^.w*
depolymerization of actin
Ca 2+
ftmmnfliiiin Inner MMM^^ membrane JJil^UUJili] [OPEN
Ca2+
.^
Ca2+
nnnnnnnnnn uuuuuuuuuu ICONTACTI
BACTERIAL CELL Fig. 5 Model of the process of Yop translocation into eukaryotic cells in the presence of calcium (right-hand side). The secretion channel, which is composed of Ysc proteins, is closed by YopN, LcrG, and TyeA. On contact with a eukaryotic cell, the channel is opened and YopB, YopD, and LcrV are released to aid in formation of a pore in the eukaryotic cell membrane. YopE, YopH, YopM, YopO/YpkA, YopP/YopJ, and YopT are translocated through this pore into the eukaryotic cell cytosol, where they disrupt normal cellular processes. Adapted with permission from Cornells and Wolf-Watz (1997) [209].
YopE was the first Yop to be shown directly to enter into eukaryotic cells. Earlier experiments had indicated that cellular cytotoxicity was induced by internalized YopE and that the internalization was dependent on other virulence plasmid-encoded proteins, including YopD [181]. The direct translocation of YopE into eukaryotic cells was shown by fractionating the cells after Yersinia infection and by using confocal immunofluorescence [180]. In addition, Sory and Comelis created a plasmid construct that fused the N terminal of the yopE gene to the cya gene and showed that infection of eukaryotic cells by Y. enterocolitica producing the YopE-Cya hybrid resulted in an increase in intracellular cAMP levels which demonstrated that YopE-Cya had been translocated into the cytosol of the eukaryotic cells [39] {cya codes for the catalytic domain of adenylate cyclase from Bordetella pertussis; this enzyme catalyzes the conversion of ATP to cAMP only in the presence of calmodulin, which is present in eukaryotic cells but not in bacteria). Using similar methods, the YopOAfpkA, YopH, YopT, YopM, and YopPAbpJ proteins have also been shown to be translocated into eukaryotic cells [19, 152, 168, 175, 183-185].
248
AoiFE P. BOYD AND GUY R. CORNELIS
2.
THE DELIVERY APPARATUS
The translocation apparatus composed of the YopB, YopD, and LcrV proteins is required for translocation of all the Yops across the eukaryotic cell membrane [19, 152, 168, 171, 175, 183-187]. Analysis of YopD and YopB suggests the presence of domains that could form coiled coils—structures commonly involved in protein-protein interactions—and suggests the presence of hydrophobic domains, thus implying they could be transmembrane proteins [155]. YopB has a moderate level of similarity with proteins of the RTX family of a-hemolysins and leukotoxins that disrupt the membranes of eukaryotic cells by forming pores, suggesting that the translocation apparatus could be some kind of a pore, where YopB would be the main element. In support of this hypothesis, purified YopB has the ability to disrupt lipid bilayers and Yersinia spp. have a YopB- and contact-dependent lytic activity on sheep erythrocytes. Protection experiments with sugars of various molecular weights suggest that the hypothetical pore has an inner diameter between 12 and 35 A [186]. YopB and YopD appear to be associated with one another in the bacterium prior to their secretion, suggesting that YopB and YopD could insert together into the eukaryotic membrane to make the translocation pore [177]. Alternatively, as YopD has been shown to be itself translocated, it has been proposed that YopD could act to guide effector Yops into the eukaryotic cytosol [171]. Other proteins are also involved in the translocation process. LcrV is involved in the process of secretion of YopB and/or YopD [187, 188]; LcrG, a protein encoded by the same operon as YopB, YopD, and LcrV, is needed for efficient translocation of the Yop effectors, but this has only been observed in K enterocolitica [169]. The degree of translocation can be controlled by changing the size of the pore, a mechanism in which YopK/YopQ is involved [189]. Some proteins have a more specific control of Yop translocation, such as TyeA, which is required for translocation of YopE and YopH but not of the effector Yops [168]. Thus, the translocation apparatus is thought to be composed of a pore formed by YopB, with essential (YopD and LcrV) and ancillary (LcrG, YopK/YopQ, TyeA) proteins also playing important roles. 3.
CONTROL OF YOP RELEASE DURING TRANSLOCATION
As described above. Yersinia spp. only secrete Yops in the absence of Ca^"^ and at 37°C. However, contact between the bacterium and the eukaryotic cell is the signal inducing Yop secretion in vivo. Transcription of the yopE gene is induced in bacteria attached to eukaryotic cells, but not in bacteria attached to glass [141]. When Yop secretion is triggered by eukaryotic cells, it is "directional" in the sense that the majority of the Yop effector molecules produced are directed into the cytosol of the eukaryotic cell and not to the external milieu [180, 185].
6.
YERSINIA
249
As described in section VLD.2, three genes are involved in the control of Yop release: yopN, IcrG, and tyeA. Bacteria carrying mutations in any one of these genes are deregulated for Yop secretion in the sense that they secrete Yops even in the presence of Ca^+ in lab media (i.e., they are calcium blind), and they secrete a large percentage of Yops into the culture media when grown in contact with cells (i.e., Yop secretion becomes nondirectional) [154, 168-170, 180, 183, 185, 187, 190]. These three proteins could form a complex on the bacterial surface that functions as a sensor and a stop valve controlling Yop secretion. YopN and TyeA are present and associated on the bacterial surface, while LcrG is primarily cytosolic, with small quantities found in the membrane and extracellular media [154, 168, 170, 191]. LcrG has been shown to bind to LcrV [188, 191], and it has been suggested that the negative effect of LcrG on secretion is counteracted by LcrV. After contact with a receptor on the eukaryotic cell, the three-member sensor would be opened to allow Yop secretion and delivery to the target cell. LcrG binds HeLa cells by interacting with heparan sulfate proteoglycans [192]. Proteoglycans are glycosaminoglycan-containing proteins found on the surface of practically all types of eukaryotic cells, and they have been shown to be receptors for a variety of microorganisms via the glycosaminoglycan moieties. Addition of exogenous heparin or treatment with heparitinase decreased the level of YopE translocation into HeLa cells. As the addition of heparin was unable to completely abolish translocation, another bacterial-eukaryotic cell interaction could also be involved. Thus, the heparan sulfate-LcrG interaction can be viewed as maximizing the efficiency of LcrG in the translocation process but may not be absolutely essential. Although Yersinia carrying mutations in yopN, tyeA, or IcrG have a similar deregulated secretion phenotype, their Yop translocation phenotypes are all different. yopN mutant Yersinia can still deliver all the Yops into the cytosol of the target cell [141, 168, 175, 185]. However, a tyeA mutant is impaired in translocation of YopE and YopH, but surprisingly not of YopM, YopO, YopP, and YopT [168, 175]. Finally, LcrG is required for efficient translocation of all the Yop effectors into eukaryotic cells [169]. These results suggest that these three proteins could form a complex at the exterior tip of the secretion apparatus that controls the passage of Yops through the translocation pore.
G. The Yop Effectors and Their Targets 1.
YOPE
YopE induces disruption of the actin microfilament structure of the host cell [181]. The host cell rounds up and detaches from the extracellular matrix, a phenomenon referred to as cytotoxicity [148, 193]. Actin is responsible for maintaining the shape of cells, and disruption of actin thus leads to cellular amorphism and the rounding up. As the actin is linked to surface-located cellular
250
AoiFE p. BOYD AND GUY R. CORNELIS
adhesion factors, there would also be subsequent detachment of cells. The actin disruption contributes to the ability of Yersinia to resist phagocytosis [181, 194]. The actual enzyme activity and the target of YopE within the eukaryotic cell remain to be identified. In addition to YopE, three other Yops have a cytotoxic effect on cultured epithelial cells, namely, YopH, YopO, and YopT (see further). 2.
YOPH
YopH is a protein tyrosine phosphatase [195-197]. Protein tyrosine phosphorylation processes form part of the signal transduction pathways that control many cellular functions, including mitogenesis and cell division. YopH inhibits phagocytosis mediated by either complement receptors or Fc receptors, and also inhibits Inv-mediated invasion [14, 17, 198]. The Yersinia antiphagocytic effect thus involves blocking of a broad-specificity phagocytic mechanism and is not restricted to the uptake of Yersinia themselves [14, 194]. YopH is able to interfere with early tyrosine phosphorylation signals that occur in the cell during phagocytosis, including phosphorylation of paxillin, focal adhesion kinase (FAK), and plSQC^^ [198-200]. YopH interacts direcdy with FAK and plSO^^^ within focal adhesions, and their subsequent dephosphorylation leads to disruption of peripheral focal complexes and impaired ability of the target cell to carry out phagocytosis by macrophages or the invasin-mediated internalization of the bacteria by nonphagocytic cells [198, 200]. YopE and YopH thus act in concert to enable Yersinia to inhibit their own uptake by macrophages and hence to be able to proliferate in the Peyer's patches as extracellular microcolonies. Integrinmediated adhesion of phagocytes to endothelia or extracellular matrix proteins plays an important role during inflammation. Interference with this process due to YopH-mediated dephosphorylation of plSO^^"^ might have an important impHcation for PMN function during a Yersinia infection. It has also been suggested that YopH is responsible for the capacity of Y pseudotuberculosis to inhibit the respiratory burst [201, 202]. 3.
YOPM
Purified YopM has thrombin-binding activity and competitively inhibits thrombin-induced platelet activation in vitro, suggesting that YopM is an extracellular effector [146, 203]. However, YopM is delivered inside eukaryotic cells [185], indicating that YopM is rather an intracellular Yop effector. YopM contains a succession of leucine-rich repeats (LRR), which are motifs for protein-protein interactions [204], but the intracellular target of YopM is as yet unknown. No effect of intracellular YopM on eukaryotic cells has yet been documented. 4.
YOPOA'PKA
YopO/YpkA is a serine/threonine kinase that induces morphological alterations in epithelial cells causing the cells to round up, but, unlike YopE or YopH, the
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cells do not detach from the extracellular matrix [151, 184, 205]. Inside the host cells, the YopOAfpkA protein is targeted to the inner surface of the plasma membrane. No target protein of YopOAfpkA has been identified yet. 5.
YOPP/YOPJ
YopPAfopJ induces apoptosis (programmed cell death) in murine macrophages [19, 120, 121, 206]. By using the mechanism of apoptosis during the infection process. Yersinia might eliminate macrophages without inducing an inflammatory response, thereby promoting Yersinia proliferation in lymphoid tissues. YopPAbpJ also inhibits the ERK2, JNK, and p38 mitogen-activated protein kinase (MAPK) activities in infected macrophages [123, 207, 208], as well as inhibiting activation of the transcription factor NF-KB [19, 206]. MAPKs and NF-KB usually become activated in response to lipopolysaccharide. YopP/YopJ inhibits NF-KB activation by preventing the phosphorylation and subsequent degradation of the NF-KB-inhibitor protein IKB, and there is a striking correlation between the abilities to inhibit NF-KB activation and to trigger apoptosis [19, 206]. These results suggest that Yersinia could trigger apoptosis by suppressing the cellular activation of N F - K B , which normally acts to help cell survival. Suppression of NF-KB activation also leads to suppression of the transcription of a number of cytokines, including the proinflammatory cytokines TNF-a, IL-8, and IL-1, and melanoma growth-stimulating activity (MGSA) [19, 123, 206]. It is easily conceivable that this effect favors Yersinia, especially during the early phase of infection, by delaying the attraction of PMNs to the site of infection. YopP activity requires a region resembling an src homology domain 2 (SH2), which is found in several eukaryotic signaling proteins [19]. Thus, YopP could interact directly with signaling proteins involved in inductive cytokine expression. 6.
YOPT
YopT induces a cytotoxic effect in eukaryotic cells that consists of disruption of the actin filaments and alteration of the cell cytoskeleton [175]. YopT does not seem to be functional in Y pseudotuberculosis, as a yopE single mutant, which would presumably still produce YopT, is not cytotoxic [181]. However, in Y enterocolitica, YopT-induced cytotoxicity is just as strong as that of YopE [175]. 7.
SUMMARY
The effector Yops cause disruption of normal cell processes. These disruptions can be visualized by morphological alterations in the target cell—cytotoxic rounding up or apoptosis. Two Yop effectors—YopE and YopT—cause disruption of the cytoskeleton, which leads, at least in the case of YopE, to inhibition of phagocytosis. YopH also inhibits phagocytosis, but in this case by dephosphory-
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lating key signaling proteins including pHO^^*^ and FAK. In contrast to the dephosphorylating YopH, YopO phosphorylates proteins, but it presumably also interferes with signaling pathways in the cell. YopP disrupts transcriptional signaling in the cell via N F - K B , leading to apoptosis and a lack of induction of cytokine release. This multipronged attack is a terribly efficient way of killing a cell. But why do Yersinia spp. produce so many effectors, when one would probably be sufficient to do the job? Perhaps the different effectors are delivered to, or are active on, different cell types. Or perhaps it is an attack strategy, with defensive Yop effectors first entering to inactivate the cell and so protecting the bacteria, and later offensive Yops entering to kill the cell.
VIL Conclusion Yersinia spp. have a number of virulence factors that contribute to their ability to establish an infection in their mammalian host. First, the two pathogens that enter via the gastrointestinal route, Y pseudotuberculosis and Y enterocolitica, must adhere to and invade M cells in the intestinal tract, a process in which Inv is particularly important but other adhesion molecules also play a role. While in the intestine, the Yst enterotoxin is released by Y. enterocolitica to cause diarrhoea. Once the three Yersinia spp., Y pestis included, have reached the reticuloendothelial system, which is their preferred niche in the body, iron acquisition becomes crucial for survival, and siderophore production and the receptor system ifyuAlpsn-irp system) are very important in this regard. The capacity of the Yersinia spp. to cause infection also depends on their ability to evade and inhibit the immune and inflammatory responses of the host, a capacity that requires the presence of the Yersinia virulence plasmid. The virulence plasmid encodes a set of secreted Yop proteins, their dedicated Ysc type III secretion system, and a regulatory network. The Yersinia first adhere to the host cells by a number of adhesion molecules. Inv and YadA are the most important, but Ail and Myf/pH 6 antigen could also be involved. This attachment induces Yop production and secretion. Several Yops (YopB, YopD, LcrV, YopK/YopQ) are involved in the formation and functioning of a pore for translocation of the effector Yops into the eukaryotic cells. The Yop effectors (YopE, YopH, YopP/YopJ, YopO/YpkA, YopM, YopT) enter into the eukaryotic cell, where, among other things, they interfere with the signaling cascades of the host cell. This Yop system allows Yersinia spp. to inhibit phagocytosis and killing by macrophages and PMNs, to reduce the inflammatory response by inhibiting the release of proinflammatory cytokines, and to induce apoptosis of macrophages.
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Acknowledgments A. P. B. was funded in part by an H. and A. Brenninkmeijer ICP fellowship and by EU TMR Programme Research Network Contract FMRX-CT98-0164. The Yersinia project is supported by the Belgian "Fonds National de la Recherche Scientifique Medicale" (Convention 3.4595.97), the "Direction generale de la Recherche Scientifique-Communaute Frangaise de Belgique" ("Action de Recherche Concertee" 94/99-172), and by the "Interuniversity Poles of Attraction Program, Belgian State, Prime Minister's Office, Federal Office for Scientific, Technical and Cultural affairs" (PAI4/03).
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195. Guan, K. L., and Dixon, J. E. (1990). Protein tyrosine phosphatase activity of an essential virulence determinant in Yersinia. Science 249, 553-556. 196. Bliska, J. B., Guan, K. L., Dixon, J. E., and Falkow, S. (1991). Tyrosine phosphate hydrolysis of host proteins by an essential Yersinia virulence determinant. Proc. Natl. Acad. Sci. U.S.A. 88, 1187-1191. 197. Bliska, J. B., Clemens, J. C., Dixon, J. E., and Falkow, S. (1992). The Yersinia tyrosine phosphatase: Specificity of a bacterial virulence determinant for phosphoproteins in the ill Ah. 1 macrophage. / Exp. Med. 176, 1625-1630. 198. Persson, C., Carballeira, N., Wolf-Watz, H., and Fiillman, M. (1997). The PTPase YopH inhibits uptake of Yersinia, tyrosine phosphorylation of pi30^"' and FAK, and the associated accumulation of these proteins in peripheral focal adhesions. EMBO J. 16. 2307-2318. 199. Andersson, K., Carballeira, N., Magnusson, K. E., Persson, C , Stendahl, O., Wolf-Watz, H., and Fallman, M. (1996). YopH of Yersinia pseudotuberculosis interrupts early phosphotyrosine signaling associated with phagocytosis. Mol. Microbiol. 20, 1057-1069. 200. Black, D. S., and Bliska, J. B. (1997). Identification of pi30^^"^ as a substrate of Yersinia YopH (Yop51), a bacterial protein tyrosine phosphatase that translocates into mammalian cells and targets focal adhesions. EMBO J. 16, 2730-2744. 201. Bliska, J. B., and Black, D. S. (1995). Inhibition of the Fc receptor-mediated oxidative burst in macrophages by the Yersinia pseudotuberculosis tyrosine phosphatase. Infect. Inmnin. 63, 681-685. 202. Green, S. P, Hartland, E. L.. Robins Browne, R. M.. and Phillips. W. A. (1995). Role of YopH in the suppression of tyrosine phosphorylation and respiratory burst activity in murine macrophages infected with Yersinia enterocolitica. J. Leukoc. Biol. 57, 972-977. 203. Reisner, B. S., and Straley, S. C. (1992). Yersinia pestis YopM: Thrombin binding and overexpression. Infect. Immun. 60, 5242-5252. 204. Leung, K. Y, and Straley, S. C. (1989). The yopM gene of Yersinia pestis encodes a released protein having homology with the human platelet surface protein GPIb. J. Bacteriol. 171, 4623-4632. 205. Galyov, E. E., Hakansson, S., and Wolf-Watz, H. (1994). Characterization of the operon encoding the YpkA Ser/Thr protein kinase and the YopJ protein of Yersinia pseudotuberculosis. J. Bacteriol. 176, 4543-4548. 206. Ruckdeschel, K., Harb, S., Roggenkamp, A., Hornef, M., Zumbihl, R., Kohler, S., Heesemann, J., and Rouot, B. (1998). Yersinia enterocolitica impairs activation of transcription factor NF-KB: Involvement in the induction of programmed cell death and in the suppression of the macrophage TNF-oc production. J. Exp. Med. 187, 1069-1079. 207. Palmer, L. E., Hobbie, S., Galan, J. E., and Bliska, J. B. (1998). YopJ of Yersinia pseudotuberculosis is required for the inhibition of macrophage TNFa production and downregulation of the MAP kinases p38 and JNK. Mol. Microbiol. 11, 953-965. 208. Ruckdeschel, K., Machold, J., Roggenkamps, A., Schubert, S., Pierre, J., Zumbihl, R., Liautard, J. P., Heesemann, J., and Rouot, B. (1997). Yersinia enterocolitica promotes deactivation of macrophage mitogen-activated protein kinases extracellular signal-regulated kinase-1/2, p38, and c-Jun NH.-terminal kinase. / Biol. Chem. 272, 15920-15927. 209. Cornelis, G. R., and Wolf-Watz, H. (1997). The Yersinia Yop virulon: A bacterial system for subverting eukaryotic cells. Mol. Microbiol. 23, 861-867.
CHAPTER 7
Molecular Pathogenesis of Salmonellae CHRISTINA A. SCHERER SAMUEL I. MILLER
I. II. III. IV.
V.
VI.
VII.
VIII.
IX.
Introduction History Taxonomy Epidemiology and Clinical Disease A. Enteric Fever B. Gastroenteritis C. Bacteremia and Other Complications of Nontyphoidal Salmonellosis D. Chronic Carrier State Clinical Course and Basic Immunology A. Disease Course B. Inbred Mouse Enteric Fever Model C. Immunology of Salmonella Infections In Vitro Models of Salmonella Virulence A. Modeling Interactions with Macrophages B. Modeling Salmonella Interactions with Epithelial Cells Virulence Factors A. Major Transcriptional Regulators B. Factors Required for the Invasion of Epithelial Cells C. Factors Required for Systemic Infection D. Salmonella Toxins E. Virulence Plasmids Antibiotic-Resistant Salmonellae A. Multi-Antibiotic Resistant Salmonellae B. Development of New Antibiotics Salmonella-Based Wsicc'mes A. Development of More Effective Typhoid Vaccines B. Salmonellae as Multivalent Vaccine Strains C. Salmonella-Based Cancer Therapy References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
9^S
266 266 267 268 268 269 271 271 272 272 274 276 280 280 286 290 290 302 308 310 310 312 312 313 314 314 315 316 316
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/. Introduction The Salmonellae are Gram-negative bacilli that cause enteric (typhoid) fever and gastroenteritis. Over 2000 serotypes of Salmonella infect humans and virtually all known wild and domestic animals, including birds, reptiles, and insects. These infections result in significant morbidity and mortality. Typhoid fever, caused by the exclusively human pathogens S. typhi and S. paratyphi, was a major cause of death throughout the world in the nineteenth and early twentieth centuries. Due to improvements in sanitation, the incidence of typhoid fever has dropped dramatically in developed nations; however, it remains a significant problem in the developing world. In contrast, human gastroenteritis caused by nontyphoidal Salmonellae is globally increasing because of zoonotic contamination of food, whose distribution and processing is now more centralized. Antibiotic resistance of both typhoidal and nontyphoidal serotypes is increasing and has magnified the public health problem of Salmonellae infections. In this chapter, the epidemiology, general disease course, and pathogenesis of Salmonella infections will be discussed. Pathogenesis involves an interaction between both host and bacterial factors. Although our understanding of the diverse and resourceful Salmonellae is far from complete, significant progress has been made in elucidating molecular mechanisms of Salmonella pathogenesis. Much of the information on the molecular mechanisms of pathogenesis has been derived from the study of tissue culture and small animal infection models. These models may not always reflect natural infection, but they do reveal important mechanisms and principles of pathogenesis. After detailed discussion of host and bacterial factors that are implicated in disease pathogenesis, this chapter considers the potential for applying pathogenic knowledge to the development of new vaccines and antibiotics.
//. History Before the nineteenth century, human enteric or typhoid fever was often confused with typhus, a rickettsial disease. The two diseases were pathologically distinguished by P. Ch. A. Louis in France (1829) and William Jenner in the United States (1850). The typhoid bacillus was first isolated in 1884, when the German microbiologist Gaffkey obtained S. typhi from human spleens. S. choleraesuis was subsequently isolated from the intestines of pigs infected with hog cholera in 1885 by the veterinary pathologist Daniel Salmon, for whom the Salmonellae are named. In 1896, Pfeiffer and Kalle introduced the first heat-killed bacterial typhoid vaccine. In the same year, Widal determined that S. typhi could be agglutinated by convalescent patient sera. This technique provided a clinical tool for the identification of Salmonellae and was used extensively by Kauffman and White in the 1920s and 1930s for classification of over 2000 serotypes. The modem era of antibiotic treatment of typhoid fever was initiated in 1948, when Woodward and colleagues successfully treated the disease with chloramphenicol.
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267
A small animal model for typhoid fever was developed in the early 1950s, when animal experiments illustrated the susceptibility of inbred mouse strains to infection by S. typhimurium. Early experiments showed that bacterial strains auxotrophic for aromatic amino acids and purines were attenuated for virulence. The discovery by Zinder and Lederberg in 1952 that S. typhimurium could be transduced by P22 bacteriophage led to the development of S. typhimurium as a model system for genetic studies [1]. The combination of a convenient animal model and the powerful genetic techniques available for study of S. typhimurium has resulted in widespread study of this model system of Salmonellae pathogenesis.
///. Taxonomy The genus Salmonella is a member of the family Enterobacteriaceae. The Salmonellae are Gram-negative bacilli, approximately 2-3 x 0.4-0.6 |Lim in size. Molecular characterization of the genus has resulted in the grouping of Salmonellae into a single species, Salmonella choleraesuis. Alternatively, to prevent confusion with the serovar choleraesuis, the species is commonly referred to as Salmonella enterica. Based on genetic similarity and host range, the species has been divided into six subspecies (ssp.): choleraesuis (or enterica, Group 1), salamae (Group 2), arizonae (Group 3a), diarizonae (Group 3b), houtenae (Group 4), and indica (Group 6). S. bongori, which was initially categorized as subspecies 5, is generally considered a separate species due to its divergence from the other Salmonellae [2]. Group 1 (enterica) includes many of the serotypes pathogenic for humans, including S. typhi and S. typhimurium. As the correct taxonomic classification for Salmonella subspecies is rather unwieldy, the common species name that prevailed before reclassification of the species is still widely used. Thus, Salmonella choleraesuis ssp. choleraesuis (or Group 1), serovar typhi, is referred to by its common name, S. typhi. Salmonellae have been grouped into over 2200 serotypes [3] according to three major antigenic determinants: the flagellar H antigen, the somatic O antigen, and the Vi antigen. The Vi antigen, a homopolymer of yV-acetylgalactosaminouronic acid, is predominantly found on S. typhi, and is considered an identifying feature of this serotype. Nontyphoidal serogroups are identified based on differential agglutination by specific antibodies to O antigen. O antigen is a polysaccharide structural component of bacterial lipopolysaccharide (LPS), the major component of the outer leaflet of the bacterial membrane. Agglutination by antibodies specific for the various O antigens is used to group Salmonellae into 6 serogroups: A, B, CI, C2, D, and E. Although these groupings can help identify pathogenic bacteria as Salmonella, crossreactivity between groups does not allow for definitive identification of the serotype. For instance, both S. enteritidis and S. typhi express O antigens of Group D. Further classification of serotypes is based on the antigenicity of the flagellar H antigen and other more specific genetic and molecular methods, such as bacteriophage typing, pulsed field gel electrophoresis, and restriction fragment length polymorphism (RFLP) analysis.
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IV, Epidemiology and Clinical Disease The two major clinical syndromes that result from Salmonella infection are typhoid (enteric) fever and gastroenteritis. Focal infections of the vasculature (endocarditis), bone (osteomyelitis), and joints (arthritis) as well as a variety of other organs can occur but are much less common and are often associated with specific immune defects. Serotypes that exhibit strict human species specificity cause enteric fever. Other serotypes are associated with specific animal populations, such as S. dublin (cattle) and S. choleraesuis (swine), but also cause disease in humans. The two predominant agents of human gastroenteritis, S. typhimurium and S. enteritidis, infect a wide range of zoonotic hosts, including poultry, catde, sheep, pigs, horses, rodents and primates. A list of common serotypes and their characteristics is provided in Table I [4-13]. A. Enteric Fever Typhoid fever is a severe systemic illness characterized by high fever, gastrointestinal symptoms, including diarrhea and constipation, and sometimes a charac-
Table I
Major Salmonella Serotypes
Serotype
Primary host
Disease in humans
Year and references of recent outbreaks (source)
5". arizonae
Reptiles
Gastroenteritis
S. choleraesuis
Swine
Gastroenteritis, bacteremia
S. dublin
Cows
Gastroenteritis, bacteremia
France. 1995 (cheese) [4]
S. enteriditis
Wide range
Gastroenteritis
United States, 1994 (ice cream) [5]
S. gal Una rum
Chickens
None
S. pullorum
Ducks
S. hadar
Poultry
Only a significant problem in immunocompromised individuals
Gastroenteritis
Northeast United States, 1991 (pet ducklings) [6]
S. Hartford
Unknown
Gastroenteritis
Orlando, Florida, 1995 (orange juice) [7]
S. marina
Iguanas
Gastroenteritis
Sporadic among iguana owners, [8]
S. paratyphi
Humans
Enteric fever
NewDehli, India, 1996 [9]
S. Stanley
Unknown
Gastroenteritis
United States, 1995 (alfalfa sprouts) [10]
S. typhi
Humans
Enteric fever
Tajikistan, 1996-97 [11], Washington State, 1990 [12]
S. typhimurium
Wide range
Gastroenteritis
Nebraska, 1996 [13]
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MOLECULAR PATHOGENESIS OF SALMONELLAE
269
teristic rash (rose spots) from which bacteria can be cultured. S. typhi and S. paratyphi are the causative agents of human enteric fever, although S. paratyphi generally produces a milder form of the disease. Both serovars are solely human pathogens, and do not exist in animal reservoirs. Infection occurs on ingestion of food or water contaminated with human waste. Onset of disease usually occurs 5 to 21 days postinfection, depending on the inoculum and the immune status of the individual. Approximately 10% of immunocompetent individuals die of typhoid fever if proper antibiotic therapy is not initiated. Most individuals recover without therapy from what is a chronic and potentially relapsing systemic febrile illness. If left untreated, complications such as intestinal perforation and hemorrhage can occur in a small percentage (0.5-1.0%) of patients [14]. Until the early 1900s, insufficient purification of the water supply was a common cause of typhoid fever outbreaks. Improved sanitation practices, including water filtration, have reduced typhoid fever in developed nations to a disease of travelers and those infected from immigrant chronic carriers. However, typhoid fever remains a disease of significant morbidity and mortality in developing nations. Incidence rates are difficult to estimate because the disease is difficult to diagnose definitively without a microbiological laboratory. The World Health Organization (WHO) estimates that 16-17 million cases occur annually, resulting in about 600,000 deaths. Approximately 400 cases are reported in the United States per year [12]. These cases are usually attributed to recent travel in endemic areas or spread by infected food handlers. Mortality rates differ from region to region, but can be as high as 5-7% despite appropriate antibiotic treatment. In some highly endemic areas, particularly Pakistan and Indonesia, disease complications such as intestinal perforation and septic shock are frequendy observed, with concomitant mortality rates of up to 7% [11]. Large outbreaks, like the 50,000-60,000 cases reported annually in Tadjikistan between 1996 and 1997, continue to be problematic [11]. In addition, antibiotic-resistant strains have been isolated in most endemic areas, particularly in Southeast Asia, India, Pakistan, and the Middle East [15]. Recent outbreaks of multi-antibiotic-resistant typhoid have also been reported in Great Britain, and have been linked primarily to travel to endemic areas [16].
B.
Gastroenteritis
Human gastroenteritis is caused by many serotypes of Salmonella, the most common of which in the United States are S. enteritidis and S. typhimurium [17]. Like enteric fever caused by S. typhi and S. paratyphi, infection occurs via ingestion of contaminated food or water. However, cases of gastroenteritis are usually due to contamination of food with animal rather than human waste. Undercooked meat, seafood, and eggs are common causes of salmonellosis.
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
although the contamination of fresh produce with animal waste is also a significant problem [18]. Disease onset is approximately 8-48 hours after ingestion, and is characterized by nausea and vomiting; diarrhea, abdominal pain, and fever often follow. Infections usually resolve within 5 to 7 days without treatment, although some infections may result in bacteremia or other complications and require treatment. Except in these rare cases, antibiotic treatment for salmonellosis is usually not advised, as it has been observed to prolong the presence of bacteria in the stool [19]. Salmonella gastroenteritis is life threatening in a small percentage (1%) of total cases, particularly infants, the elderly, and immunocompromised individuals [3, 20]. Worldwide, the incidence of acute gastroenteritis due to Salmonella is estimated at 1.3 billion cases per year, resulting in about 3 million deaths [21]. In the United States, approximately 2-4 million cases of Salmonella-relaiQd gastroenteritis occur per year, causing about 500 deaths per year, mosdy in children or immunocompromised individuals (CDC data). In the 1990s, S. enteritidis surpassed S. typhimurium as the most prevalent source of gastroenteritis. In 1980 and 1995, S. enteritidis accounted for only 8 and 25% of total cases of salmonellosis, respectively. In 1998, however, the U.S. Department of Agriculture (USDA) attributed approximately 80% of current cases of salmonellosis to contaminated shell eggs, presumably with S. enteritidis [22]. The major increase of 5. enteritidis cases in humans is due to its ability to cause ovarian infections in egg-laying hens, thus contaminating the contents of intact shell eggs, which cannot be conveniendy pasteurized [17]. This serotype is easily transmitted vertically from breeding flocks to egg-laying hens, and is difficult to eliminate because of its ability to survive in rodents and in manure. Recendy, the emergence of antibiodc resistant S. typhimurium strains, particularly the penta-resistant strain DTI04 (which is also thought to be more virulent than sensitive strains), is troublesome [23, 24]. DT104 appears to have arisen in catde and has been associated with outbreaks related to contaminadon of beef. The increased occurrence of nontyphoidal salmonellosis observed in the industrialized world may in part be due to the centralization of food processing and the increased range of food distribution. Tradidonally, Salmonella outbreaks have been linked to focused gatherings such as weddings or other social events. However, large-scale distribudon of prepared food from industrial production facilities has resulted in much more diffuse patterns of infection that can be difficult to trace epidemiologically [18]. For instance, in 1985, contaminadon of milk in a large dairy resulted in approximately 250,000 cases of salmonellosis [25]. In 1994, a similar-scale outbreak occurred when ice-cream premix was delivered in contaminated tanker trucks [5]. Contamination of raw produce, such as alfalfa sprouts and cantaloupe, by manure or water sources has also led to wide-scale outbreaks of salmonellosis (reviewed in [18]). Thus, the potential for large-scale infecdon due to contaminadon at one site is a serious side-effect of industrialized and centralized food processing.
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271
C. Bacteremia and Other Complications of Nontyphoidal Salmonellosis Approximately 8% of untreated cases of salmonellosis result in bacteremia, a serious condition in which bacteria enter the bloodstream after passing through the intestinal barrier. Higher incidence rates of bacteremia have been documented in patients with immune deficiencies such as acquired immune deficiency syndrome (AIDS) [26], for whom recurrent bacteremias can be a significant problem. Patients with bacteremia and other complications should be treated with antibiotics, as potentially lethal infections, including endotoxic shock, can ensue. This is a particular problem in neonates and the elderly. Other rare complications of salmonellosis include focal infections of the bone (osteomyelitis), heart (endocarditis), joints, kidney, and arteries (for an in-depth review, see [27]). Nontyphoidal infection of the vasculature is a well-recognized clinical syndrome. Endovascular infections are often the result of abnormalities in the vasculature that disrupt blood flow; thus, bacteria are more likely to colonize developing aneurysms or arteriosclerotic areas of blood vessels. Children with sickle cell anemia are particularly susceptible to osteomyelitis, probably due to splenic dysfunction and actively dividing bone marrow. Reiter's syndrome, characterized by inflammation in joints, ocular tissues, and the urethral tract, is a postinfectious complication of nontyphoidal salmonellosis. It is more common in males with HLA-B27. This autoimmune disease occurs after a variety of gastrointestinal illnesses, including Salmonella, Shigella, and Yersinia infections. D.
Chronic Carrier State
Salmonella infections can be spread by chronic carriers, especially those who work in food-related industries. Nontyphoidal serotypes on average persist in the gastrointestinal tract, depending on the serotype, from 6 weeks to 3 months. However, persistence beyond 6 months is rare. Only about 0.1% of nontyphoidal Salmonella cases are shed in stool samples for periods exceeding 1 year, the clinical definition of chronic carriage. Approximately 2-5% of untreated typhoidal infections result in a chronic carrier state [28]. Bacteria can persist in several reservoirs, including the urinary tract and bile duct [29]. Factors contributing to the chronic carrier state have not been fully elucidated, although anatomical abnormalities such as gallstones and kidney stones have been implicated. Some individuals can harbor asymptomatic infections without their knowledge, often from a low initial inoculum (reviewed in [30]). The most famous chronic carrier is Mary Mallon, otherwise known as Typhoid Mary [31]. A New York City cook, she was held responsible for transmitting typhoid fever to at least 22 individuals (3 of whom died) between the years 1900 and 1907, although she herself never had any symptoms. After being apprehended in 1907 by public health officials, she was confined to an isolation cottage for 3 years. Although she was released with the stipulation that she never cook again, she was unable to keep this
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
promise, and is thought to be responsible for at least 25 more cases of typhoid fever at a Manhattan maternity hospital, where she was employed as a cook in 1915. She was subsequently confined to isolation until her death in 1938.
V. Clinical Course and Basic Immunology A. Disease Course Salmonellae are ingested via contaminated food or water. The minimum infectious dose (ID50) for human pathogenic serotypes in human volunteers is approximately 10^ organisms. However, single-food-source outbreaks indicate that lower doses can cause disease [32, 33]. Furthermore, 16,000 people were infected in a 1965 California outbreak by contaminated municipal water containing 10^ S. typhimurium per liter [34]. Some of the individuals who presented symptoms after small inoculi may have had impaired gastrointestinal defenses when compared with human volunteers. Conditions that increase susceptibility to lower inoculi include decreased stomach acidity (e.g., in infants, or due to achlorhydial disease or antacid ingestion), chronic gastrointestinal diseases such as inflammatory bowel disease, gastrointestinal surgery, and alteration of the intestinal flora by antibiotic administration [35, 36]. Immunocompetency also affects the ID50, as those with immune disorders, such as AIDS, are approximately 20 times more likely to contract symptomatic salmonellosis than the general population, even when presented with a low infectious dose (WHO). A schematic diagram of Salmonella infection is shown in Figure 1. Bacteria that survive the low pH of the stomach quickly colonize the lumen of the small intestine, where they predominandy localize to the Peyer's patches [37-39]. Passage of Salmonella through the intestinal epithelial barrier most likely occurs through specialized microfold (M) enterocytes, which overlay the Peyer's patches [40]. The primary function of M cells is to sample intestinal antigens (reviewed in [41]). This function is aided by the characteristic "pocket" containing lymphocytes and macrophages that extend from the basolateral layer into the M cell and allow for transcytosis of phagocytosed antigens and particles to the immune cells (see Fig. 1). Invasive Salmonellae enter M cells via a process that induces membrane ruffling and endocytosis and gain access to the underlying lymphatic tissue either by transcytosis across the M cell or by lysing the M cell [42-45]. Several groups utilize ligated ileal loops to model the interaction of Salmonella with intestinal epithelia. In mice, infection of ileal loops has shown that M cells are preferentially invaded in vivo and are killed when exposed to high numbers of bacteria [43]. However, the observed cytotoxicity may be a nonspecific effect of the high multiplicity of infection in a closed system, since M-cell cytotoxicity has not been documented during infections of mice, rabbits, and cows. Infection of cow ileal loops is a useful system because multiple loops can be infected in the same cow. In this model, infection with S. typhimurium or S. dublin does not result in preferential invasion of M cells, but does produce an
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inflammatory response that might represent an appropriate model for human gastroenteritis [46]. Salmonellae enter normally nonphagocytic epithelial cells by a process known as bacterial-mediated endocytosis, or BME. Early EM studies of guinea pig enterocytes showed that Salmonella first destroy the enterocyte microvilli and then induce the formation of large membrane ruffles in the enterocytes [47]. Bacteria are internalized when the ruffles fuse to form phagosomes. After a period of time, the enterocytes eventually repair the brush border and return to normal. The ability to invade epithelial cells may enable Salmonella to colonize and cross epithelial barriers with greater efficiency, as noninvasive mutants are somewhat attenuated on oral inoculation [48, 49]. Once bacteria have crossed through the intestinal epithelium, they enter phagocytic cells (macrophages) in the underlying lymph tissue. Salmonellae are able to survive and replicate within macrophages, a feature that is correlated with pathogenesis in the mouse typhoid model of S. typhimurium infection [50]. Little is known about the molecular mechanisms whereby Salmonella serovars cause typhoid fever versus gastroenteritis in humans. Typhoid fever is a severe systemic illness characterized by dissemination of bacteria from the intestinal submucosal tissue through the lymphatic system to organs rich in reticuloendothelial tissues such as the liver, spleen, lymph nodes, and bone marrow. Nontyphoidal strain infection, on the other hand, is limited to the intestine. Infection with nontyphoidal Salmonellae results in an influx of neutrophils characteristic of acute inflammation followed by self-limited inflammatory diarrhea. This is in contrast to typhoidal infections, where gastrointestinal symptoms are unusual and lymphoid tissues throughout the body, including the Peyer's patches, contain organisms and a mononuclear infiltrate of lymphocytes and macrophages characteristic of chronic inflammation.
B. Inbred Mouse Enteric Fever Model Infection of BALB/c and C57B1/6 mice with Salmonella typhimurium is widely used as a model system for human enteric fever. Oral infection of these inbred strains with 10,000-100,000 colony forming units (CPUs) of S. typhimurium results in a systemic infection that results in death. The dose required to cause death in 50% of infected mice (LD50) is less than 10 when bacteria are administered intravenously (iv) or through the peritoneum (ip). On oral inoculation, bacteria quickly cross the epithelial layer to colonize the Peyer's patches by infection of macrophages in this lymphatic tissue. Bacteria survive and replicate within macrophages, and usually migrate (via the lymphatic system) to the spleen and liver within 3 to 4 days. Figure 2 (see color plate) shows two intracellular bacteria found in a macrophage within the liver of an infected BALB/c mouse [51]. Bacterial replication within reticuloendothelial tissues and the influx of inflammatory cells (e.g., macrophages and neutrophils) results in hepato-
7.
MOLECULAR PATHOGENESIS OF SALMONELLAE
275
splenomegaly, focal necrosis, and bacteremia, followed by death of the mouse within 5-8 days. Sublethal doses of attenuated bacteria can provide protection against subsequent infection. Such sublethal infections or "vaccinations" are commonly used to investigate the acquired immune response to Salmonella infection. Because inbred mice are so susceptible to Salmonella infection, this model is a very specific, but not necessarily sensitive, system for identification of virulence factors. The majority of strains highly attenuated for virulence in the mouse model contain mutations in regulatory genes or secretory genes that influence expression of multiple factors. In addition, it must be kept in mind that these mice have a genetic defect that alters macrophage function (see the next paragraph), and hence mutants attenuated in this model system are more likely to have mutations that affect survival after macrophage phagocytosis. This association has been noted in genetic studies performed by Fields, Heffron, and coworkers in which in vitro survival within macrophages correlated with mouse pathogenesis [50]. The susceptibility of BALB/c and other inbred mice (C57B1/6, DBA/1, and BIO) to Salmonella infections has been linked to a single locus, Bcg/Ity/Lsh, which mediates innate resistance to Mycobacteria, Salmonellae, and Leishmania, respectively [52, 53]. Bcg^lIty^lLsh^ phenotypes are all linked to the same gene, Nrampl [52, 54], which encodes a macrophage-specific phosphoglycoprotein that is recruited to the phagosomal membrane during phagocytosis [55, 56]. The allele linked to susceptibility to all three bacteria contains a single inactivating mutation at amino acid 169 (Nrampl^^^^^"^). Nrampl is hypothesized to have a transport function that promotes killing of intravacuolar microorganisms, either directly or indirecdy. Other loci that mediate resistance to Salmonella infection have also been described. CBA/N mice, which succumb to Salmonella infection about 3 weeks after infection, carry a mutation in the X-linked Xis locus, which results in a defect in antibody formation. This defect can be rescued by reconstituting the mice with immunologically normal bone marrow cells [57]. Genes within the major histocompatibility complex (MHC) have also been shown to influence the ability to clear Salmonella from infected mice [58, 59]. The Lps locus governs the ability to respond to bacterial lipopolysaccharide (LPS, or endotoxin) [60]. Mice homozygous for the recessive Lps^ (defective) allele (i.e., C3H/HeJ mice) are hyporesponsive to LPS, and are therefore completely resistant to the toxic effects of LPS. At the same time, they are extremely susceptible to Salmonella infection [60-62]. Although this effect is seemingly paradoxical, it presumably occurs because the mice are unable to activate an appropriate innate immune response when challenged with Salmonella. In 1998, at least two more independent loci that mediate innate resistance to infection with S. typhimurium have been identified in MOLF/Ei mice, a wild-derived inbred strain that is also extremely sensitive to Salmonella infection [63]. Thus, susceptibility of mice to lethal S. typhimurium infection is governed by multiple loci. Although these specific
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defects have led to a better understanding of the immune responses necessary to combat Salmonella infections in mice, it is unclear to what extent these findings will apply to human disease susceptibility and host specificity.
C. Immunology of Salmonella Infections Little information is available about human and inbred murine antigen-specific immune responses to Salmonella infection. It is clear that protective immunity can be achieved against typhoidal Salmonellae infection in humans and a variety of experimental and farm animals. Immunity requires both humoral and cellular responses, but the most important protective antigens are not known. The role of cytotoxic T lymphocytes in human and mouse disease and immunity is also unknown. It is known that flagella, LPS, and Vi antigen are recognized by the immune system and that THl-type lymphocytes that recognize these antigens are promoted by Salmonellae infection. The role of innate immune responses in promoting specific immune responses as well as in disease pathogenesis is an important area for future research that may define important principles of bacterial interactions with immune cells.
1.
INNATE IMMUNE RESPONSE TO SALMONELLA INFECTION
Innate immunity to bacterial pathogens, including Salmonella, has received considerable attention in recent years. By definition, innate immune responses are host defense mechanisms that are not acquired on exposure to infectious agents. These mechanisms do not involve clonal lymphocyte proliferation in response to antigen and do not require a prolonged activation period. Innate immune mechanisms relevant to Salmonellae infection include gastric acidity, peristalsis, complement, opsonins, antimicrobial peptides, cilia, mucin, lysozyme, and the intestinal cell glycocalyx. Innate immune mechanisms relevant after invasion include phagocytosis, antimicrobial activity within phagosomes (antimicrobial peptides, nitrates, oxygen radicals, acidity), and secretion of chemokines and cytokines in response to signature bacterial molecules such as LPS. Chemokines and cytokines secreted by activated phagocytic cells elicit multiple responses, including recruitment of additional phagocytic cells to the site of infection (host inflammatory response) and modulation of the subsequent acquired immune response. A list of a number of innate immune mechanisms and corresponding bacterial properties that allow Salmonellae to evade or interact with the host immune system is provided in Table II. Innate immunity plays an important role in controlling Salmonella infections, both in mice and in humans. Macrophages are a crucial component of this response, as illustrated by the susceptibility of Ity^ mice to infection by S.
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Table II
Innate Immunity and Salmonella Infection Host immune mechanisms
Bacterial properties/factors
• Stomach acid • Extracellular matrix (glycocalyx) barrier • Antimicrobial peptides — lysozyme — defensins and other cAMP — complement
» Envelope modifications — outer membrane proteins, lipoproteins — lipid A modifications — transporters
•Phagocytosis — mannose binding protein — lectins — complement
»Vi antigen — inhibition of phagocytosis • Macropinocytosis/spacious phagosomes — unique phagosome trafficking • Intracellular survival
• Cytokine and chemokine production • Immune cell chemotaxis — PMN transmigration
• Stimulators of innate immunity — lipid A — flagellin — Type III secretion
typhimurium. Wild-type Salmonellae capable of surviving within macrophages will cause a lethal disease in Ity^ mice, but mutant strains defective in macrophage survival are avirulent in this model. A number of mouse studies have suggested that several inflammatory cytokines, including interleukin-12 (IL-12), interferongamma (IFN-y), and tumor necrosis factor-alpha (TNF-a), also play an important role in the early response to Salmonella infections. For instance, IFN-y-receptor null mice, which cannot respond to IFN-y, are hypersusceptible to infection with normally avirulent S. typhimurium [64]. Likewise, depletion of IL-12 with specific antibodies exacerbates Salmonella infections [65, 66]. Genetic evidence in humans also points to a role for IL-12 in the immune response to Salmonella, Individuals with a mutant form of the IL-12 (31-receptor (which is present on T cells and NK cells) suffer from severe and recurrent mycobacterial and Salmonella infections [67].
2.
ACTIVATION OF INNATE IMMUNE RESPONSES BY BACTERIAL FACTORS
The major bacterial factor responsible for stimulation of the innate immune system during Salmonella infection is lipopolysaccharide (LPS), or endotoxin (reviewed in [68]). Several different mechanisms for the recognition and response
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to endotoxin exist. First, binding of LPS by macrophage membranes sensitizes them to the action of IFN-y, which results in macrophage activation independent of T-cell stimulation. Second, opsonization of bacteria with LPS-binding protein (LBP), a serum glycoprotein that binds to LPS with high affinity, mediates the adhesion of coated bacteria to macrophages. A third mechanism for endotoxin recognition takes place when LBP interacts with soluble LPS in the serum. This complex binds to the CD 14 receptor on macrophages and monocytes [69] and results in secretion of TNF-a by macrophages [70]. This process is thought to require a coreceptor to activate the signal transduction cascade necessary for TNF-a secretion. A candidate for this coreceptor, the Toll-like receptor-2, has been identified in humans [71]. LBP null mice exhibit the same phenotype as LPS^ mice, in that they are hyporesponsive to LPS [72, 73]. Localized secretion of TNF-a is an important mediator of the host inflammatory response, as it can induce expression of E-selectin on endothelial cells. E-selectin promotes the adherence of neutrophils to endothelial cells and their subsequent migration to sites of infection [74]. Although small amounts of LPS promote a beneficial host inflammatory response, large amounts of serum LPS (in bacteremic individuals, for instance) can induce overproduction of inflammatory cytokines, which results in serious tissue damage and septic (or endotoxic) shock [75]. Although LPS is the major inflammatory agent produced by Salmonella, other bacterial factors have been shown to induce expression of inflammatory molecules in vitro. Expression and production of the flagellar filament protein FliC (either monomers or filaments), for instance, correlates with the induction of TNF-a expression by infected human monocytes [76]. The alternate filament protein FljB induces TNF-a expression to a lesser extent. The significance of these findings has not yet been determined, but they indicate that unique protein structures, such as flagellin, may be recognized by specific innate immune receptors and contribute to inflammatory responses.
3.
ANTIMICROBIAL PEPTIDES
Antimicrobial peptides constitute an important component of innate immune defense against bacterial infection, including Salmonella (reviewed in [77]). Cationic antimicrobial peptides (CAMPs) have been found in a wide variety of animal tissues, including neutrophil granules, phagosomes, mucosal epithelia, and skin. These peptides are amphipathic, cationic molecules that bind to the bacterial surface, at least in part through electrostatic interactions with the negative charges of LPS. Once bound, the peptides permeabilize the bacterial membrane, leading to cell death. Inducible resistance to cationic antimicrobial peptides by Salmonella is required for virulence in the BALB/c mouse model, indicating that wild-type
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Salmonella can at least partially avoid the action of these potent bactericidal compounds [78-81]. Resistance to a variety of other membrane-active molecules, including complement, is likely important to Salmonellae pathogenesis. Such molecules are important both at mucosal surfaces as well as within phagosomes. Which of the myriad of known and unknown membrane-active compounds Salmonellae must resist within host tissues is unknown. It seems likely that the ability to regulate the permeability of the outer membrane to such compounds is an important virulence property, as discussed in more detail in section VILA.
4.
ACQUIRED IMMUNE RESPONSE
Although the innate immune response is crucial for containing Salmonella infections early in disease, protection against subsequent infection, and the ability to cure late or latent infection is related to the development of an antigen-specific (or adaptive) immune response. Both the humoral and cell-mediated arms of the immune response are necessary, as both immune serum and immune T cells are required for protection of susceptible mice [82]. The requirement of T cells for the development of acquired immunity has been further demonstrated in experiments with nude mice or mice artificially depleted of T cells. Infection of such mice results in increased colonization of the liver and spleen, the inability to clear Salmonella infections, and eventual death [83]. Like most intracellular pathogens. Salmonella induces primarily an inflammatory helper T-cell response, designated the THI response. Development of naive CD4-positive T cells into THI cells is induced by secretion of IL-12 by infected macrophages and IFN-y by NK cells during the early (or acute) phase of infection. THI cells help eradicate bacterial infections by activating the microbicidal properties of uninfected macrophages and by inducing production of circulating antibodies that can opsonize extracellular bacteria to maximize phagocytosis by activated macrophages. Vaccination of mice with attenuated Salmonella-Qxpressing heterologous antigens also can result in induction of cytotoxic (008"^) T cells (CTLs) that recognize these heterologous antigens. It is unknown whether CTLs can be induced to native Salmonella proteins. Litde is known about the antigen specificity of immune responses generated during Salmonella infections. However, CD4 helper T cells have been shown to recognize specific flagellin epitopes [84]. It has been known for many years that humans and animals produce antibodies to the major cell surface components: LPS, Vi polysaccharide, and flagella. Neither the diversity of antigens nor the role of specific antigens in immunity to salmonellosis has been defined. Although typhoidal Salmonellae are primarily intracellular pathogens, humoral immunity does play a role, as evidenced by the fact that CBA/N mice, which are unable to mount a normal antibody response to infection, eventually succumb to
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Salmonella infections [57]. This indicates that extracellular Salmonella can be targeted by antibodies, perhaps preventing spread or initial invasion through the intestinal barrier [85]. The production of secretory IgA antibodies is an important component of the humoral response. These antibodies have been shown to play a protective role against Salmonella infection [86], perhaps by contributing to the development of mucosal immunity. The predominant antigen recognized by Salmonella-induced sera is LPS; however, many other antigens, including outer membrane proteins (Omps), are recognized by immune sera and appear to be important in developing protective immunity [87, 88].
VL In Vitro Models o/Salmonella Virulence A. Modeling Interactions with Macrophages Infection of cultured macrophages is widely used as a model pathogenic system. Such studies have been performed using both primary macrophages and immortalized cell lines from a variety of species (including mouse and human) and using a variety of Salmonella serotypes. Most studies use S. typhimuriiim to infect the susceptible BALB/c mouse-derived J774 and RAW264.7 cell lines, which contain an Nrampl mutation, or primary bone marrow and peritoneal macrophages from BALB/c mice. S. typhi infection has been modeled using primary human peripheral blood monocytes as well as cell lines from a variety of human cells. The standard assay to calculate the ability to survive within macrophages is the gentamicin protection assay. After incubation of cultured cells with bacteria, gentamicin sulfate is added to the extracellular medium. As the eukaryotic membrane is impermeable to gentamicin, internalized bacteria are protected from the antibiotic, whereas extracellular bacteria are killed. The number of internalized bacteria is calculated by releasing intracellular bacteria with a nonionic detergent such as Triton X-100 and plating dilutions of the lysate onto appropriate plates. Strains capable of surviving and replicating within macrophages will show a small increase in the number of intracellular bacteria over a period of 3-24 hours. These numbers must be interpreted with caution, as Salmonella can also exert a cytotoxic effect on infected cells, as will be discussed in more detail below. Despite this concern, this assay was used in a seminal study to demonstrate that survival within macrophages was a useful screening technique that predicted the mouse virulence phenotype [50]. The ability to survive within macrophages depends on the serotype of Salmonella and the source of macrophage, indicating that macrophages may be a
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component of Salmonellae host specificity. For example, S. typhimurium survives better in mouse splenic macrophages than in peritoneal macrophages [89]. In addition, S. typhi is able to survive in human monocytes, but does not survive as well within murine macrophages [90, 91 ]. Specific bacterial mutations that reduce survival within macrophages will be discussed in a later section.
1.
MACROPINOCYTOSIS AND SPACIOUS PHAGOSOME (SP) FORMATION
Interaction of cultured macrophages with S. typhimurium results in immediate formation of large membrane ruffles that are dependent on actin polymerization. Bacteria are taken up in vacuoles formed from these membrane ruffles. As these vacuoles resemble the macropinosomes formed on induction of membrane ruffling in cells stimulated with growth factors, the process of Salmonella uptake by macrophages has also been termed macropinocytosis [92]. Phagosomes containing wild-type S. typhimurium have been termed "spacious phagosomes" because of their unusually large size (approximately 2-6 |im) and because bacteria appear to be swimming freely within them. An example of spacious phagosomes formed by infected bone-marrow-derived macrophages is shown in Figure 3. Unlike phagosomes containing dead bacteria or non-Salmonellae, spacious phagosomes do not shrink immediately after internalization, but maintain their size for approximately 4-6 hours, after which they decrease in size. The ability to form and/or maintain spacious phagosomes in cultured macrophages correlates
Fig. 3 Spacious phagosomes formed by mouse bone-marrow-derived macrophages. Macrophages were infected with wild-type Salmonella typhimurium and filmed using a Metamorph video imaging system (lOOx phase objective). Several large (spacious) phagosomes are apparent, including one containing a bacterium (arrow). Arrowhead shows site of membrane ruffling. Image reprinted through the courtesy of C. Alpuche-Aranda.
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with virulence in the BALB/c mouse model [90]. For instance, infection of BALB/c bone-marrow-derived macrophages with serotypes that are not pathogenic for mice, including S. typhi, S. arizonae, and S. pullorum, results in formation of fewer spacious phagosomes that appear to shrink faster than S. typhimurium-conimmng phagosomes. 2.
TRAFFICKING OF SALMONELLA-CONTAINING VACUOLES (SCVS)
Pathogenic microorganisms employ various strategies to survive within the macrophage phagosome, an intracellular membrane-bound compartment containing a variety of antimicrobial factors. Strategies utilized by bacteria to combat this host defense mechanism include: (1) escape from the phagosome to the host cell cytoplasm {Shigella and Listeria; reviewed in [93]); (2) inhibition of phagolysosomal fusion (Legionella); and (3) survival in a novel phagosome that fuses with the lysosomal compartment (Mycobacteria; reviewed in [94]). The strategies that Salmonellae use to survive within macrophages have not yet been fully elucidated, but results to date suggest two general mechanisms: (1) that Salmonellae traffic to the phagolysosome and synthesize factors, including those that remodel the bacterial surface, that promote resistance to microbicidal activities within the phagosome; or (2) that Salmonellae alter host cell processes that modify the SCVs and promote bacterial survival. Though both factors may contribute to survival of Salmonellae, conflicting reports exist in the literature on whether the trafficking of the SCVs to the lysosomal compartment is altered. At least two different phases of SCVs have been observed: initial spacious phagosomes, which appear to fuse with the lysosomal compartment within 15 minutes and which, at least in some cases, take a longer time to acidify due to their large volume, and a later (V2 to 4 hours) maturing acidified phagosome in which the phagosomal membrane is tightly adherent to the bacteria. Although some studies have addressed the composition of later phagosomes, the majority of studies have focused on the biochemical properties of early (spacious) phagosomes.
3.
ACIDIFICATION OF SALMONELLA-CONTAINING VACUOLES
Two studies have utilized modem methods to measure the pH of SCVs (see [95] for a review of methodology). Using fluorescein-isothiocyanate-conjugated dextran (FITC-Dx) as a probe, one group measured the pH of individual spacious phagosomes formed in bone-marrow-derived macrophages. They observed that within the first 2 hours of formation the mean pH of the SPs was 5.5. The mean pH of SPs that persisted for 4-6 hours subsequently decreased to approximately 4.9 [96]. In contrast, phagosomes containing killed bacteria (which were taken up
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by receptor-mediated phagocytosis) rapidly acidified to a pH of less than 4.9 within the first 2 hours. This study indicated that acidification of the SPs during the time of phagolysosomal fusion might be delayed due to the presence of a large volume of fluid of neutral pH. Another study of this type used a different pH-sensitive probe, DM-NERF dextran, to measure the pH of SCVs [97]. In this study, the pH of SCVs was observed to decrease more rapidly than previously observed. The pH of SCVs formed within bone-marrow-derived macrophages decreased to approximately 4.5 in 50 minutes, whereas phagosomes formed within RAW 267.4 macrophages dropped to an approximate pH of 4.0 within 10-20 minutes. Phagosomes containing latex beads or killed bacteria acidified to a pH of 4.0 within 15 min. Despite the conflicting data, the results of Alpuche-Aranda et al (see [96]) are consistent with delayed acidification of some of the larger SPs formed. The discrepancies described above could be the result of differences in experimental protocol. First, it is possible that the measurement of pH may depend on the population of phagosomes observed. In the first study, the pH of the most spacious phagosomes was reported. Because of their large volume, it is likely that acidification of this population of phagosomes is retarded and some vacuoles measured had neutral pH. As the number of spacious phagosomes drops significantly within the first 2 hours postinfection [92], continued observation of spacious phagosomes might bias the measurement toward a higher pH. Second, the composition of the bacterial inoculum may have played a role in determining the fate of the SCVs. The first group used opsonized stationary-phase bacteria to infect macrophages, whereas the second group used log-phase bacteria grown under limiting oxygen conditions. As bacterial growth state and oxygen limitation can affect infection of epithelial cells [98, 99] and expression of various virulence genes [100], it is reasonable to hypothesize that phagosome development in the macrophage might be similarly affected by alterations in virulence gene expression. In order to resolve the conflicting data, infection of macrophages and measurement of intravacuolar pH will have to be normalized so that these results can be direcdy compared.
4.
PHAGOLYSOSOMAL FUSION
Phagosome maturation is normally accomplished by fusion with endosomal and lysosomal compartments. This results in acidification of the vacuole and digestion of phagocytosed particles by lysosomal enzymes. Phagolysosomal fusion is usually monitored by either the fusion of phagosomes with fluid phase markers from the lysosomal compartment (discussed in [95]) or by colocalization of phagosomes with endosomal- or lysosomal-specific markers (reviewed in [94]). Fusion with the early endosome results in delivery of the GTPase Rab 5 to the phagosome, whereas fusion with the late endosome is characterized by the presence of the mannose 6 phosphate receptor and Rab 7. Lysosomal markers
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include the lysosomal-associate membrane proteins (LAMPs), lysosomal-associated proteins (LAPs), and cathepsin L. These markers are not entirely restricted to lysosomes, however, as LAMPs can also be found in the late endosome compartment. At least three groups have reported that SCVs display incomplete fusion with lysosomes [97, 101, 102]. One of these studies utilized modem immunofluorescence techniques to determine the extent of phagolysosomal fusion [103]. On infection of both primary and cultured macrophages with wild-type S. typhimurium, the lysosomal markers LAMP-1 and LAP were detected in over 90% of SCVs 30 minutes after infection. The mannose-6-phosphate receptor and cathepsin L were only detected in a small proportion of phagosomes, even after a 10-hour incubation. In contrast, 80% of vacuoles containing latex beads colocalized with the latter two markers. In addition, SCVs appeared inaccessible to subsequently loaded endocytic markers, including rhodamine-labeled transferrin (which should traffic to early endosomes) and the fluid phase lysosomal marker fluorescein dextran. Conversely, another group, using similar techniques, found that, in addition to LAMP-1 and LAP, most SCVs also contained cathepsin L within 20 minutes of phagocytosis [104]. Observation of SCVs in this study was enhanced by pulse-chase labeling of phagosomes with fluorescent dextran during infection so the exact age of the vacuole could be determined. In addition, this group showed that SCVs readily fused with a fluid-phase lysosomal marker, Texas Red-ovalbumin, which was preloaded into infected macrophages. It is clear that SCVs have a novel morphology on formation. The delayed shrinkage of these vacuoles is also novel compared to growth-factor-induced macropinosomes. Given the above results, it seems likely that SCVs can undergo differential trafficking. Factors affecting trafficking might include bacterial growth conditions (which promote expression of different virulence factors) or the number of bacteria in contact with macrophages or within a vacuole, which may effect initiation of apoptosis in macrophages. Given the results of Oh et al. [104], it is clear that SCVs can rapidly fuse with the fluid phase of the lysosomal compartment and that bacteria can survive in this environment. However, since not all endocytic and lysosomal markers are known or have been tested, and given the morphologic novelty of the SPs, it would seem likely that SCVs have some biochemical novelty. The uniqueness of the more mature, acidified postspacious SCV remains to be better defined. Discovery of this novelty and the biochemical mechanism by which Salmonellae induce alterations of the phagosome are required to resolve these issues.
5.
CYTOTOXICITY OF SALMONELLAE ON MACROPHAGES
Several groups have observed that Salmonella infection of macrophages results in cytotoxicity, either in the form of necrosis (generalized cell death) or apoptosis (programmed cell death). Apoptotic death is characterized by membrane blebbing.
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cell shrinkage, and chromatin condensation and fragmentation. Chromatin fragmentation is usually visualized using the fluorescent-based TUNEL assay, in which labeled dUTP is incorporated into nicked DNA by the enzyme terminal deoxynucleotide transferase (TdT). Necrotic death, on the other hand, is characterized by cytoplasmic swelling, membrane disruption, the disappearance of nuclear chromatin, and eventual lysis of the cell. Necrotic death usually produces an inflammatory response in the area, whereas ingestion of apoptotic cell fragments by neighboring cells prevents this response. Cytotoxicity of Salmonella on macrophages has been observed under several different situations. In one situation, infection of macrophages with relatively low numbers of bacteria- (a multiplicity of infection [moi] of 10-20 bacteria per cell) induced apoptosis within 45 minutes to 2 hours [105, 106]. Initiation of apoptosis required a functional type III secretion system (or TTSS) from Salmonella pathogenicity island 1, a specialized secretion apparatus that mediates the translocation of bacterial effector proteins into the host cell cytosol (described in §VII.B). Although both macrophage cell lines and bone-marrow-derived macrophages underwent apoptosis in these studies, primary macrophages appear to be much more sensitive to killing than the cell lines [106]. The requirement for bacterial entry is uncertain at this time, as there are conflicting results as to the effect of Cytochalasin D on induction of apoptosis. Apoptosis of cultured macrophages has also been observed when cells are infected with a high moi of stationary phase bacteria [107]. In this case, apoptosis is not apparent until 10-12 hours after infection. Induction of apoptosis in this system does not require the TTSS, but does require genes in the ompR/envZ regulon. Infection of macrophages with a high moi of 5". typhimurium has also been shown to induce necrotic death of these cells [106]. The differential induction of apoptosis and necrosis in culture may represent similar situations during in vivo infections. For instance, the type III secretion-dependent induction of apoptosis by small numbers of bacteria might mimic the situation during early stages of infection. Conversely, necrotic and apoptotic death in response to large numbers of bacteria might be indicative of late-stage infections, when the reticuloendothelial tissues contain high bacterial loads and undergo major inflammatory and necrotic responses. Nevertheless, it is unclear if activation of apoptotic pathways in cultured cells is physiologically significant. For instance, although induction of apoptosis might help bacteria escape from macrophages in the Peyer's patch, the TTSS associated with apoptosis in vitro is not required for pathogenesis in vivo. In one study, confocal microscopy of livers and spleens isolated from infected BALB/c mice has shown that, not only does S. typhimurium reside mostly within macrophages in vivo, but also that many of these macrophages display the hallmarks of apoptotic death [51]. Interestingly, a number of apoptotic macrophages did not actually contain bacteria, indicating that 5. typhimurium can induce apoptosis both direcdy and indirectly. Thus, it is possible that apoptotic macrophages are not responding to infection with Salmo-
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nella per se, but rather to the secretion of cytokines and chemokines by infected (and dying) macrophages as a result of the acute inflammation of these tissues.
B. Modeling Salmonella Interactions with Epithelial Cells 1.
BACTERIAL-MEDIATED ENDOCYTOSIS
Although the main portal of entry for Salmonella into the gut epithelia is thought to be M cells, Salmonellae are also capable of entering normally nonphagocytic epithelial cells. A small percentage of these bacteria also transcytose from the apical to the basolateral surface, effectively crossing the epithelial barrier. A number of different cultured cell lines have been used to study this process, including nonpolarized epithelial cell lines such as Hela, Hep-2, and Henle-407 cells, and the polarized cell lines Caco2 and T84. As with macrophages, Salmonella induces membrane ruffling in epithelial cells that is morphologically similar to membrane ruffling induced by growth factors [108-110]. However, these ruffles are localized to the site of bacteria-cell interaction. Bacteria are taken up within vacuoles formed from the ruffles, a process known as bacterial-mediated endocytosis (BME). A scanning electron micrograph of Salmonella interacting with membrane ruffles is shown in Figure 4. Bacterial-mediated endocytosis is accompanied by major cytoskeletal rearrangements, in particular the accumulation of actin filaments around the bacteria [111]. Actin reorganization is required for bacterial entry, as the addition of cytochalasin B and cytochalasin D, actin filament inhibitors, greatly decreases the proportion of internalized bacteria. In addition to actin, a number of other cytoskeletal components, including a-actinin, tropomyosin, tubulin, and vincuHn, are observed around the bacterial vacuole. Interestingly, despite the presence of tubulin around the vacuole, microtubule inhibitors have no effect on bacterial invasion [HI, 112]. Invasion-defective mutants do not induce membrane ruffling or BME. Induction of membrane ruffling appears to be sufficient to allow bacterial entry, as passive entry of noninvasive bacteria has been induced by addition of either growth factors (such as EGF) or wild-type bacteria [108, 113]. A number of bacterial factors are required for epithelial cell invasion. Most invasion-defective strains contain mutations in the type III secretion system located in Salmonella pathogenicity island 1, which will be discussed in detail later in this chapter. Bacterial-mediated endocytosis in epithelial cells results in the formation of spacious phagosomes similar to macropinosomes in the host cell [114]. Salmonella are capable of replicating within these vacuoles, a feature which in some cases correlated with disease in the inbred mouse enteric fever model [115]. Replication within epithelial cells does not require the same gene products as survival within macrophages, as mutants that cannot replicate within epithelial
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Fig. 4 Scanning electron micrograph of polarized MDCK cells infected with wild-type Salmonella typhimurium. Two bacteria are attached to a large membrane ruffle; one bacterium appears to be in the process of internalization (white arrow). Note that the other adherent bacteria exhibit multiple surface appendages. Magnification 8400x. Photo reprinted through the courtesy of M. A. Clark, T. A. Booth, and M. A. Jepson.
cells can survive within macrophages. Like the phagosomes formed in macrophages, Salmonella-conidiXmng spacious vacuoles persist within epithelial cells and are still visible 2 hours after invasion [114]. The vacuoles appear to be mildly acidic [116], and contain some markers of the lysosomal compartment. However, fusion with lysosomes appears to be incomplete, as most vacuoles contain the lysosomal membrane glycoproteins LAMP-2 and LAP, but do not contain the mannose 6-phosphate receptor and associated lysosomal hydrolytic enzymes [117]. In addition, fusion with fluid lysosomal tracers appears to be minimal. Interestingly, BME of Salmonella also induces the formation of a filamentous network of lysosomal glycoprotein-containing tubular lysosomes that extend from Salmonella-conidiming vacuoles [118]. The formation of this network requires bacterial protein synthesis, vacuole acidification, and an intact microtubule network. Unlike Listeria and Yersinia, Salmonella enters cells by induction of macropinocytosis rather than receptor-mediated endocytosis. Therefore, inhibitors of phosphatidylinositol (PI)-3-kinase do not prevent Salmonella invasion [119]. The
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molecular mechanism by which Salmonella induces membrane ruffling and BME in epithelial cells has not yet been fully elucidated. Induction of membrane ruffling is an extremely fast event (within seconds), and is likely to take advantage of cellular signal transduction pathways that can be immediately activated. Although early experiments suggested a role for tyrosine phosphorylation of the epidermal growth-factor receptor (EGF-R) during invasion of Henle-407 cells by Salmonella [113], subsequent experiments by other groups were unable to reproduce this result, and entrance does not require host cell tyrosine phosphorylation [120]. Investigation of the activation of membrane ruffling by Salmonella has focused on the small GTP-binding proteins that modulate actin rearrangements in eukaryotic cells, including Ras, Rac, Rho, and CDC42. Expression of constitutively active alleles of these proteins result in various actin-based cytoskeletal changes: expression of activated Rac induces the formation of lamellipodia, expression of activated Rho results in the formation of stress fibers, and activated CDC42 induces filopodia formation (reviewed in [121]). Although uptake of Shigella flexneri into epithelial cells has been shown to require Rho [122], Salmonella enters epithelial cells by a Rho-independent process [110] that requires CDC42 and, to a lesser extent, Rac [123]. Expression of a dominant negative CDC42 mutant interferes with bacterial invasion, whereas expression of a constitutively active CDC42 allele facilitates internalization of noninvasive strains. The mechanism by which Salmonella activates CDC42 and Rac is not yet known, but it is likely that they are activated by translocated bacterial effector proteins, as at least one bacterial protein (SopE) has been shown to interact with CDC42 and Rho and induce ruffling when expressed at high levels in mammalian cells [124].
2.
CELLULAR RESPONSES TO SALMONELLA INFECTION
Invasion of epithelial cells by Salmonella results in initiation of a complex cellular signal transduction cascade. One of the first documented cellular responses was an apparent increase in free intracellular calcium (Ca^"^) [125]. The ability to mobilize Ca^"*^ is necessary for the process of invasion, as Ca^"^ channel antagonists such as lanthanum or cadmium chloride block invasion. Interestingly, the Ca^"^ response is not blocked by cytochalasin D, and therefore does not appear to require bacterial entry. Ca^"^ fluxes have never been documented in individual infected cells, so it is unclear if the observed changes are a direct effect of bacteria-cell communication or if they are due to decreased membrane integrity as a result of bacterial protein translocation or cytotoxicity. In addition to Ca^"^ fluxes, increased levels of other second messenger molecules, including phospholipase A and arachidonic acid metabolites, appear to contribute to invasion [125].
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Studies in the late 1990s have shown that three members of the mitogen-activated protein (MAP) kinase family—Jun kinase (JNK), p38, and ERK—are activated on internalization of wild type, but not invasion-defective, Salmonella [126]. These kinases appear to activate a signaling cascade that eventually results in the expression and secretion of IL-8, an inflammatory chemokine, as a specific inhibitor of p38 MAP kinase prevents IL-8 secretion. Activation of IL-8 expression is mediated by the transcription factors AP-1 and NF-KB, and is in part due to degradation of the NF-KB inhibitor, I-KB, and an increase in intranuclear c-Jun concentrations. These effects are not immediate; rather, they occur approximately 2 hours after infection. Activation of this signaling cascade by Salmonella requires a functional type III secretion apparatus and translocase, indicating that these effects might be mediated by translocated effector proteins. However, there is a high degree of crosstalk between mammalian signal transduction molecules, and paths of activation are quite difficult to elucidate. Cytokines other than IL-8 are also secreted by infected epithelial cells. In vitro studies have documented secretion of IL-6 by human small intestinal epithelial cells (lECs) infected with S. typhi [127]. The induction of IL-6 secretion is cytochalasin D independent, indicating that only bacterial adherence is required.
3.
INTERACTIONS WITH POLARIZED EPITHELIAL CELLS IN VITRO
Polarized tissue culture cells have been used to study the interaction of Salmonella with epithelial cells under slightly more physiological conditions. When grown on permeable filters, some cell lines will form polarized epithelial monolayers. Salmonellae can enter polarized epithelial cells from the apical side via formation of large membrane ruffles. Although bacterial invasion generally does not affect monolayer integrity [128], infection of polarized epithelial cells with high doses of bacteria can result in a decrease in transepithelial electrical resistance, which might be the result of major remodeling of the cytoskeleton at intercellular junctions at the apical (but not basolateral) pole [129]. In addition. Salmonella typhimurium does not stimulate chloride secretion at the apical pole of polarized T84 cells, indicating that it is unlikely that human gastroenteritis is a result of Salmonella secretion of a cholera-toxin-like activity [128]. Association of Salmonella with the apical side of T84 monolayers results in basolateral secretion of IL-8 and induction of neutrophil transmigration across the monolayer, both hallmarks of the host inflammatory response [128]. Basolateral secretion of IL-8 directs neutrophils to a subepithelial (basolateral) position, but transmigration of neutrophils across the monolayer appears to occur in response to a novel cytokine signal [130]. The induction of IL-8 secretion and transepithelial migration requires bacterial invasion and subsequent protein synthesis in both bacteria and cells. This in vitro system may be a model for human gastroenteritis, as the ability to induce an inflammatory response in vitro
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correlates with the abihty of strains to cause human gastroenteritis [131]. Serotypes such as S. typhimurium that cause gastroenteritis in humans also induce transepithelial migration in cultured T84 cells. Conversely, host-restricted serotypes that do not cause gastroenteritis, including S. typhi, S. pullorum, and S. arizonae, cannot induce neutrophil migration in vitro.
VIL Virulence Factors In this section we will discuss a number of factors that contribute to pathogenicity in in vitro and/or in vivo assays. A list of many proposed virulence factors and their in vitro and in vivo phenotypes is also provided in Table III. The interaction of Salmonellae with their hosts is complex and requires many different bacterial factors. Approximately 4% of the Salmonella genome has been estimated to be required for virulence in the BALB/c mouse model system [132]. A number of virulence factors map to regions of the genome known as Salmonella pathogenicity islands (SPIs). Pathogenicity islands contain large segments of DNA that appear to have been acquired by horizontal transmission from an exogenous source, as the ratio of GC to AT basepairs in these regions differs from that of the rest of the Salmonella chromosome [133]. To date, five such islands have been described [134-139], at least two of which are specific for Salmonella species.
A. Major Transcriptional Regulators 1.
THE PHOP/PHOQ REGULON
One of the best-characterized transcriptional regulons required for Salmonella pathogenesis is made up of the two-component regulatory system [187] PhoPand PhoQ, which controls expression of more than 40 genes [159, 161]. PhoP/PhoQ is required for virulence in mice and humans, survival within macrophages, growth on succinate as a sole carbon source, and growth in the presence of magnesium limitation. PhoQ is a sensor histidine kinase [188] that phosphorylates PhoP, a response regulator, in response to environmental conditions. PhoQ activity is repressed by the divalent cations magnesium and calcium. PhoP-P04 activates expression of a set of genes arbitrarily designated pags (for PhoP-activated genes), which promote Salmonella survival within host tissues. Proteins encoded by ^pag include a nonspecific acid phosphatase [189], cation transporters, outer membrane
7.
MOLECULAR PATHOGENESIS OF SALMONELLAE
291
proteins, and enzymes important for lipopolysaccharide modification. In addition, PhoP-P04 activates expression of the pmrCAB (also known as pagBpmrAB) operon encoding another two-component regulatory system that is activated in response to an acidic environment [190, 191]. PhoP-P04 represses transcription of another set of genes found on SPI1 (designated /7rgs, for PhoP-repressed genes) required for epithelial cell invasion and spacious phagosome formation, including hilA, which encodes a transcriptional regulator, and the prgHIJKorgA operon, which encodes components of a type III secretion system [48, 92, 171, 192]. Regulation and function of SPIl will be discussed in the following section and in section VLB. In addition to regulating a large number of genes, iht phoPQ operon is autoregulated, as full expression requires both PhoP and PhoQ [193]. A summary of the known components of the PhoP regulon is shown in Figure 5. Expression of PhoP-activated genes is maximally induced within nonspacious acidified phagosomes as much as several hours after phagocytosis [96, 194]. Although the in vivo signals for PhoQ activation are not fully defined, activation can be induced in vitro by low pH or growth in media containing low (micromolar) concentrations of the divalent cations Mg^"^ and Ca^"*" [96, 195, 196]. PhoQ contains distinct binding sites for Mg^+ and Ca^^, and is maximally repressed in the presence of both cations [197, 198]. Therefore, the best-defined signal in vitro is depletion of the divalent cations Mg^"*" and Ca^"^. This has led to the hypothesis that PhoQ is also activated by limiting concentration of divalent cations in vivo, and that expression of a subset of pags in response to low pH is mediated by PmrA and PmrB. Mild acidic growth conditions have been shown to promote transcription of the subset of PhoP-activated genes that are also PmrA-dependent [199]; in addition, transcriptional activation of psiD (also called pmrC or pagB) by mild acidification is independent of the PhoQ protein [196]. However, a recent report indicates that the expression of several proteins induced upon acid shock is dependent on PhoP/PhoQ but not PmrA [195]. Though posttranscriptional effects cannot be ruled out, this suggests that PhoQ or some unidentified target of PhoP can respond to low pH. In addition, as pH can affect the effective concentration of cations in solution, it is difficult to definitively rule out the possibility that PhoQ is affected by pH in vivo. Since the complex cationic milieu of the phagosome, other than pH, has not been directly measured, this question cannot be completely resolved. Two classes of mutations within phoPQ have been particularly useful in the study of this regulon. Mutations which inactivate PhoP (phenotype PhoP-null or PhoP") cannot repress prgs and are phenotypically similar to wild-type bacteria grown in high Mg^"^. Conversely, the phenotype of a phoP constitutive mutation (pho-24 or phenotype PhoP), in which a mutation in the periplasmic domain of PhoQ results in increased phosphorylation of PhoP, is similar to that of bacteria grown in micromolar concentrations of Mg^"^ [188]. The PhoP phenotype is also thought to mimic in part the environment within host phagosomes, where PhoP is activated. Both PhoP-null and PhoP mutants are avirulent in the mouse typhoid
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
Table III
Salmonella Virulence Factors
Protein/ locus
Function
Phenotype in vitro
Phenotype in vivo (mouse, unless specified)
Slightly attenuated on oral inoculation [141]
agf
Thin aggregative fimbriae (or curli)
aw
Aromatic amino acid synthesis
Avirulent in mice [142] and humans (5. typhi) [143]
Crp, Cya
Cyclic AMP receptor, adenylate cyclase
Avirulent in mice [144] and humans {S. typhi) [143]
fim
Type I fimbriae
Adherence to (and invasion of) HeLa cells [145]
Slight decrease in LD50 on oral inoculation [141]
GalE
UDP-galactose-4epimerase, LPS synthesis
Galactose sensitive and rough phenotype
Avirulent in mice [146]; S. typhi mutant still causes disease in humans [147]
HilA
Transcriptional regulator of SPIl
Epithelial cell invasion defect [148]
InvABCEFGHIJ
Type III secretion components (SPI1)
Secretion, translocation, and invasion defects [49, 149-152]
Attenuated by oral inoculation [49]
Ipf
Long polar fimbriae
Adherence to (and invasion oO Hep-2 cells [145]
Adherence to murine Peyer's patch [153]; slight decrease in LD50 on oral inoculation [154]
MetL
Methionine biosynthesis (homocysteine production)
Resistance to host nitric oxide [155]
Attenuated in mice [155]
MgtC
Unknown
Intracellular survival [137, 156]
Avirulent [137, 156]
OmpR/EnvZ
Transcriptional regulators (osmolarity)
Intracellular survival [157]
Avirulent [157]
pef
Plasmid-encoded fimbria
Adherence to murine small intestine [158]
Reduced fluid accumulation in infant mice [158]
PhoPQ
Transcriptional regulators (pH, Mg-^
Intracellular survival, CAMP resistance [78, 80, 159], stimulation of cytokine secretion [160]
Avirulent in mice [159, 161]; PhoP" mutants are immunostimulatory compared to wild type [162]; S. typhi PhoP null mutants are avirulent in humans [163, 164]
PmrAB
Transcriptional regulators (pH)
Polymyxin resistance [165]
PrgHIJK
Type III secretion components (SPI 1)
Invasion defect [48]
pur
Purine biosynthesis
Adherence to cultured mouse small intestinal epithelial cells [140]
Attenuated by oral inoculation [48, 90] Avirulent [166]
continued
7.
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MOLECULAR PATHOGENESIS OF SALMONELLAE
Protein/ locus
Function
Phenotype in vitro
Phenotype in vivo (mouse, unless specified)
Rck
Resistance to complement
RpoS
Transcriptional regulator (stationary phase, stress response)
Sip/SspBCD
Translocase (SPIl)
Invasion and translocation defect, decreased macrophage cytotoxicity [105, 171-173]
SlyA
Transcriptional regulator
Intracellular survival [174, 175]
SopB
Inositol phosphate phosphatase [176]
Fluid secretion and inflammation in cow ileal loops [177]
SopD
Unknown
Fluid secretion and inflammation in cow ileal loops [178]
SopE
Guanine exchange factor
Slight invasion defect iS.dublin, [179]), actin rearrangements when expressed exogenously [124]
SpaOPQRS
Type III secretion components (SPI1)
Secretion, translocation and invasion defects [180]
Attenuated by oral inoculation
SPI3
Unknown {mgtQ
Macrophage survival [137]
Avirulent [137]
SPI4
Type I secretion system?
Survival within macrophages [139]
SPI5
Unknown
SptP
Protein tyrosine phosphatase
SpvABCD
Unknown (sopB)
Growth within reticuloendothelial system [183]
SpvR
Transcriptional regulator of spvABCD
Growth within reticuloendothelial system [183]
SsaBCDE,G-V
Type III secretion apparatus of SPI2
Intracellular survival [135, 184]
Avirulent [184]
SseBCD
SPI2 translocase?
Intracellular survival [185]
Avirulent [185]
SsrAB
Transcriptional regulators of SPI2
Intracellular survival [135, 184]
Avirulent [184]
TolC
Unknown
Increased sensitivity to complement and detergents
Attenuated by oral inoculation [186]
Prevents complementmediated lysis [167] Avirulent in mice [168, 169] and humans (S. typhi) [170]
Attenuated by oral inoculation [48]
Effectors of gastroenteritis in mice and cows [138] Actin rearrangements when exogenously expressed [181]
Slight decrease in colonization of liver and spleen [182]
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fever model and display pleiotropic mutant phenotypes [159, 161, 200]. The avirulence of the PhoF mutant indicates that proper timing of prg and pag expression is important for virulence.
2.
RESISTANCE TO ANTIMICROBIAL PEPTIDES
The ability of Salmonella to induce resistance to cationic antimicrobial peptides (CAMPs) is correlated with virulence in the mouse typhoid fever model and requires PhoP/PhoQ [78-80]. While PhoP-null mutants exhibit increased sensitivity to CAMPs compared to wild-type Salmonella, PhoF mutants are more resistant to most CAMPs tested (defensins being one of the exceptions). This differential sensitivity to CAMPs is at least in part due to modifications in the structure of lipopolysaccharide (LPS), in particular the lipid A moiety. Analysis of lipid A isolated from PhoP^ bacteria has revealed remarkable differences compared to PhoP-null bacteria, including the addition of an aminoarabinose group, hydroxylation of the second acyl group (2-OH-myristate), and the addition of a seventh acyl chain, palmitate [160]. Similar structural changes are observed in bacteria grown in low Mg^"^, when expression of the PhoP regulon is induced. Genetic analysis of the PhoP regulon has resulted in identification of pags that appear to mediate resistance to specific CAMPs by introducing distinct lipid A modifications. Inducible resistance to polymyxins, for instance, requires the PhoP-regulated locus pmrCAB [190, 191, 201]. PmrA and PmrB are two-component regulators that are activated in response to mildly acidic environments [191]. It is believed that phosphorylation of PmrA by PmrB allows activation of a number of genes, including several that were also defined as pags. Autoregulation of pmrAB by PmrA further enhances expression of this regulon. Two PmrA-regulated loci, pmrE (or ugd) 2ind pmrF (or pbgP), have been shown to directly affect polymyxin resistance (but not resistance to p-sheet or a-helical peptides) and are also necessary for addition of aminoarabinose to lipid A [202, 203]. The pmrE gene, which was previously identified as a pag expressed in acidic environments (pagA or ugd) [159, 191, 204], is predicted to encode a UDP glucose dehydrogenase, an enzyme that catalyses the formation of an aminoarabinose precursor. The pmrF gene is part of an operon that is predicted to encode other enzymes involved in aminoarabinose biosynthesis, such as glycosyl-transferases, or addition of this sugar to lipid A. Another PhoP-activated locus, pagP, has been shown to mediate lipid A modifications that promote resistance to different structural classes of CAMP, including a variety of a-helical CAMPs and protegrin [205]. Expression of pagP is required for increased acylation of lipid A through the addition of palmitate. The addition of palmitate is regulated in a PhoP-dependent manner and is induced by growth in low Mg^+. pagP mutants exhibit increased outer membrane permeability in response to a variety of a-helical CAMPs. Addition of the
296
CHRISTINA A. SCHERER AND SAMUEL I. MILLER
palmitate group protects Salmonella from a-helical CAMPs, but does not promote resistance to polymyxins, indicating that specific changes in Upid A structure are required for resistance to different CAMPs. This work suggests that a major function of PhoP activation may be to increase the barrier function of the outer membrane as well as increasing the transport of specific molecules (e.g., cations, discussed below). Mechanisms for increasing the barrier function include the generation of a less fluid membrane outer leaflet by the addition of the extra acyl chain to lipid A and modification of lipid A phosphates to reduce the charge of the bacterial surface. When coupled to expression of a different set of outer membrane proteins (many pags encode predicted OMP) and other LPS modifications, the net result is a bacterium that is more impermeable to antimicrobial peptides. It has been suggested that attenuation of the phoP mutant in mice might be due to increased susceptibility to CAMPs [78]. To date, no pags implicated in mediating resistance to CAMPs have been shown to contribute to the mouse virulence phenotype of phoP mutants. This could be due to the fact that the strains tested do not have a CAMP-sensitivity phenotype of the magnitude and breadth (i.e., classes of peptides) of the PhoP-null mutant. Although resistance to CAMPs has been most studied in relation to PhoP, Salmonella also utilize PhoP-independent mechanisms for antimicrobial peptide resistance. The sapABCDF operon, for instance, promotes protamine resistance, survival within macrophages, and virulence in the mouse model [80]. It is hypothesized to encode a peptide transporter that mediates resistance to CAMPs [206]. Expression of two additional genes, sapG and sapJ, is thought to be required for protamine resistance. SapJ is required for proper membrane localization of SapG, which is predicted to be part of a K+ transporter [207]. Together, the various sap loci may encode a potassium-dependent peptide transporter. A general pathogenic process utilized by Salmonellae, and perhaps a variety of bacteria, to survive in host tissues may be alteration of cell envelope components, such as LPS and membrane proteins, to decrease membrane permeability while selectively transporting important nutrients. While PhoP/PhoQ is clearly an important player in this task, a variety of PhoP/PhoQ-independent processes have been identified as important to resistance to complement. These include the outer membrane proteins TolC [186] and Rck [167], as well as genes important to the synthesis of the 6>-polysaccharide polymer component of LPS [208].
3.
LIPID A MODIFICATIONS AND INNATE IMMUNITY
In addition to promoting resistance to CAMPs, PhoP-dependent modifications of lipid A have been shown to affect the host inflammatory response to Salmonella infection [160]. LPS from PhoP-null bacteria induces higher levels of E-selectin expression from human endothelial cells and higher TNF-a expression from
7. MOLECULAR PATHOGENESIS OF SALMONELLAE
297
human monocyte-derived macrophages relative to wild type. LPS isolated from PhoF bacteria is significandy less inflammatory. The structural feature of modified LPS most likely to be responsible for these observadons is the acylation state of the LPS, specifically the presence of 2-hydroxy-myristate in lipid A. Though a variety of bacteria can modify lipid A in response to environmental condidons, the 2-OH-myristate modification appears unique to Salmonellae [205]. These results are significant because they suggest that Salmonella can dampen the host inflammatory response, which may allow bacteria to quickly colonize the host and promote extended survival in host tissues. This hypothesis is consistent with the fact that PhoP-null mutants are immunosdmulatory in vivo in mice and humans [209] and that antigen processing of these mutants is more efficient [162]. These results suggest that the use of PhoP-null mutants as live vaccines may result in increased inflammatory response and immunogenicity with limited in vivo survival, an ideal quality for a vaccine strain (immunostimuladon and safety).
4.
REGULATION OF ALTERNATIVE MAGNESIUM TRANSPORTERS
Given the possibility that PhoPQ is regulated by Ca^"^ and Mg^^ levels in vivo, it is interesUng to note that expression of three putative magnesium transporters is regulated by PhoP Transcription of the mgtA and mgtCB loci occurs in a PhoP-dependent manner and is repressed by Mg-"^ and Ca-"^ [196, 199]. Expression of mgtCB, which is located on a 17-kb pathogenicity island known as SPI3 [137], is also induced by exposure to acid, even in the presence of high concentradons of Mg^"^; this response is also PhoP dependent [195, 210]. Although mgtA and mgtB are not required for intracellular survival or for virulence, mgtC is essential for both functions [137, 156]. The functions of mgtA, mgtB, and mgtC are currently unclear. MgtA and MgtB are homologous to P-type ATPases, which use the energy from ATP hydrolysis to move molecules across cell membranes. Therefore, these proteins have been proposed to act as magnesium transporters for intracellular Salmonella. This is a reasonable supposition, as both MgtA and MgtB can transport magnesium (and other divalent cadons) and are regulated by magnesium and PhoP, and because of the reliance of PhoQ on magnesium. However, this hypothesis has been challenged for several reasons [211]. First, a consdtudvely acdve magnesium transporter (CorA) has a much higher affinity for magnesium, and should be able to provide ample amounts of magnesium for intracellular bacterial growth. Second, it seems somewhat unusual that MgtA and MgtB should have to hydrolyze ATP in order to move magnesium across the membrane with its gradient, instead of against its gradient. Third, although MgtC has also been hypothesized to be a magnesium transporter, there is no evidence that this protein can actually funcdon as a magnesium transporter [156]. It has therefore been proposed that these three proteins might function as a counter- or cotransporter
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
for some other molecule (e.g., an antimicrobial peptide), using magnesium as a signal. Although there is no direct evidence for this hypothesis, it is an intriguing model that is consistent with the role of PhoP in defending Salmonella against the host immune response. 5.
NONSPECIFIC ACID PHOSPHATASE
All Salmonella species tested contain a gene (phoN) encoding a nonspecific acid phosphatase (reviewed in [212]). The expression of this phosphatase requires PhoP and is induced on nutrient limitation. This protein is localized to the periplasm and may function to scavenge phosphate from periplasmic substrates within acidified phagosomes. Consistent with this, PhoN demonstrates a pH optimum of 5.5. However, phoN mutants are not attenuated in the BALB/c typhoid fever model [78], and thus the function of PhoN remains elusive.
6.
VIRULENCE PHENOTYPES OF PAGS
While phoP mutants display significant virulence defects, deletions within individual pags (either alone or in combination with other pag deletions) fail to attenuate virulence in the mouse model (the exception being the mgtC mutant) [213]. It is attractive to hypothesize that these genes contribute to virulence in ways that cannot be tested or measured by the currently available models. Alternatively, many of these genes may have redundant or complementary functions. The complexity of the PhoP regulon suggests that the regulation of many factors, including expression of outer membrane proteins and modification of LPS, contributes to its virulence role. These factors may need to act in concert to promote virulence through resisting the action of host innate immune processes. As the functions of many pags are still unknown, more detailed knowledge of the genes and functions regulated by PhoP/PhoQ should reveal interesting aspects of host-pathogen interactions.
7.
HILA AND THE REGULATION OF EPITHELIAL CELL INVASION
Invasion of epithelial cells, which requires the type III secretion system encoded in SPIl, is under complex regulatory control. Initial studies demonstrated that invasion is controlled by numerous environmental conditions, including growth phase [98, 99], DNA structure or supercoiling [214], osmolarity [98], and oxygen availability [45, 100, 215]. At least one transcriptional regulator from SPIl, HilA, responds to environmental conditions to control expression of the TTSS genes. Mutants in hilA, which encodes a member of the OmpR/ToxR family of transcriptional regulators, are severely defective in epithelial cell invasion [148], whereas overexpression of HilA results in a hyperinvasive phenotype [216].
7.
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MOLECULAR PATHOGENESIS OF SALMONELLAE
HilA has been shown to be coordinately regulated by oxygen, osmolarity, pH, and PhoP (which negatively regulates it) [100]. Maximal transcription from HilAregulated genes, and thus maximal invasion of epithelial cells, occurs under conditions of high osmolarity and low oxygen, and in a wild-type PhoP background. If any one condition is repressing (i.e., low osmolarity, aerobiosis or a PhoP background), expression of HilA-regulated genes is significandy decreased, resulting in concomitant decreases in epithelial cell invasion. HilA regulates a number of genes in SPIl, including those that encode the TTSS structural components PrgHIJK and OrgA, the secreted proteins Sip/SspBCDA, and a putative transcriptional regulator, InvF. InvF is homologous to transcriptional regulators from the AraC and PulD families [149], and preliminary reports indicate that it has the ability to regulate expression of the sspBCDA operon [217]. The signal transduction pathways leading to activation of hilA expression and transcription of SPIl genes are still being elucidated. A summary of known interactions is shown in Figure 6. Transcription oihilA is repressed in the presence of PhoP-P04 but appears to be induced by several other transcriptional regulators. The SirA protein, which belongs to the response regulator family of proteins, positively regulates hilA in response to an unknown signal and sensor molecule [218]. Several unlinked loci (also termed sir) that suppress a sir A mutation and activate SPIl gene expression have also been identified [218,219]. It is interesting
Environmental signals Phagosome,
SirA
other Sir
prgHIJK Fig. 6 Regulation of SPIl genes. HilA coordinately regulates invasion of eukaryotic cells by sensing environmental signals either by itself or via a number of other upstream regulators (Sir), as well as SirA and PhoP. Sir may also directly regulate transcription of SPIl genes. Negative regulation by PhoP is represented by the open arrowhead. Putative regulatory steps are indicated with dashed arrows.
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
to postulate that each Sir transduces information about a single environmental condition. This suggests a model in which HilA functions as one central "receiving" center, where various different environmental signals are processed. However, this does not rule out the possibility that other regulators can directly activate transcription of SPIl genes independently of HilA [219]. 8. RpoS: STATIONARY
PHASE AND THE ACID
TOLERANCE RESPONSE
The alternate sigma factor RpoS (also called KatF or a^) is required for expression of over 30 genes during stationary phase in Salmonella. RpoS also plays an important role in Salmonella pathogenesis, as genes regulated by RpoS protect the bacteria against a variety of stressful conditions that might be encountered within the host, including anaerobiosis, nitrogen and phosphate starvation, acid shock, and osmotic and oxidative stress (reviewed in [220]). In addition, RpoS regulates expression of several genes on the virulence plasmid that contribute to efficient systemic infections (reviewed in [221]). Mutations within rpoS have been associated with attenuation of virulence of both S. typhimurium in the mouse model system [168, 169] and S. typhi in humans [170].
9.
THE ACID TOLERANCE RESPONSE
One of the better-characterized bacterial responses to environmental stress is the induction of the acid tolerance response (ATR) (reviewed in [222]). Exposure to low pH (i.e., 4.4-5.8) for a short period of time initiates a bacterial response that increases resistance to even more acidic conditions (pH 3.3-3.0). Activation of the acid tolerance response also induces crossprotection to other environmental conditions, including heat, osmotic, and oxidative stress. Bacterial response to acid shock differs depending on the growth state of the bacteria, that is, stationary phase or log phase, and requires at least three different transcriptional activators, including RpoS, PhoP, and Fur. Activation of RpoS during the acid tolerance response is regulated by the mviA gene product, which decreases the turnover rate of RpoS when the intracellular pH decreases, presumably by regulating expression of a specific protease [223]. Although RpoS is not required for initiation of the ATR during log phase growth, it is required for maintenance of acid tolerance [224]. Initiation of the ATR during log phase requires the ferric uptake regulator Fur, which also regulates genes required for the acquisition of iron (reviewed in [225]). The role of Fur during ATR is not entirely understood, although the domains required for the ATR are distinct from those required for iron uptake [226]. The PhoP protein is also induced during acid shock and is itself responsible for expression of three other acid shock-induced proteins [195].
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301
Although the acid tolerance response is thought to contribute to the ability of Salmonella to survive acidic environments in vivo, there is no direct evidence to date for a specific role. Investigations of the importance of the acid tolerance response in vivo have been hampered by the pleiotropic effects of mutations within the global regulators RpoS and PhoP. Furthermore, multiple mechanisms for induction of the acid tolerance response indicate that there is some functional redundancy in the ATR [227, 228]. Mutations within a number of individual acid-induced genes that disrupt the ATR in the avirulent S. typhimurium laboratory strain LT2 have little effect on either acid resistance or virulence in the mouse model when transduced into wild-type Salmonella [228]. However, strains carrying multiple mutations are no longer acid tolerant and are attenuated in vivo, implicating acid tolerance as an important virulence mechanism.
10.
OMPR/ENVZ: OSMOLARITY
Responses to external osmolarity are controlled by the two-component regulators OmpR (response regulator) and EnvZ (sensor kinase). OmpR reciprocally regulates the expression of the outer membrane porins OmpC and OmpF (reviewed in [157]). When Salmonella is exposed to conditions of high osmolarity, the level of OmpC in the membrane is increased while the level of OmpF is decreased. As OmpC has been shown to form smaller pores than OmpF, the preferential expression of OmpC in the membrane when bacteria are exposed to high osmolarity medium has been hypothesized to decrease the permeability of the membrane to potentially harmful substances. Single mutations in ompC or ompF do not attenuate the virulence of S. typhimurium [157]; however, a double mutant is attenuated when administered orally [229]. As ompR mutants are severely attenuated on both oral and intravenous inoculation [157], there must be other members of the regulon that contribute to virulence; however, these have not yet been identified. Interestingly, mutants that are required for the late-onset apoptosis described in section VI. A have been linked to the OmpR/EnvZ regulon [107]. In addition, OmpR and EnvZ have been shown to be required for the formation of filamentous tubular lysosomes described in section VLB [230], raising the hypothesis that OmpR/EnvZ-regulated genes may affect trafficking of Salmonella-conidAmng phagosomes.
11.
CYA/CRP
The Cya/Crp regulon is also required for Salmonella virulence in the mouse typhoid fever model [144], although its function is poorly characterized, cya and crp encode adenylate cyclase and the cAMP receptor, respectively. Cyclic AMP and its receptor are required for the transcription of many genes required for transport and breakdown of catabolites. Mutations in cya and crp have pleiotropic
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
effects, including reduced synthesis of flagellae and fimbriae. However, their avirulence in the mouse model may be due to the fact that in vivo replication requires cya and crp at some point in the infectious cycle to overcome carbon limitation.
B. Factors Required for Invasion of Epithelial Cells Invasion of epithelial cells by S. typhimurium requires a large number of gene products that enable both adhesion and invasion. A number of these proteins comprise a specialized secretion system, known as a type III secretion system, which is required for the secretion of bacterial invasion proteins into the extracellular milieu and transport of effector molecules directly into the host cell cytosol.
1.
ADHESION FACTORS
Adherence of Salmonella to epithelial cells has been shown to be mediated by at least four different fimbriae, including type I fimbriae (fim, [154]), thin aggregative fimbriae, or curli {agf, [140, 231]; csg [232]), plasmid-encoded fimbriae (pef, [158, 233]), and long polar fimbriae {Ipf, [145]). Expression of various fimbriae mediates adherence and invasion to different epithelial cells [145], and possibly to the extracellular matrix (fibronectin [234]). Although individual fimbriae are not required for virulence in the mouse model, a quadruple mutant is attenuated by oral inoculation [141], indicating that fimbriae contribute to the ability of Salmonella to cross the epithelial barrier. Individual host receptors for fimbrial binding have not been identified, although a glycoconjugate receptor present on Caco-2 cells has been shown to be important for bacterial adherence [235].
2.
SALMONELLA PATHOGENICITY ISLAND 1
A number of mutations that prevent or decrease invasion of epithelial cells have been linked to a 40-kb region at centisome 60 of the Salmonella chromosome termed Salmonella pathogenicity island 1, or SPIl [134]. Horizontal acquisition of this segment from an exogenous source is suggested by the fact that the ratio of GC to AT basepairs in this region is lower (approximately 47% compared to 51-53%) than that of the rest of the Salmonella chromosome [180]. This region is present in all Salmonella serotypes tested to date. Proteins encoded in this pathogenicity island include components of a type III secretion apparatus, targets of the secretion apparatus, regulatory proteins, and specific chaperone or escort proteins. In addition to playing a role in epithelial cell invasion in vitro, gene
7. MOLECULAR PATHOGENESIS OF SALMONELLAE
303
products encoded in this island contribute to the induction of macrophage cytotoxicity (apoptosis) in vitro, appear to help initiate infection by the oral route in the mouse model, and mediate inflammatory responses that may contribute to diarrhea in humans and cows. 3.
TYPE III SECRETION SYSTEMS
The type III secretion system (or TTSS) encoded in SPIl is made up of structural proteins encoded in the mv, spa, and prg loci (see also [236] for a comprehensive review). Type III secretion systems are highly conserved in a wide variety of plant and animal pathogenic bacteria, including Salmonella, Shigella, Yersinia, Pseudomonas, enteropathogenic E. coli (EPEC), Erwinia, and Rhizobium. Salmonella is unique because two separate type III secretion systems are encoded on the chromosome; the second TTSS, found on Salmonella pathogenicity island 2 (SPI2), is described in section VI.C. Because expression of these systems is often induced on contact with mammalian cells, the type III secretion systems have also been termed "contact-dependent" secretion systems [237]. Type III secretion systems secrete proteins via a Sec-independent process into the extracellular milieu; however, their main function appears to be direct translocation of bacterial effector proteins into the host cell cytosol. Although components of the secretion apparatus are highly conserved among Gram-negative pathogens, the secreted and translocated effector proteins vary greatly. They induce a variety of phenotypes in cultured cells (and presumably in vivo as well), including cytoskeletal rearrangements, inhibition of phagocytosis, and the plant hypersensitivity response. 4.
STRUCTURE OF THE
TTSS
The TTSS is composed of more than 20 proteins that are thought to assemble into a supramolecular structure spanning both the inner and outer bacterial membranes. A number of TTSS structural proteins are homologous to proteins that comprise the cytoplasmic and membrane-spanning portions of the bacterial flagella. A schematic view of the homology of type III secretion proteins encoded by Salmonella (in both SPIl and SPI2) to flagellar proteins is shown in Figure 7 (see color plate). By homology to flagellar components, topological and structural predictions have been made for some of the Salmonella TTSS proteins. For instance, invC is predicted to encode an FQEI ATPase that might provide the energy required to transport proteins through the apparatus [152]. PrgK, which is similar to FliF, a component of the MS Ring, is predicted to be localized in the inner membrane [192]. InvA, SpaP, SpaQ, SpaR, and SpaS, which are all homologous to the flagellar export apparatus, are predicted to be localized in the inner membrane [151, 180]. Interestingly, SpaO, which shows some homology to FUN (a component of the flagellar C ring), has been reported to be secreted, and
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
is most likely not associated with the inner membrane [238]. Finally, InvG, which is homologous to the PulD family of secretory proteins [239], is localized in the outer membrane [149]. InvH, which is also located in the outer membrane, is required for the proper localization of InvG [240, 241]. Research in the 1990s has been focused on visualization and purification of TTSS structures from Salmonella in order to determine their molecular composition. Although the appearance of transient surface appendages (called invasomes) has been previously reported for Salmonella [242], it is only recently that a supramolecular structure was purified and visualized by electron microscopy [243]. Electron micrographs of nonflagellated Salmonella reveal dense basalbody-like structures in the inner and outer membranes with thin needle-like appendages extruded through the membrane and outside the bacterium (see Fig. 8A). As these macromolecular complexes were visualized in osmotically shocked bacteria, it is formally possible that the needle structures that are seen to protrude through the outer membrane do not extend through the outer membrane in normal bacteria. Membrane invaginations are observed at the base of the needle structure, indicating that this structure spans the periplasmic space. These structures can be distinguished from flagella by the needle filaments, which are much thinner than the flagellar filaments. Electron microscopy of needle complexes purified through cesium chloride density gradients shows cylindrically symmetrical structures with two inner and two outer rings, like those seen in the flagellar basal body (Fig. SB). Molecular analysis of the purified needle complexes demonstrated that the predominant protein components included PrgH, PrgK, and InvG. A diagram of the needle complex and putative protein components is shown in Figure 8C.
5.
PROTEINS SECRETED BY THE
TTSS
At least 25 proteins are secreted by the TTSS encoded in SPIl [192]. Proteins secreted by the TTSS are unusual in that they do not contain obvious signal sequences and are not processed on secretion. The exact recognition sequence for targets of the TTSS has not been identified, as the various secreted proteins do not show any significant homology that would indicate a signal sequence. The most prevalent proteins seen in culture supematants have been variously named Salmonella invasion proteins (Sips) [172], or Salmonella secreted proteins (Ssps) [171]. Sip/Ssp A, B, C, and D are homologous to the Shigella flexneri secreted proteins (IpaA-D) required for epithelial cell invasion [244]. Sip/SspB, C, and D are all required for induction of Salmonella-mt&i^Xtd endocytosis of epithelial cells [171, 172]. In addition, SipB and SipC have been observed within the cytosol of infected cells by indirect immunofluorescence [173]. Cytosolic localization requires a functional TTSS but not bacterial invasion, suggesting that these proteins are translocated by the secretion apparatus. Sip/SspB, C, and D have also been shown to participate in the translocation of other bacterial effector proteins (which will be discussed below) [124, 173, 179,
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245], and are thus sometimes referred to as "translocase" proteins. Based on homologies to proteins in Yersinia and Shigella, it is thought that the translocase proteins interact with the TTSS and each other to faciUtate formation of a pore in the eukaryotic cell membrane. For instance, there is some evidence that IpaB and IpaD can associate in the bacterial membrane, while IpaB and IpaC can associate in bacterial supematants (this interaction is thought to be prevented by the IpgC chaperone in the bacterial cytosol) [246, 247]. IpaB, YopB (from Yersinia), and SipB all contain several potential membrane-spanning domains and exhibit some homology to the RTX family of pore-forming toxins. These proteins have been hypothesized to make pores in the eukaryotic plasma membrane [248]. SipC and SipD may help form the pore or a channel through which effector proteins are translocated or, alternatively, could help link SspB to the TTSS structure. Instability or shearing of the translocase once it is inserted into the host plasma membrane might explain the resultant presence of SipC and SipB inside the cell. A diagram of this model is shown in Figure 9 (see color plate). In addition to its role as a translocase, Sip/SspB is required for induction of TTSS-dependent apoptosis in macrophages [105]. Interestingly, IpaB has been shown to be sufficient to induce apoptosis in macrophages [249]. The current hypothesis for the role of IpaB is that it direcdy binds to interleukin 1 p-converting enzyme (ICE, also known as Caspase-1) to activate the apoptotic pathway [250]. ICE, which cleaves IL-lp to its mature form, is a natural inducer of apoptosis in cells. Recent data indicate that SipB plays a similar role in the induction of apoptosis by S. typhimurium [251].
6.
TRANSLOCATED PROTEINS (PUTATIVE EFFECTOR PROTEINS)
To date, six Salmonella proteins (excluding SipB and SipC) have been shown to be translocated into the eukaryotic cell cytosol in an SPIl-dependent manner. These include SptP [182, 245], AvrA [252], SopE [124, 179, 181], SopB [177], SopD [178], and Ssp/SipA [253]. The AvrA protein is homologous to the avirulence factor AvrRxv from Xanthomonas campestris pv. vesicatoria and the apoptosis-inducing protein YopJ from Yersinia pseudotuberculosis [252]. No virulence defects are associated with an avrA mutant, and no known function has been assigned to this protein at this time. The SopB protein, which is present on SPI5 in S. dublin [138], has been shown to promote fluid secretion and an inflammatory response in infected catde [177]. Recendy, it has been demonstrated to be an inositol phosphate phosphatase [176]. Mutations in the S. typhimurium sopB homolog sigD has been implicated in invasion of epithelial cells [254]; this phenotype is not observed in S. dublin sopB mutants. S. typhi and S. dublin infection of human polarized epithelial cells results in an increase in intracellular levels of inositol 1,4,5,6-tetrakisphophate
7.
MOLECULAR PATHOGENESIS OF SALMONELLAE
307
[Ins(l,4,5,6)P4], a signaling molecule that induces secretion of chloride in Salmonella-inftcicd cells [255]. SopB inositol phosphate phosphatase activity has been implicated in this induction, which may contribute to fluid secretion and diarrhea [176]. Another translocated protein from Salmonella dublin, SopD, has also been shown to act in concert with SopB to promote an inflammatory response in bovine hgated ileal loops [178]. Since SopB and SopD have been shown to play a role in intestinal inflammation and fluid secretion in cattle, and since SPIl is required for inflammatory responses that result in neutrophil transmigration in a human model system for gastrointestinal inflammation [126, 131], it is attractive to hypothesize that SPIl-translocated effector molecules promote responses that lead to bovine and human gastroenteritis. Several translocated effector proteins have been associated with cytoskeletal rearrangements in eukaryotic cells. Recent evidence indicates that Sip A binds actin, and may be directly involved in Salmonella-induced membrane ruffling [253]. SptP and SopE have both been shown to induce actin rearrangements when expressed exogenously in eukaryotic cells [124, 245]. The Salmonella protein tyrosine phosphatase (SptP) is an interesting protein that appears to be a chimera between two Yersinia proteins, YopE and YopH [182]. The amino terminus of SptP (residues 107-290) is homologous to two bacterial cytotoxins: Exotoxin S from Pseudomonas aeruginosa and YopE from Yersinia spp. The carboxyl-terminal portion of SptP (residues 340-513) is homologous to the catalytic portion of the Yersinia protein tyrosine phosphatase YopH. SptP appears to be required for full virulence of Salmonella in mice, as sptP mutants display a slight defect in the ability to colonize spleens [182]. SopE was first described in S. dublin, where mutants displayed mild defects in epithelial cell invasion [179]. Recendy, SopE from S. typhimurium has been shown to bind to and induce GDP/GTP exchange by the small GTP-binding proteins CDC42 and Racl, resulting in their activation [124]. SopE also facilitates activation of downstream signal transduction pathways, as expression of SopE resulted in the increased phosphorylation of c-Jun. This is the first example of a Salmonella effector protein that directly modulates a cellular factor to induce actin rearrangements and nuclear responses. The role of SopE, SptP, SipA, and other translocated effector proteins in promoting invasion is unclear, as mutants are still able to enter epithelial cells (albeit with slightly lower efficiency, at least in the case of the sopE mutant). It seems most possible, given the identified phenotypes of expression in host cells, that multiple effectors are involved in redundant functions to induce actin rearrangements and membrane ruffling in infected cells; thus, individual mutants would only display mild or no defects in invasion. Alternatively, the effects observed are due to high nonphysiological expression of these proteins, and other unidentified factors with different functions promote membrane ruffling and BME.
308
C.
CHRISTINA A. SCHERER AND SAMUEL I. MILLER
Factors Required for Systemic Infection
Initiation of systemic infections by Salmonellae requires that bacteria survive various severe environments, including the low pH of the stomach and phagosome, oxidative stress within the phagosome, and the relative unavailability of nutrients such as carbon and iron. As discussed before, the acid tolerance response is highly regulated and is probably necessary for Salmonella to survive in vivo. In this section, we will discuss other systems required for virulence in the mouse model, including metabolic pathways and the TTSS encoded in another Salmonella pathogenicity island.
1.
SALMONELLA PATHOGENICITY ISLAND 2
Salmonellae are unique among the Enterobacteriaceae in that they appear to utilize two separate type III secretion systems. Recent studies have defined a second type III secretion system in Salmonella that is required for growth within macrophages and virulence in the mouse model [135, 184]. Genes encoding this apparatus are located in a 25-kb region at centisome 30 in Salmonella pathogenicity island 2 (diagrammed in Fig. 7, see color plate). Sequences in SPI2 are conserved throughout the Salmonellae, with the exception of S. bongori [133, 256], but are not found in other enteric bacteria [136]. Thus, it is thought that this pathogenicity island contributes to Salmonella-specific aspects of infection. Like SPIl, SPI2 contains a number of genes predicted to encode a secretion apparatus (designated ssa by [185]), as well as chaperones (ssc), transcriptional regulators (ssr), and putative effector proteins (sse). Expression of genes within SPI2 is regulated by the two-component regulators SsrA (or SpiR) and SsrB. Regulation of these proteins has been difficult to study in vitro because optimal conditions for gene expression have not been determined. However, both apparatus genes and putative secreted effector genes are expressed when bacteria are internalized within cultured macrophages [194, 257]. Expression is dependent on SsrAB and acidification of vacuoles, as bafilomycin treatment prevents expression of SPI2 genes. 2.
SPI2 EFFECTOR
PROTEINS
Six potential secreted effector proteins have been identified in SPI2 [185]. These proteins are minimally expressed in vitro in an SsrAB-dependent manner. Secretion of these proteins has not yet been observed during in vitro growth, and confirmation that the putative effector proteins are secreted and/or translocated awaits further characterization of infected cells. Nevertheless, the Sse proteins do appear important for virulence, as sseA, sseB, and sseC mutants are avirulent, and sseF and sseG mutants are attenuated in the mouse model. It is likely that some
7. MOLECULAR PATHOGENESIS OF SALMONELLAE
309
of these proteins assemble into a functional translocase associated with the TTSS from SPI2. Several Sse proteins contain homologies to proteins involved in translocation by other bacteria [185]. SseC, for instance, is homologous to YopB from Yersinia and EspD from enteropathogenic E. coli (EPEC), and contains three predicted membrane spanning alpha helices that might enable its insertion into host cell membranes. In addition, predicted transmembrane helices are present in all of the Sse proteins except SseA. SseB and SseD are predicted to encode proteins that are similar to EspA and EspB of EPEC. This is quite interesting, as EspA, B, and D have previously been shown to be required for induction of host cell signaling and translocation of the intimin receptor Tir [258-260]. In addition, EspA has been shown to form extracellular filaments that contact host epithelial cells and might represent a translocation channel [261]. As Salmonella have been shown to form many pili and fimbrial-like filaments that are not SPIl dependent [262], it is possible that one of the filaments is formed by proteins in SPI2, and the Esp-like proteins in particular.
3.
METABOLIC MUTANTS
A number of mutations within metabolic loci have been reported to attenuate Salmonellae in vivo, presumably because of the scarcity of required nutrients in the intracellular environment. Auxotrophic mutants with defects in the biosynthetic pathways of aromatic amino acids {aw) [142], purines (pur) [166], pyrimidines, histidine, and methionine [50], or defects in ethanolamine utilization, through which carbon and nitrogen might be acquired [263], are all attenuated in the mouse model system. In addition, the simultaneous prevention of glutamine synthesis and transport also results in bacterial attenuation [264]. Because of their inability to persist in vivo, auxotrophic strains have been investigated as potential vaccine strains [87, 142, 143, 166, 265-267], as will be discussed later.
4.
SURVIVAL WITHIN MACROPHAGES
Although little is known about the mechanisms utilized by Salmonella to survive within macrophage phagosomes, a few genes that affect this process have been identified. For instance, recombination-deficient mutants, which are hypothesized to be unable to repair DNA damaged by the macrophage oxidative burst, do not survive within macrophages and are avirulent in the mouse model [268]. Another gene product that appears to be required for resistance to oxidative stress in macrophages is the Sly A transcription factor [174], which is induced during stationary phase and required for survival within macrophages [175]. The ability to survive within macrophages may be due in part to resistance to host nitric oxide (NO). The production of nitric oxide is a host defense mechanism associated with broad-spectrum antimicrobial activity, especially important dur-
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
ing intracellular infections. Resistance to nitric oxide in macrophages appears to be mediated at least in part by the metL gene product, which controls the production of homocysteine in the methionine biosynthetic pathway. Salmonella with a mutation in metL are hypersensitive to 5-nitrosothiol NO donor compounds and are attenuated for virulence; this is apparently because homocysteine can act as an endogenous antagonist of NO, thus protecting the bacteria [155].
5.
Vi ANTIGEN
The major surface antigen of 5. typhi, Vi antigen, appears to protect these bacilli from some host innate immune mechanisms [269]. Expression of the Vi antigen correlates with prevention of antibody-mediated opsonization, increased resistance to host peroxide, and resistance to complement activation by the alternate pathway and complement-mediated lysis. Vi antigen thus may function to inhibit phagocytosis of typhoidal Salmonella by neutrophils while not interfering with the induction of phagocytosis by more permissive macrophages and epithelial cells.
D. Salmonella Toxins Several cholera-like toxins [270] and enterotoxins [271, 272] have been described in Salmonella, but none have been isolated or molecularly characterized. Interestingly, a 27-kb pathogenicity island (designated SPI4) that is predicted to encode proteins with homology to type I secretion systems has been identified [139]. Genes within this locus display considerable homology to proteins involved in the secretion of several members of the RTX toxin family, including the Bordetella pertussis CyaA protein, the Serratia marcescens 8000 lipase LipA, and the E. coli hemolysin HlyD. Preliminary evidence indicates that genes within this operon may encode another secretion system, perhaps utilized for secretion of a Salmonella toxin. A transposon insertion within SPI4 prevents secretion of at least one uncharacterized protein and is required for survival within macrophages.
E. Virulence Plasmids A number of Salmonella strains contain large (50-100 kb) virulence plasmids (reviewed in [221]). Although they are present in other host-adapted serotypes such as 5. dublin, S. choleraesuis, S. gallinarum/pullorum, and S. abortusovis.
7. MOLECULAR PATHOGENESIS OF SALMONELLAE
311
they have not been detected in the human pathogen S. typhi. They are also present in the broad-host range serotypes S. typhimurium and S. enteritidis. In strains that do harbor virulence plasmids, their presence has been associated with full virulence in vivo. Phenotypes attributed to genes present on the virulence plasmid include increased intracellular growth, resistance to complement, and macrophage cytotoxicity [221, 273]. Plasmid-cured strains colonize the Peyer's patch and reticuloendothelial systems with wild-type efficiency; however, subsequent growth in the reticuloendothelial system is impaired, and bacteria are eventually cleared by the host. A single 8-kb region, which contains the Salmonella plasmid-virulence genes {spv), is sufficient to return full virulence to plasmid-cured strains of S. dublin [183]. The spv locus consists of five genes, the first of which, spvR, encodes a transcriptional regulator required for expression of the spvABCD operon. The spv genes are expressed during stationary phase in an RpoS-dependent manner [274]. In addition, expression of spvR has been reported to be autoregulated in a positive manner and negatively regulated by the spvA and spvB gene products [275]. Although one group has reported that expression of a promoter trap IVET {in vitro expression technology) vector-generated gene fusion to spvB in S. typhimurium was regulated by PhoP [276], another group has shown that spv expression in S. dublin is not dependent on the phoP, ompR, or cyalcrp regulatory loci [277]. Analysis of the predicted amino-acid sequence of SpvB revealed homology between the amino-terminal region and the CatM repressor of Acinetobacter calcoaceticus [111]. Mutations in either spvR or spvB result in the attenuated phenotype associated with plasmid loss. Mutations in spvC or spvD are partially virulent, but plasmid maintenance in vivo is impaired. Specific functions of the spv gene products have not yet been elucidated. Although plasmid-cured strains appear wild type in their ability to resist complement-mediated lysis, four loci on the virulence plasmid have been reported to affect complement lysis (reviewed in [278]). These include Rck (resistance to complement killing), an outer membrane protein that prevents polymerization of C9, the pore-forming component of the classical pathway of complement lysis. A surface-exposed loop appears to mediate this ability [279]. Rck is similar to two other virulence proteins, the Ail invasin from Yersinia enterocolitica and the S. typhimurium virulence protein PagC [167, 280]. Like Ail, Rck confers on E. coli the ability to adhere to and invade epithelial cells, but the molecular mechanisms for this are unknown. PagC, Ail, and Rck are members of an interesting family of outer membrane proteins that are present in all Gram-negative bacteria. Family members are present in bacteriophages, plasmids, and chromosomal regions that indicate their acquisition by horizontal transmission. All the phenotypes for these proteins, which include complement resistance, antibiotic resistance, and invasion, have been defined on the basis of multicopy expression. Since multicopy
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
expression of these proteins results in more global effects, including transcription and expression of unlinked genes, their function remains to be defined. Other virulence plasmid proteins that may modulate Salmonella virulence have also been described. A protein homologous to the LuxR family of quorum sensors that controls expression of genes encoded on the S. typhimurium virulence plasmid was discovered in 1998 [281]. SdiA is the first quorum-sensing protein to be identified in Salmonella. SdiA regulates at least one uncharacterized operon on the virulence plasmid, but its contribution to virulence is unknown at this time. Another interesting protein encoded on the virulence plasmid is TlpA, an autoregulatory coiled-coil transcriptional repressor. TlpA is unique in that its structure is modified by temperature, which in turn affects its transcriptional activity. Increased temperature results in a more unfolded (monomeric) state, which decreases repressor activity. It has been proposed that TlpA might act as a thermosensor to help regulate changes in gene expression on entry into hosts [282]. Although this is an intriguing hypothesis, the role of TlpA in host-induced gene expression is unknown at this time.
VllL Antibiotic-Resistant Salmonellae
A. Multi-Antibiotic Resistant Salmonellae The recent emergence of Salmonellae carrying stable resistance to multiple clinically relevant antibiotics is a significant health problem worldwide. Antibiotic resistance of S. typhi has been an issue since 1950, when strains resistant to chloramphenicol were isolated in Great Britain, only 2 years after the successful use of chloramphenicol in treatment of typhoid fever [283]. Currently, S. typhi isolates resistant to six different antimicrobial agents prevail in highly endemic typhoid areas, particularly China, Pakistan, and India. These strains of S. typhi carry a 120-kb plasmid that encodes resistance to ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycHne, and trimethoprim. In addition, S. typhi strains isolated from recent outbreaks in Tadjikistan and Pakistan have also acquired resistance to ciprofloxacin (a fluoroquinolone), one of the preferred antibiotics for treatment of typhoid fever [16]. As all of the above antibiotics, except streptomycin and tetracycline, are clinically relevant for the oral treatment of typhoid fever, the existence of a hepta-resistant agent for typhoid fever is a serious health problem. In addition, multi-antibiotic-resistant (MAR) typhoid has been a significant cause of death in children; the mortality rates of children
7.
MOLECULAR PATHOGENESIS OF SALMONELLAE
313
infected with MAR S. typhi range from 7 to 16%, compared to a rate of 2% for children infected with susceptible strains of Salmonella [284]. Resistance of nontyphoidal Salmonellae is also a growing health problem. Particularly troubling is the penta-resistant strain of S. typhimurium known as DTI04 (definitive phage type 104), which emerged in Great Britain in 1984 and was reported in 1997 to have been isolated in the United States [23]. This strain has been isolated from numerous species of animals (wild and farm) and is resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (R type ACSSuT). In addition, there have been reports of resistance to two other antibiotics, trimethoprim and fluoroquinolones, in Great Britain [285]. The veterinary use of antibiotics, such as ciprofloxacin and trimethoprim, to treat DTI04 infections in cattle has been proposed as a factor in the acquisition of resistance to these (and other) antibiotics by Salmonellae. Interestingly, resistance to ciprofloxacin has not been observed in the United States yet, possibly because fluoroquinolones are only licensed for use in poultry, where DTI04 may not yet be established as a pathogen [24]. Reports documenting the emerging resistance to fluoroquinolones by Salmonellae is worrisome, as ciprofloxacin is the antibiotic of choice for treating human salmonellosis; resistance to this drug will leave few available options. In addition to the problematic treatment of DTI04 infections, there are reports indicating that DTI04 may be more virulent for humans. In a study performed in the United Kingdom, 41% of patients infected with DTI04 were hospitalized, and 3% of culture-confirmed patients died (compared to an average death rate of 0.1%) [286].
B. Development of New Antibiotics The emergence of multidrug resistant Salmonella (and other bacterial pathogens) underlines the necessity for the development of new antibiotics. Salmonella is an ideal organism for the development of new drugs because of the readily available small animal model (mouse), in which antibiotics can be quickly and cheaply tested. In addition, as a number of the molecular mechanisms contributing to pathogenesis have been elucidated, the opportunity exists to develop antibiotics that target specific virulence mechanisms in Salmonella (i.e., pathogenesis-based antimicrobial therapy). As many of these mechanisms are likely to be utilized by other Gram-negative bacteria as well, the development of new drugs targeting Salmonella should also be beneficial for treatment of other pathogens. Such antibiotics might reduce the acquisition of antibiotic resistance while at the same time preserving normal bacterial flora. One active area of research is the development of new cationic antimicrobial peptides. Specific bacterial mechanisms that could be targeted include the PhoP-regulated modifications of the
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CHRISTINA A. SCHERER AND SAMUEL I. MILLER
bacterial outer membrane and LPS that mediate resistance to CAMPs and the type III secretion system through which bacterial effector proteins are translocated to the eukaryotic cell cytosol.
IX. Salmonella-Bosed Vaccines
A. Development of More Effective Typhoid Vaccines As new antibiotics are still in the developmental stage, it is also important to continue the effort to produce effective vaccines for typhoid fever. Three vaccines are currently available for typhoid fever, none of which are 100% effective, even when tested on endemic populations [287]. The live attenuated Ty21a strain (manufactured by the Swiss Serum and Vaccine Institute), which is orally administered, requires at least four doses to achieve 51-76% protective efficacy [288]. The heat- and phenol-inactivated typhoid vaccine (manufactured by Wyeth) has similar efficacy, and requires at least two doses (by injection). However, this vaccine has a variety of severe local and systemic side effects that limit its use. Finally, the cell-free, parenteral Vi-antigen vaccine produced by Pasteur Merieux (ViCPS) requires only a single dose, but most likely has a shorter duration of protection. A number of new live strains have been evaluated in human volunteers for their vaccine potential. Live attenuated vaccine strains may be preferable for typhoid fever because they can induce a wide spectrum of protective immunity, including mucosal, humoral, and cell-mediated immunity. Ideal vaccine strains should be genotypically stable (containing at least two nonreverting deletions) and should offer long-term protection after one or two initial doses. Data generated in the S. typhimurium inbred mouse model of typhoid fever are used to identify candidate vaccine strains. Unfortunately, although mutations in single pathways can effectively attenuate S. typhimurium in mice, they are not always adequate in humans. For instance, galE mutants, which are rough, galactose sensitive, and defective in LPS synthesis, are avirulent in mice, but S. typhi galE mutants can still cause typhoid fever [147]. Other more promising vaccine strains include aw deletion mutants [142, 143], crplcya deletion mutants [143, 144] and the phoPlphoQ deletion mutants [163, 164]. aw and crplcya mutants are equally attenuated in mice and humans, and produce vigorous mucosal, humoral, and cellular immune responses on oral inoculation. The PhoP/PhoQ null strain is also highly immunogenic after a single dose in human volunteers. This strain is also promising because it does not persist for long periods of time in vivo, produces very few
7. MOLECULAR PATHOGENESIS OF SALMONELLAE
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side effects, and has been shown to be more immunostimulatory compared to wild type [162]. Although there is little requirement for human vaccines for nontyphoidal Salmonellae, as the infections are usually self-limiting, various vaccines for use in animals have been developed. Such vaccines would be quite useful if they could eliminate the source of most Salmonella infections without increasing the potential for additional antibiotic resistance. Both aro-negative [289] and crp/cya mutant [290] S. typhimurium strains have been shown to be effective vaccine strains in chickens. These strains offer long-term protection against infection with both S. typhimurium and 5. enteritidis. In addition, immunization with the crp/cya strain in ovo (up to 7 days before hatching) protected chicks from infection [290], a fact that is particularly important in light of the ability of S. enteritidis to colonize intact shell eggs.
B. Salmonellae as Multivalent Vaccine Strains Induction of mucosal immunity is a hallmark of Salmonella infection, and has driven the development of multivalent Salmonella vaccines that can induce immunity not only to Salmonella but also to heterologous antigens. Antigen delivery to the gut-associated lymphoid tissue (GALT) by Salmonellae results in the eventual secretion of IgA antibodies at a number of sites, including the respiratory system, gastrointestinal tract, genitourinary tract, mammary glands, and salivary glands. The development of effective multivalent vaccine strains is dependent on several factors (reviewed in [291]). First, it is imprudent to introduce antibiotic resistance genes on plasmids carried by the vaccine strain. Therefore, heterologous antigens must either be integrated into the chromosome in single copy or they must be expressed on plasmids that do not utilize selectable antibiotic resistance markers. Expression of antigens from the chromosome is also problematic, as the level of expression may be too low for adequate immunogenicity; therefore, the use of in v/v(9-induced bacterial promoters to maximize expression has been investigated [292, 293]. An alternative approach is to express antigens on balanced lethal plasmids, which are required for bacterial survival in the host but do not require antibiotic selection for maintenance in vivo [294, 295]. Using the techniques described above, a number of studies have shown that immunity to foreign antigens, including tetanus toxin [292, 296], can be attained in mice. An interesting twist on the use of live attenuated Salmonella strains for delivery of heterologous antigens is the use of an aroA strain to transfer eukaryotic expression vectors to host cells [297]. Oral immunization of mice with S. typhimurium strains containing plasmids expressing portions of the Listeria monocytogenes protein listeriolysin resulted in transfer of these plasmids to host cells and subsequent protective immunity to infection with Listeria.
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Investigators have utilized the type III secretion system encoded in SPIl to induce MHC Class I-restricted T-cell responses to heterologous antigens [298]. Fusion of an amino-terminal domain of the translocated protein SptP to various viral antigens allowed translocation of these antigens to the cytosol of infected cells and induced a protective Class I-restricted CTL response. Mice infected with the vaccine strains were completely protected from infection by murine lymphocytic choriomeningitis virus (LCMV), a normally lethal virus. C. Salmonella-Based Cancer Therapy Salmonella vaccine strains have also been investigated as potential cancer treatment vectors. Interestingly, some attenuated Salmonella strains have been shown to preferentially colonize tumors (rather than liver and spleen) and suppress tumor growth when inoculated into mice [299]. As Salmonella could be engineered to express and translocate (via the type III secretion system) anti-tumor prodrugs, such schemes appear to be worth investigating further.
Acknowledgments We would like to thank former and present members of our laboratory for helpful discussions, in particular C. Lesser, M. Hantman, and J. Gunn for critical reading of this manuscript. We also thank R. Valdivia, D. Holden, A. Zychlinsky and S. Falkow for communicating results prior to publication, and C. Alpuche-Aranda, S.-I. Aizawa, B. Finlay, and M. Jepson for providing photos. Work in our laboratory was supported by grants AI30479 (S.I.M.) and AI09312 (C.A.S.) from the National Institutes of Health.
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CHAPTER 8
Shigellosis: From Disease Symptoms to Molecular and Cellular Pathogenesis PHILIPPE J. SANSONETTI COUMARAN EGILE CHRISTINE WENNERAS
I. II. III. IV. V.
VI. VII. VIII. IX.
X.
Introduction Bacteriology The Somatic Antigen Epidemiology and Transmission Disease Symptoms and Complications: Orientations for Future Research? A. A Spectrum of Disease Symptoms from Diarrhea to Dysentery B. Acute Complications of Shigellosis C. Long-Term Complications of Shigellosis Histopathology of Shigellosis: A Window on Pathogenesis Animal Models: Strengths and Weaknesses Cellular Models of Infection: The Contribution of Shigella to the Concept of Cellular Microbiology Pathogenic Mechanisms: In Vitro Expression of the Invasive Phenotype A. Molecular and Cellular Mechanisms of Shigella Invasion of Epithelial Cells: Basic Principles and Reviews B. Molecular and Cellular Biology of the Entry Process into Epithelial Cells C. Escape of Shigella into the Cell Cytoplasm D. Intracellular Motility and Cell-to-Cell Spreading of Shigella E. Actin-Based Intracellular Motility of Shigella E Apoptotic Killing of Macrophages and Induction of the Release of Mature IL-1 p by Shigella G. Activation of PMNs Also Reflects the Shigella Invasive Phenotype Pathogenic Mechanisms: In Vivo Expression of the Invasive Phenotype A. Does Shigella Express Organ-Specific Adhesins?
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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B. Crossing the Epithelial Lining by Shigella: Which Route is Best? C. Intestinal Inflammation during Human Infection XI. Role of Chromosomally Encoded Genes in the Virulence of Shigella A. Regulation of Plasmid Virulence Genes B. Lipopolysaccharide C. Toxins D. Other Virulence Factors XII. Conclusions References
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/. Introduction Enteric bacterial pathogens may be divided into two pathovars: (1) The noninvasive pathovar, exemplified by Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC), cause disease by adhering to the apical side of the small intestinal epithelium and by secreting enterotoxins, thereby causing massive water and electrolyte secretion (i.e., watery diarrhea). Likewise, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) colonize and partially destroy the epithelium. EHEC may even cause bloody diarrhea due to the production of Shiga-like toxins. However, here again, in spite of significant epithelial alterations (i.e., attaching-effacing effect on epithelial villi), these bacteria are not significantly invasive and the syndrome they cause is still dominated by watery diarrhea [1]. (2) The invasive pathovar is exemplified by microorganisms such as Shigella and Salmonella, it is characterized by invasion of the intestinal mucosa. In the case of Salmonella typhi infection, the intestinal phase of the invasive process may lead to limited symptoms, but crossing of the epithelial barrier allows the microorganisms to enter a septicemic phase that causes the systemic symptoms and complications characteristic of typhoid fever [2]. In the case of Shigella, the invasive process remains localized to the colonic and rectal mucosa, thereby causing major inflammatory destruction that accounts for a dysenteric syndrome, thus the name bacillary dysentery [3]. In many cases, however, shigellosis causes only a watery diarrhea similar to that observed with noninvasive pathogens. Shigellosis is a disease of the poor, primarily affecting young children in the developing world. Epidemic outbreaks also occur in industrialized countries following accidental breaches in hygiene or sanitation. The term "dysentery" was introduced by Hippocrates, who noted the seasonal pattern of the disease. The disease entity and its probable bacterial cause were described by Widal and Chantemesse. One of the four etiological species, Shigella dysenteriae (Shiga bacillus), was first identified by Shiga a century ago. The global burden of Shigella infection has recendy been reevaluated [4].
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//. Bacteriology The shigellae are Gram-negative, nonsporulating, facultative anaerobic bacilli belonging to the family Enterobacteriaceae. The genus Shigella comprises four different species: S.flexneri (6 serotypes), S. dysenteriae (16 serotypes), S. sonnet (1 serotype), and S. hoydii (8 serotypes). Shigella are identified based on their capacity to ferment various sugars and on the antigenic specificity conferred by LPS 0-sidechains (i.e., somatic antigen), which accounts for their specific serotype. Shigella is a close variant of Escherichia coli since Shigella species have more than 80% nucleotide sequence identity with E. coli [5]. Conjugation between Shigella and E. coli has been carried out successfully [6], thereby facilitating genetic studies, particularly those investigating pathogenesis. Shigella is a nonmotile bacterium, although the genes encoding the flagellar apparatus are present [7]. The significance of the crypticity of flagellar expression is presently unknown. However, it should be noticed that careful electron microscopic analysis has allowed identification of one to three polar flagella on certain fresh clinical isolates [8]. As described below, what really characterizes Shigella and maintains it as an independent genus is its invasive phenotype. This invasive phenotype reflects, in cell assay systems of infection, the ability of Shigella to enter into, invade, and destroy the colonic and rectal epithelial tissues. The invasive phenotype is encoded by a large 200-kb virulence plasmid found in all Shigella species. Coevolution of the chromosome with the virulence plasmid has led to coadaptation, and genetic modifications have progressively accumulated in the Shigella chromosome that enhance the efficiency of the plasmid-encoded invasive phenotype. Plasmid invasion genes falling under chromosomally encoded regulatory loops, acquisition of pathogenicity islands, and the absence of certain metabolic functions constituting "black holes" in the chromosome [9] support this concept, which is reinforced by the observation that enteroinvasive E. coli (EIEC), an E. coli pathovar considered an intermediate in evolution between E. coli and Shigella, is less virulent in humans since the oral infectious doses required in human volunteers to cause dysentery are 100 colony forming units (cfu) for Shigella and 10^ cfu for EIEC [10]. Shigella is present in the stools of patients at a concentration of 10^ to 10^ cfu per gram of feces during the first days of illness. Then, the number of colony-forming units decreases dramatically, and diagnosis may become difficult, especially because the microorganisms are fragile and no enrichment medium exists. Large numbers of polymorphonuclear leukocytes (PMNs) are present in stools at the early stage of the disease, reflecting the intense inflammation caused by the pathogen. Careful examination of the stools can rule out the presence of trophozoites of pathogenic Entamoeba histolytica, which, unlike Shigella, do not elicit massive luminal release of PMNs. Bacterial identification is confirmed by seroagglutination with sera elicited against the various Shigella somatic antigens corresponding to the 0-sidechains of the LPS.
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///. The Somatic Antigen LPS is the major constituent of the bacterial outer membrane. It is composed of three covalently Hnked moieties: lipid A, core, and 0-sidechains. The Shigella lipid A is identical to that of E. coli and mediates the endotoxicity of LPS. The core region consists of an oligosaccharide similar but not identical to that of E. coli. The 0-sidechains are composed of repeated sugar subunits which vary in their composition, thereby contributing to the serotypic diversity. For example, the tetrasaccharide 3P-A^-acetyl-glucosamine-a 1 -2-rhamnose-a 1 -2-rhamnoseal-3-rhamnose-l represents the basic tetrasaccharide repeating subunit of S. flexneri Y. Further changes, such as glycosylation of one of the rhamnoses, produce the serotype specific variations that yield serotypes 1-5 [11]. This explains the high level of crossreactivity among the S. flexneri serotypes, which extends in some cases to E. coli strains. On the other hand, S. sonnei does not crossreact with E. coli, but with one serotype of Pleisiomonas shigelloides [12]. In S. flexneri, the genes involved in the biosynthesis of LPS are mainly chromosomal, but some of them have been identified on the virulence plasmid (C. Parsot, unpublished data, 2000); however, whether these plasmid genes are functional has not yet been addressed. Some of the chromosomal genes that determine 0-sidechain specificity are carried by lysogenic bacteriophages [13]. In S. dysenteriae 1, the LPS biosynthesis genes are located both on the chromosome and on a 9-kb plasmid [14]. In S. sonnei, the genes encoding LPS 0-sidechains are all located on the virulence plasmid, which is easily lost on subculturing, thus generating stable rough Form II colonies from the unstable smooth Form I colonies carrying the plasmid [15, 16].
IV. Epidemiology and Transmission The most common Shigella species in the developing world are S. flexneri and S. dysenteriae 1. They accounted for 66 and 16% of hospitalized cases of shigellosis in Bangladesh, respectively, in the early 1980s [17]. S. flexneri is primarily responsible for the endemic form of the disease, whereas S. dysenteriae accounts for the epidemic form of the disease. S. boydii is rarely encountered and seems essentially associated with cases of shigellosis on the Indian subcontinent. In industrialized areas. Shigella epidemic outbreaks are dominated by S. sonnei. The transition from S. flexneri to S. sonnei is associated with economic development. The reasons underlying this interesting association between socioeconomic context and prevalence of a particular species of Shigella are currently unknown.
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Also, the mechanisms that cause severe dysenteric forms of the disease in some patients and mild purely diarrheic forms in others are likely to respond to the nature and size of the infecting inoculum, as well as to the host immune status and the host genetic background. The exact incidence of shigellosis is difficult to assess. Recent extrapolations made from reliable epidemiological data selected on a worldwide basis indicate that there are about 145 million cases every year, 99% of which take place in the developing world, with children under the age of 5 years being the principal victims [4]. For instance, in a poor suburban area of Santiago (Chile), a child had a 67% chance of developing shigellosis in the first year of life when this extensive study was carried out in 1991 [18]. Mortality due to shigellosis reaches between 500,000 and 1.5 million every year [4] and is particularly associated with epidemics developing in a dramatic public health context. Rapid constitution of refugee camps such as those that recently formed in the Central Lake area of Africa is a typical example of such emergency situations. Lack of sanitation and of a safe water supply, absence of personal hygiene, stress, malnutrition, concurrent infections, and antibiotic resistance are likely to explain the high rate of mortality in those situations in which S. dysenteriae 1, the most virulent Shigella, is the etiological agent. Accurate mortality data have been obtained in Bangladesh, where Shigella infection may account for up to 20% of the total mortality among children between the ages of 1 and 4 years [19]. Studies carried out in the 1980s showed that the mortality rate in hospitalized Bangladeshi children with Shigella dysentery was 10%, with a surprisingly similar rate of death regardless of species or serotype [19]. During epidemics, which are often due to S. dysenteriae 1, attack rates have been calculated to range from 1 to 50%, and the mortality rate from 6 to 70 per thousand. The only natural hosts of Shigella are humans and monkeys. Most of the disease transmission occurs via person-to-person contact, the bacteria being able to survive on the skin. Shigella is also often transmitted by contaminated food and water. Flies can transmit Shigella from human feces to food [20]. Due to oral/anal and oral/genital sexual practices, shigellosis is also considered a potentially sexually transmitted pathogen that became very prevalent in some homosexual communities in the 1980s [21]. As already mentioned, Shigella is highly infectious. Striking examples are the occurrence of large outbreaks of shigellosis following accidental contact between a sewage pipe and an urban water supply system in Haifa (Israel), thereby causing 8,000 cases of shigellosis within a week [22], or a food contamination in the United States in which more than 50% of the 12,700 persons attending a mass gathering contracted S. sonnei infection. These striking epidemiological observations reflect the results of experimental infections carried out with volunteers in the United States, during the course of which it was established that as few as 10
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microorganisms administered orally could cause infection [10], and a few hundred induced a high attack rate that was not increased in frequency and severity by further increasing the infectious dose [23]. Acid resistance may be part, but not all, the explanation for the high infectivity of Shigella, which is not seen with other enteric pathogens, such as V cholerae, which requires much higher doses (i.e., 106-10^ cfu).
V. Disease Symptoms and Complications: Orientations for Future Research!?
A. A Spectrum of Disease Symptoms from Diarrhea to Dysentery As already mentioned, there are essentially two patterns of illness caused by Shigella. The first is the classical bacillary dysentery, characterized by fever, major intestinal discomfort with intestinal cramps and tenesmus, as well as permanent emission of a fecal, bloody, mucopurulent stools. This clinical form is characteristic of infections caused by S. dysenteriae 1 and S. flexneri. Second is a more benign episode of watery diarrhea that may last several days, which is typically associated with S. sonnei. There are, however, severe cases of S. sonnei infection [19] and mild cases of S. flexneri infection; therefore, the intrinsic capacity to cause dysentery seems to be present in all strains, regardless of species and serotype. The watery diarrhea that often precedes the dysenteric form of the disease, as well as the purely diarrheic form of the illness, raise the yet-unsolved problem of the pathogenesis of the diarrheal component of this disease. It is well established that shigellosis leads to colonic dysfunction, which is characterized by net decreased absorption of water, increased secretion of potassium ions, and decreased absorption of chloride ions [24]. There are two, possibly complementary, explanations for these symptoms. First, diarrhea represents the minimal disease symptom if the invasive process remains limited in severity and extension. This situation is classically observed in inflammatory bowel diseases such as ulcerative colitis. Second, diarrhea reflects the production of one or several "classical" enterotoxins that have been recognized in S. flexneri and will be mentioned later in this chapter. It should also be emphasized that about 50% of Shigella infections are asymptomatic [25]. In experimental infections of volunteers, the classical "symptomatic triad" of shigellosis—fever, abdominal pain, and bloody mucopurulent stools—was seen in fewer than 50% of the volunteers who developed the illness [26].
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Acute Complications of Shigellosis
The most frequent causes of death among hospitaHzed children during shigellosis are septicemia, primarily seen in malnourished children [27], and hypoglycemia. Septicemic strains are not necessarily Shigella, because other Gram-negative microorganisms are observed in about half of cases [19]. This is due to rupture of the epithelial barrier facilitating passage of other bacteria from the intestinal flora. The hypoglycemia may result in blood glucose concentrations as low as 1 mM, regardless of the nutritional status of the child. As insulinemia is normal in this situation, a block in gluconeogenesis is likely to occur, as the concentrations of glucose-releasing hormones such as glucagon, epinephrine, and norepinephrine are high [28]. Relevant animal models mimicking these processes are required in order to study these situations at the experimental level. Toxic megacolon, a major widening of the colon that becomes atonic, possibly leading to intestinal perforation with peritonitis and severe sepsis, is of poor prognosis. The pathogenesis of this complication is not understood. It occurs regardless of the infecting species, thereby ruling out a major role for Shiga toxin. It is likely that severe underlying inflammation caused by mucosal invasion largely accounts for this complication. The relationship existing between inflammation and malfunction of the autonomous colonic nervous system needs to be further investigated. Among the classical complications of shigellosis are seizures. They may appear in the absence of hypoglycemia [28, 29], and their pathogenesis is not clear. They are seen in children regardless of the infecting strain, thus ruling out an exclusive role of Shiga toxin, which has been shown to be neurotoxic, in their pathogenesis. The hemolytic and uremic syndrome (HUS) occurs essentially as a complication of infections by S. dysenteriae 1, thereby suggesting a major role played by Shiga toxin in this process (see below), and in agreement with the phenotypes reported for its homologs in EHEC infections [30]. Ongoing experiments carried out in animal models of HUS should soon unravel the molecular and cellular mechanisms underlying this complication and possibly indicate prophylactic and therapeutic approaches [31]. Similarly, the pseudo-leukemoid syndrome in which white blood cells may reach 40 x 10^/L can be observed either in the presence or the absence of an HUS. It has no consequence in itself but reflects a serious condition and is associated with a poor prognosis [32]. It is likely to reflect a major inflammatory status, possibly with high circulating levels of cytokines (such as IL-6, G-CSF and GM-CSF) stimulating leucopoiesis. In the mouse model of pulmonary infection by Shigella, a strict correlation exists among the severity of the inflammatory process, the numbers of colony-forming units in lungs, and the levels of circulating IL-6 [33].
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C. Long-Term Complications of Shigellosis Malnutrition is a severe consequence of serious cases of shigellosis. Shigellosis has been recognized as a major cause of chronic malnutrition in children [19]. The pathogenesis of this condition is probably multifactorial, associating lack of appetite and limited malabsorption, the latter having a weak impact as the small intestine is not affected by Shigella. However, it is likely that the persistence of intestinal inflammation with colonic protein loss and the systemic effect of elevated levels of TNF-a accounts for this condition. This is supported by the observation that the number of cells producing proinflammatory cytokines remains identical in rectal biopsy samples taken at the acute phase of shigellosis and a month later [34, 35]. Reactive arthritis following shigellosis is a condition more frequently observed in patients expressing the HLAB27 haplotype.
VL Histopathology of Shigellosis: A Window on Pathogenesis Colonoscopic examination of patients at the acute phase of shigellosis shows that the rectosigmoid area is constantly affected by the inflammatory process. The proximal segments of the colon, and the ileum, are less often affected. Segmentous localization is another characteristic that is shared between shigellosis and ulcerative colitis. However, the reason for this rather selective localization of the lesions is unknown. The intestinal lesions are diffuse and continuous; they comprise edema, erythema, focal hemorrhages, and often a white mucopurulent layer of adherent exudate resembling false membranes [36]. On histopathological examination of these lesions, it appears that the mucosa is primarily affected, whereas the submucosa is rather spared by the inflammatory infiltrate. When seen, the bacteria are usually localized to the epithelium of the surface and upper thirds of colonic crypts. The relationship between the degree of bacterial invasion of colonocytes and colonocyte damage is not obvious. In addition, when rectal biopsies are performed during the early stages of infection, the epithelium overlying lymphoid follicles (i.e., follicle-associated epithelium or FAE) is damaged by Shigella infection [37]. The corresponding macroscopic lesions may be aphthoid ulcers, as observed in Crohn's disease. These observations raise two major points that will be considered later: (1) the route of Shigella translocation through the epithelial lining at the initial stage of the disease that seems to correspond to the FAE, and (2) the mechanisms of the destructive process that extends far beyond the sites of
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bacterial invasion, thus indicating that the development of shigellosis is characterized by an uncontrolled and diffuse inflammatory process responding to a cascade of amplification signals that still needs to be characterized. An exudate made of colonocytes, PMNs, bacteria, and red blood cells in a fibrin layer is observed in the intestinal lumen, the epithelium showing major signs of degeneration with damaged cells, sometimes in the process of shedding, and empty goblet cells. The epithelial lining is often infiltrated by white blood cells—particularly PMNs and lymphocytes [38]—and in some places it undergoes complete detachment. In the crypt epithelium, activated intraepithelial lymphocytes are seen as well as numerous PMNs. Monocytes and eosinophils can be seen, although at a later stage of the disease. The lamina propria is infiltrated by PMNs, monocytes/macrophages, and plasma cells. Widespread vascular lesions are seen, from swollen or pyknotic endothelial cells to total destruction of capillaries. Thrombi may be observed in larger local vessels [38]. At a later stage of the disease, dilated, elongated, and branched crypts may be observed, some of them heavily infiltrated by PMNs, thus forming crypt abscesses [36].
VIL Animal Models: Strengths and Weaknesses The oldest animal model for shigellosis is the keratoconjunctivitis assay or Sereny test [39]. This assay consists of instillating a suspension of bacteria in the keratoconjunctival sac of a guinea pig. Pathogenic shigellae invade the conjunctival layer, causing an acute destructive conjunctivitis that is characterized by redness of the eye followed by a keratitis with massive migration of inflammatory cells, particularly PMNs, which cause corneal turbidity and purulent discharge closing the eyelids [40]. Bacterial mutants that cannot invade epithelial cells, are unable to spread from cell to cell, or express a rough LPS, are negative in the Sereny test. This assay reflects the invasive phenotype of Shigella both in terms of invasion of cells and cell-to-cell spread, and in terms of triggering an inflammatory response. It does not reflect, however, the specificity and complexity that can be observed in histopathological studies in humans. During the 1950s, an impressive series of studies was carried out in order to identify animals (essentially mammals) that would develop a typical dysenteric syndrome in the presence of an oral or a gastric inoculum of Shigella [41]. Only monkeys (particularly macaques), young bears, and kittens developed the disease when orally infected with 10^^ microorganisms. From these studies, only macaque monkeys, who develop an invasive rectocolitis when orally or intragastrically infected with Shigella, have "survived" as one of the most faithful animal models.
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This model has been extensively used for pathogenesis studies and for testing of live oral attenuated vaccines [42]. Yet, ethical and experimental drawbacks make it unsuitable for routine studies. These drawbacks include the high cost of animals and their housing. In addition, the infectious doses required for the animals to develop dysentery are so high (i.e., 10 million to 100 million times greater than the infectious dose in humans) that the relevance of the model can be questioned, particularly in the context of testing the tolerance of an attenuated vaccine candidate, or doing challenge experiments in vaccinated animals. Guinea pigs are infectable orally or intragastrically; however, they develop a dysentery-like syndrome only if starved 4 days before the inoculation, which must be preceded by administration of streptomycin in order to disrupt the barrier effect of the resident intestinal flora, and morphine in order to block intestinal peristaltism. Two models are of value for routine pathogenesis studies. The first is a mouse model in which the Shigella inoculum is administered intranasally, resulting in invasion of the tracheobronchial tract that causes a massive inflammatory bronchotracheal alveolitis [43]. Although this model is not relevant with regard to the organ specificity of Shigella infection, it has the advantage of making use of an animal in which the immune system has been explored in such detail that most of the tools are available to study the immunoinflammatory components of the disease, as well as some aspects of the systemic and local immune protection against Shigella infection [33,44,45]. Alternatively, the rabbit ligated-loop model of infection consists of using intestinal loops that are ligated after laparotomy under general anesthesia, the vasculature being carefully preserved. A large inoculum of bacteria is then injected into the loops, and the invasive and inflammatory processes can be followed by sacrificing animals at given times (usually between 2 and 16 hr). Histopathological studies can then be carried out, and proinflammatory cytokines dosed. This model is particularly useful for studying the role of specific cytokines, such as interleukin-1 (IL-1) and interleukin-8 (IL-8), in the development of intestinal invasion, mucosal inflammation, and tissue destruction [46, 47], and to better analyze the role of the follicular-associated epithelium in the initial steps of epithelial translocation [48, 49].
VIIL Cellular Models of Infection: The Contribution of Shigella to ttie Concept of Cellular Microbiology In v/Yro-cultured cells (e.g., HeLa cells, Henle cells, Hep-2 cells) have been used to study the capacity of Shigella to penetrate into cells that are not professional
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phagocytes [42]. In the 1980s, these tests were refined and became quantitative. The "gentamicin protection assay," in which the antibiotic remains extracellular, thereby killing bacteria that have not entered cells but sparing intracellular bacteria, allowed to quantitate bacterial entry into host cells. The "plaque assay" [50] and the "infectious focus assay" [51 ] both allowed the study of cell-to-cell spread on confluent cell monolayers. Combined with molecular genetic analysis of bacterial pathogenicity, these tests have allowed identification of the bacterial factors involved in entry, escape into the cytoplasm, and intracellular motility/cellto-cell spread. They have also uncovered the major eukaryotic cell components supporting these processes, particularly the actin cytoskeleton [52]. These approaches helped establish the concept of cellular microbiology that analyzes microbial pathogenesis at its intersection between the prokaryotic and eukaryotic worlds [53]. According to this concept, virulent Shigella are characterized by expression of an "invasive phenotype" whose effect depends on the target cell: entry/escape into cytoplasm/intracellular motility/cell-to-cell spread in the presence of epithelial cells; the complete invasive phenotype being best observed when polarized epithelial cells such as Caco-2 or T84 cells are infected basolaterally [54]; apoptotic killing and maturation/release of IL-ip in macrophage [55, 56]; activation of PMNs adherence; and release of granule contents [57]. The cell-dependent aspect of this "invasive phenotype" is summarized in Figure 1. More recently, these cell assays have been made even more sophisticated by growing the epithelial cells on filters in order to induce their polarity, and by establishing complex systems combining epithelial cells and PMNs in order to mimic some aspects of the inflammatory response [58, 59].
IX. Pathogenic Mechanisms: In Vitro Expression of the Invasive Phenotype A. Molecular and Cellular Mechanisms of Shigella Invasion of Epithelial Cells: Basic Principles and Reviews The pathogenic factors involved in Shigella invasion of epithelial cells have been extensively studied and reviewed [52, 60-65]. These factors participate in entry into nonphagocytic cells by a macropinocytic event requiring massive cytoskeletal rearrangements [66], escape of the bacterium into the cytoplasm [67], intracellular multiplication, and cell-to-cell spread [51, 68, 69]. Shigella can enter several cell lines in vitro, regardless of the species and organ of origin.
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+++ (CYTOPLASMIC)
HOST CELL RESPONSE
^„.^„^,... ^.... EPITHELIAL CELL
INTERNALISATION PRODUCTION OF INFLAMMATORY CYTOKINES/CHEMOKINES (IL-8..) MONOCYTE/MACROPHAGE INTERNALISATION RELEASE OF ILlp APOPTOSIS
+++ (CTTOPLASMIC)
POLYMORPHONUCLEAR LEUCOCYTE INTERNALISATION RELEASE OF GRANULES (INTRAVACUOLAR)
ENHANCED EXPRESSION OF ADHERENCE MOLECULES
Fig. 1 Differential expression of the Shigella invasive phenotype depending on whether epithehal cells, macrophages, or polymorphonuclear cells are infected.
B.
Molecular and Cellular Biology of the Entry Process into Epithelial Cells 1.
THE PARADIGMS OF BACTERIAL ENTRY INTO EPITHELIAL CELLS
Two paradigms of the entry process have been described. First, the "zippering" process, which involves intimate contact between the bacterial surface and the host cell membrane, is mediated by a bacterial surface ligand binding with high affinity to a cell membrane receptor involved in cell adherence [70]. The Y. pseudotuberculosis invasin, which binds with high affinity to integrins of the (31 family [71], and Listeria monocytogenes intemalin A, which binds to E-cadherin [72], illustrate this "zippering" phagocytic process. Second, the "triggering" process, which is closely related to macropinocytosis, results in bacterial internalization in a vacuole that initially is loosely associated to the bacterial body. Shigella and Salmonella, which both secrete a set of homologous invasion proteins on contact with their cellular targets, induce important but localized rearrangements of the cell cytoskeleton at their site of interaction. Actin polymerization is essential for bacterial entry since it is abrogated by cytochalasins. Cellular extensions rise to 10 |Lim over the cell surface, forming a flower-like
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Fig. 2 Entry of Shigella into epithelial (HeLa) cells, (a) Scanning electron microscopy showing an ongoing entry focus characterized by membrane projections getting organized in multiple ruffles that eventually form a macropinocytic vacuole. Reprinted through the courtesy of Ariel Blocker and Roger Webf, EMBL (Heidelberg, Germany), (b) Transmission electron microscopy showing the section of an entry focus with multiple projections characterized by massive rearrangements of the cell subcortical cytoskeleton. Reprinted through the courtesy of R Gounon and P. Sansonetti, Station Centrale de Microscopic Electronique, Institut Pasteur. Bars = 1 |im.
Structure (Fig. 2), both in the case of Salmonella [73] and Shigella. These projections merge and engulf the bacterial body within 5 to 10 minutes [74]. These membrane projections are supported by newly formed actin filaments. These filaments are oriented with their fast-growing "barbed" end facing the inner face of the cell cytoplasmic membrane, and bundled by plastin, which is necessary to
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Stabilize the projections, and required for efficient bacterial entry [74]. The signals eliciting these massive cytoskeletal rearrangements are the result of a complex crosstalk between the bacterium and its target cell. In confluent, polarized epithelial layers (i.e., Caco2 or T84 colonic cell lines of human origin) reconstituted in vitro on a tissue culture dish, or on a filter. Shigella is unable to significantly enter cells. Only direct contact with the basolateral pole of these cells allows efficient entry [54]. This paradox has been solved in complex cellular models of infection, or in in vivo studies in which the invading bacteria and their respective mutants are traced, according to kinetics of infection of the intestinal mucosa.
2.
GENES AND GENE PRODUCTS REQUIRED FOR SHIGELLA ENTRY
Invasive strains of S.flexneri harbor a 220-kb plasmid that contains most of the identified virulence genes of this microorganism [15]. A 30-kb locus in this plasmid contains all the genes necessary for entry [75-77]. This region is divided into two divergently transcribed operons that encode two classes of proteins (Fig. 3). The mxi-spa locus comprises about 20 genes specifying a type III secretory apparatus [78-81], which is expressed and assembled at 37°C and activated on contact with the target cells [82, 83]. Homologs of this system are present in many Gram-negative pathogenic bacteria [84]. These systems seem essentially devoted to allow translocation of bacterial effectors (i.e., toxins) directly into the cytosol of the eukaryotic target cells; they are functionally conserved among these different species. In the presence of proper chaperones, heterologous secretion of virulence proteins has been shown in Shigella, Salmonella, and Yersinia [85, 86]. One of the best-characterized translocation processes is the Yersinia Ysc type III secreton, which, on contact with macrophages, allows secretion of Yop proteins through the Ysc complex. One of them, YopB, forms a pore across the host cell membrane through which other secreted proteins such as YopE and YopH are translocated into the cytoplasmic compartment of the host cell, resulting in
• t virB
^fe
ipgD
a M ipaA
ipaD
ipaC
ipgF mxiH mxU mx'iK mxiM mxiD ipgE mxiG mxU mxiL mxiE
ipaB ipgC ipgA icsB ipgB
mxiC
mxiA
T^ ipgD
spa47 spa32 spa33 spa9 spa40 spa 15 spalS spa24 spa29
Fig. 3 Genetic map of the Shigella flexneri 5 entry locus. This 30-kb region is located on the large virulence plasmid of this species, pWRlOO. It comprises two subloci transcribed in opposite directions. (Top) The ipa operon encodes the entry effectors genes (shaded in gray). (Bottom) The mxi and spa operons encoding the components of the type HI secretory apparatus (shaded in gray).
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inhibition of phagocytosis [87]. In Shigella, a complex formed by the IpaB and IpaD proteins regulates secretion of the Ipa proteins [83]. Optimal secretion occurs during the exponential phase of growth [88]. The host compounds that activate the Mxi-Spa secretory system are now being identified. Fibronectin has been proposed as a possible candidate [89]. In addition, dyes such as Congo red and some of its structural equivalents are able to induce active secretion of the Ipa proteins, and may provide some insight into the structure of the components that are able to induce secretion [90]. The Mxi-Spa system of Shigella secretes about 15 proteins that share such common features as the lack of signal peptide and the capacity to aggregate in an extracellular milieu in large supramolecular structures [91]. The ipa locus, which essentially consists of an operon, encodes four proteins— IpaB (62 kDa), IpaC (42 kDa), IpaD (37 kDa), and IpaA (70 kDa)—which are secreted by the type III Mxi-Spa system on contact of bacteria with host cells. It also encodes IpgC, an 18-kDa cytoplasmic chaperone that binds IpaB and IpaC in the bacterial cytoplasm, preventing their aggregation and proteolytic degradation [92]. In-frame deletions of the ipaB, ipaC, and ipaD genes lead to complete inactivation of the bacterial entry phenotype, indicating their essential role in entry [93], whereas inactivation of the ipaA gene leads only to partial inactivation of entry [94]. Once secreted, IpaB and IpaC form a complex [92] interacting with the epithelial cell membrane [94a]. Latex beads coated with the IpaB-C complex are internalized by HeLa cells, indicating that this molecular complex acts as an entry effector [95]. However, the surface rearrangements observed during internalization of the beads are not as strong as those observed during bacterial entry, suggesting that the entry mechanism is only partially reproduced by the IpaB/C complex. There are two major possible explanations for these results: either some secreted proteins are missing from the complex, or immobilization of the complex on the bead surface allows only partial interaction of the secreted proteins with their cellular targets. The mode of action of these proteins is still unclear. The Ipa complex has been shown to bind a fibronectin receptor, a5pi integrin [96], but integrins do not appear to be exclusive receptors for the IpaB-C complex. Another cell surface receptor, CD44, the hyaluronate receptor, binds IpaB [96a]. These interactions may contribute to an adherence stage, which represents only a preliminary step in the entry process. It is possible that, after transient binding to one or several receptors, such as the a5pl integrin and/or CD44, the IpaB-IpaC complex inserts into the epithelial cell membrane, forming a pore or translocator structure that allows further injection of other effector proteins into the cell. This is based on analogies with the Ysc type III secretion system of the Yops and with the ability of IpaB and IpaC to insert into and destabilize lipid bilayers [97, 97a]. Insertion of this IpaB-IpaC complex may cause two different effects: (1) induction of nucleation and polymerization of actin filaments supporting membrane projections, and (2) translocation of other Ipa proteins such as IpaA and other Shigella proteins secreted on cell contact. Ipa proteins are likely to bypass the usual processes of outside signaling in which extracellular ligands such as growth
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factors bind the extracellular domain of a transmembrane receptor that becomes activated and initiates a signal cascade causing cytoskeletal rearrangements. A current hypothetical model for the secretion and translocation of the Ipa proteins is shown in Figure 4. 3.
SIGNALING PATHWAYS INVOLVED DURING SHIGELLA ENTRY
Observation by transmission electron microscopy after SI-myosin decoration of actin filaments shows progressive accumulation of dense dots underneath the cytoplasmic membrane of the epithelial cell at the site of interaction with the bacteria. These dots correspond to actin nucleation zones from which filaments
0 Fig, 4 Hypothetical scheme showing activation of the Mxi-Spa apparatus in the presence of the target eukaryotic cell membrane, formation of a pore by the IpaB-IpaC complex, and injection of proteins such as IpaA. There is no current evidence that the IpaB-IpaD complex, which controls protein secretion, is located at the tip of the secretory apparatus.
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further extend [74]. These filaments support the formation of microspikes that progressively fuse to form membrane leaflets that engulf the microorganism in a large vacuole. As maturation of this entry focus proceeds, a dense meshwork of polymerized actin accumulates around the vacuole, forming a cup that may represent an essential step for internalization of the macropinocytic vacuole. These steps are summarized in Figure 5. The recruitment of most of the cytoskeletal-associated proteins seen in focal adherence complexes indicate that there is significant analogy between these structures and the actin cup. If one considers the subdomains of an entry focus, the cytoskeleton-associated proteins can be classified into two major categories: (1) proteins required for signaling such as the three small GTPases of the Rho family—Cdc42, Rac and Rho itself [98, 99, 99a]—and the protooncogene protein p60^"^'^^ [100], and (2) proteins likely to be required for structuring and stabilizing the entry focus architecture (i.e., scaffolding proteins), particularly those forming F-actin bundles such as plastin [74], a-actinin [94], ezrin [96a], and vinculin and talin, which are both involved in focal complexes formation [94]. The location of these structural, motility, scaffolding, and signaling molecules in the two major substructures of an entry focus is shown in Figure 6. 4.
INVOLVEMENT OF SMALL GTPASES
The Rho subfamily of small GTPases regulates specific rearrangements of the cytoskeleton [101]. In Swiss 3T3 fibroblasts, Cdc42 induces the formation of mi-
Filopodes Lamellipodes Adherence-plaque like structure
Fig. 5 The four steps of Shigella remodeling of the eukaryotic cell cytoskeleton leading to a fully functional entry focus. Initial filopodial projections supported by extension of actin filaments undergo a transition to the formation of curtains (ruffle-like) structures. An actin cup eventually forms around the entering vacuole.
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n In Oil W ff r
actin myosin n a-actinin plastin ezrtn RhoB/C Cdc42
Rac focai-piaqiie like stmctiire
,^^
actin myosin 11 >v a-actlnin Ji::> vinculin • paxiiiin \ talin 1 src RhoA
structure motiliiy/scalfoldiiif scaffdidiiig
-
slgita!]zati0&
• stmctiire iiMrtility/scalfoidiiig scaffc^ding
-
signalizatlon
-
Fig. 6 Localization of structural, motility/scaffolding, and signalization-related cytoskeletal proteins to subdomains of the Shigella entry focus.
crospikes and filopodia; Rac is mostly responsible for lamellipodial formation, and Rho is implicated in the formation of focal adhesion complexes and stress fibers. Members of the Rho family act as molecular switches and cycle between their active GTP-bound form and their inactive GDP-bound form. These GTPases can be inhibited by overexpressing an inactive, dominant negative form of the GTPase bearing a mutation in the GTPase domain (asparagine 17 for Cdc42 and Rac, and asparagine 19 for Rho). Rho can also be inactivated by the C3 exoenzyme of Clostridium botulinum that ADP-ribosylates the 21 -kDa Rho molecule on asparagine 41 [102]. Using these approaches, Cdc42 and Rac were shown to be required for entry of Shigella into HeLa cells [99a]. Transient expression of a dominant negative form of Rac or Cdc42 inhibited Shigella entry approximately 70%, due to a dramatic decrease in actin polymerization at the level of entry foci. Rho is also required [98, 99] because both C3 treatment and transient expression of Rho N19 inhibited Shigella entry by 90 and 70%, respectively [99a]. Decoration of actin filaments with S1 -myosin in samples treated with C3 suggests that Rho is involved in the bundling, and possibly also in the elongation of actin filaments rather than in the nucleation of foci [98]. These observations are in contrast with the Salmonella entry process, in which Cdc42, but not Rho, appears to be required [103]. The pathway(s) by which the IpaB-IpaC complex activates the three GTPases is yet unknown. A current model is shown in Figure 7 along with other factors that are presented below. It is likely that the various small GTPases are used in a stepwise process providing initial actin polymerization (i.e., Cdc42 mediated) followed by dynamic remodeling of the actin-made entry focus (i.e., Rac mediated), which ends up in a mature structure (i.e., focal-plaque-like) able to efficiently internalize the bacterial body (i.e., Rho mediated).
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IpaB/C
I
IpaB/C
f
LAMELLBPODIA
IpaA
\TNCUr.IN
FOCAL PLAQUE
Fig. 7 Signaling cascade causing the serial events of cytoskeletal remodeling that lead to a mature Shigella entry focus. Activation of the cascade of small GTPases initiates the actin polymerization process and remodeling of the entry focus. Both cellular and bacterial proteins participate in the remodeling, particularly ezrin, which increases filament extension, and IpaA, which participates in formation of the actin cup.
5.
INVOLVEMENT OF PP60^-^^^
Substrates of the src tyrosine kinase family and src itself are recruited at the site of Shigella entry. Current evidence indicates that c-src is activated since one of its major substrates, cortactin, becomes tyrosine phosphorylated as entry of Shigella proceeds [100]. It is not yet clear whether the cortactin-mediated kinase activity of c-src is required for entry, or if c-src simply recruits focal adhesion components such as FAK and/or paxillin through its SH2 and SH3 domains. Recent demonstration that expression of a dominant negative form of c-^rc inhibited cortactin phosphorylation as well as the efficiency of Shigella entry via a significant reduction of
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the actin rearrangements in the entry focus would indicate that c-src activation is required for bacterial entry [104]. Src could therefore activate cytoskeletal components, or coordinate some key steps leading to the remodeling and maturation of the entry focus. Src and Rho are two major regulators of focal adhesions, consistent with the fact that the formation of SL Shigella entry focus shares similarity with focal adhesion plaque formation. Substrates of the tyrosine kinase Src, such as cortactin, which is the major tyrosine-phosphorylated substrate during Shigella entry, accumulate in the entry focus [100]. Other substrates found in focal adhesion complexes, such as paxillin and pl25FAK, are also present in the entry focus [96] (J. Mounier and P. J. Sansonetti, unpublished results, 1998). Finally, Rho is known to recruit c-src at the cell periphery via remodeling of the cytoskeleton [105]. Phosphorylation of pl25FAK has been shown to occur in conjunction with the interaction between the Ipa complex and a5pl integrins [96]. How important this signal is in the development of the entry focus remains to be determined. 6.
INVOLVEMENT OF "SCAFFOLDING" PROTEINS
Several cytoskeletal proteins are recruited at the entry site of Shigella. These include actin-bundling proteins, which may stabilize the actin extensions that support the entry focus, and has been demonstrated for plastin [74]. In addition, ezrin, which is a member of the ERM (ezrin, radixin, moesin) family is essential to the maturation and functional efficiency of the foci elicited by entering shigellae [96a]. A common characteristic of ERM proteins is to accumulate underneath the plasma membrane in subcellular structures such as microvilli, at cell-to-cell contact sites, and in structures that are dynamically regulated such as filopodia and lamellipodia [106]. In the dynamic contact of Shigella entry, ezrin seems to act not only as a membrane-cytoskeleton linker but also as a molecule mediating extension of cellular projections. 7.
THE "PSEUDO-FOCAL ADHESION" MODEL OF SHIGELLA ENTRY: A ROLE FOR VINCULIN
Several focal adhesion proteins are present in the entry focus [94, 107]. In addition, the Rho GTPase and src are both regulatory components of the architecture of focal contacts. Therefore, it is likely that formation of this "pseudo-focal plaque" is essential for Shigella entry. Vinculin shows a dense pattern of recruitment in close contact with the forming vacuole during the early stages of internalization. A similarly dense recruitment of vinculin can be observed at the level of contact points between the basal surface of the cell and the matrix in the focal adhesions, where the cytoskeleton is anchored to the membrane [108]. Vinculin is thought to play a major role in anchoring the cytoskeleton to the cell membrane. This molecule exists in two conformations: a folded conformation that represents the major pool of vinculin in the cytosol, and an active, open configuration, able to interact with F-actin and actin-binding proteins such as talin and a-actinin, which simultaneously bind the cytoplasmic domain of (31 -integrins [108]. IpaA is able to interact direcdy with
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vinculin during Shigella entry into cells [94], presumably after translocation from extracellular bacteria into the cell. IpaA-vinculin interaction increases entry by a factor of 10. This interaction does not seem to play a role in the induction of the entry foci, but rather in their maturation. Entry foci induced by wild-type Shigella are characterized by a dense network of vinculin and F-actin, localized at the interface between the cell membrane and the bacterial body. This is in contrast to the foci promoted by a AipaA mutant that show massive and prolonged actin polymerization, and diffuse recruitment of vinculin, but no cup structure. It is possible that IpaA binding leads to vinculin activation, thus consolidating the formation of the pseudo-focal adhesion structure that could organize the actin network necessary to anchor the bacterial body in the entry focus. It may also allow efficient recruitment of a-actinin and other constituents that further participate in the assembly of the entry focus. It is likely that several bacterial components act in concert with cytoskeletal proteins or regulatory components of the host-cell cytoskeleton to build up the entry focus and achieve its maturation. C. Escape of Shigella into the Cell Cytoplasm Once intracellular. Shigella lyse the membrane-bound vacuole and gain access to the cytoplasm [67]. Ipa proteins account for the membrane-lysis process [109]. Both purified IpaB [109a] and IpaC [97] are independendy able to lyse lipid vesicles in a pH-dependent manner. Lysis of the vacuole is followed by bacterial multiplication at a rate similar to that observed in broth, thereby reaching a number of several hundred microorganisms per infected epithelial cells. This behavior differs from that displayed by Salmonella that remains intravacuolar, having to adapt to the hostile metabolic conditions that prevail inside this vacuole before they remodel this compartment and acquire the capacity to grow at a rate similar to that of Shigella [110]. Aside from rapid intracellular multiplication, another major consequence of establishing direct contact with cytosolic components is intracellular motility.
D. Intracellular Motility and Cell-to-Cell Spreading of Shigella 1.
The OLM PHENOTYPE
This phenotype is visible when fibroblasts are infected by Shigella [111]. After their escape from the vacuole, shigellae move along the stress cables that radiate from adhesion plaques toward the edge of the nucleus. In this area, they grow as microcolonies. This movement is similar to that of cell organelles and has thus been termed organelle-like movement (Olm). It is clearly visible in cells with a highly organized cytoskeleton such as fibroblasts. The molecular basis of this movement is still unknown. In epithelial cells, Olm movement is hard to detect, with bacteria moving almost immediately by a process in which they induce actin polymerization as shown below.
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E. Actin-Based Intracellular Motility of Shigella 1.
MORPHOLOGICAL ASPECTS OF ACTIN-BASED MOTILITY
Although many bacterial pathogens are able to enter host cells by inducing cytoskeletal rearrangements, few of them invade the cytosol and subvert the host cytoskeleton in order to colonize the entire tissue. Shigella, Listeria, and Rickettsia are three bacterial species that escape from the phagocytic vacuole and use cytoplasmic cytoskeletal components to propel themselves inside the first infected cell before they reach the cell membrane and induce cellular protrusions. Engulfment of these cellular extensions that contain bacteria by neighboring cells leads to cell-to-cell spread of the pathogen [112, 113]. Among these three bacterial species, Shigella was the first pathogen identified as an intracellular spreading bacterium inside infected cells. Using time-lapse video microscopy, Ogawa and colleagues [114] showed that intracellular shigellae were able to move independently of cellular organelles and to induce formation of cellular extensions at the cell surface. Since this pioneering work, it has been shown that Shigella intracellular motility is associated with formation of actin tails at one pole of the bacterium (Fig. 8, see color plate) [69]. Most of the thermodynamic studies of bacterial intracellular motility have been originally done with Listeria and confirmed with Shigella. Using video microscopy and microinjection of labeled actin monomers, it has been shown that the actin tail remains stationary in the cytoplasm, trailing behind the moving bacterium, and that the rate of incorporation of actin monomers correlates directly with the speed of bacterial movement, suggesting that continuous actin polymerization at one pole of the bacterium is itself sufficient to generate the motile force [115]. Shigella movement inside the cytoplasm is random and rapid (6-60 |Lim/min) [116]. It occurs optimally at the stage of bacterial division [117]. Ultrastructural analysis of Shigella actin comet tails by transmission electron microscopy after SI myosin decoration reveals two types of comets. Intracellular comets are composed of short, crosslinked, randomly organized filaments with their fast growing (barbed) ends always oriented toward the bacterial surface. The density of actin filaments is high in the vicinity of the bacterial body and decreases at the distal part of the comet (Fig. 8, see color plate). Comets present in the protrusions are similar to the intracellular ones with a third supplemental distal portion mainly composed of long axial filaments [117a].
2. IcsA, A
SHIGELLA SURFACE PROTEIN INVOLVED IN
ACTIN-BASED MOTILITY
Genetic analysis of Shigella virulence factors has allowed the identification of icsA, a gene that is responsible for F-actin comet tail formation and intracellular movement [69]. The IcsA protein, also named VirG [68, 118], is required for inter-
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cellular spread in in vitro assays and dissemination in the Sereny test. Thus, IcsA is critical for Shigella virulence, and AicsA mutants have been used for the development of live attenuated Shigella vaccine strains [119]. The icsA gene is present on the pWRlOO virulence plasmid of S. flexneri 5a strain outside the entry locus and 15 kb downstream from the spa operon [107]. The G+C content of the icsA gene, 41%, is different from the G+C content of the entry genes, 34%, suggesting independent acquisition of the icsA gene by Shigella. IcsA is an outer membrane protein. Translocation across both membranes and surface anchoring is independent of the Mxi/Spa type III secretion apparatus and is mediated by an autotransporter secretion pathway, similar to the one responsible for IgA-protease secretion [120]. The IcsA autotransporter domain, IcsAP (344 carboxy terminal residues), may form a p-barrel-like structure composed of antiparallel hydrophobic stretches allowing translocation and anchorage of the N-terminal IcsAa domain (710 residues) at the outer membrane [120] (Fig. 9). Apart from a series of 6 glycine-rich repeats (GRRs) of 32 to 34 residues at the N terminus, IcsAa does not exhibit any specific feature [121], nor does it exhibit sequence similarity to any known protein, and this includes cytoskeletal proteins, or the ActA protein that mediates actin-dependent bacterial motility in Listeria [122, 123]. The surface distribution of IcsA is unusual among bacterial proteins. In wild-type bacteria, IcsA is asymmetrically distributed, being present exclusively at the bacterial pole opposite to the septation furrow in dividing bacteria [121]. Inside infected cells, IcsA colocalizes with the base of the actin tail and determines the site of actin assembly and direction of movement. Two factors are required for polar localization of IcsA. A yet uncharacterized intrinsic property of IcsA may privilege addressing of the protein to the bacterial pole, although a significant proportion of it is still expressed over the entire cell surface. In addition, cleavage of 50% of the total amount of IcsA removes the fraction of this protein expressed laterally and leads to exclusive polar distribution. Cleavage occurs at the junction of the IcsAa and IcsAp domains [124] on a sequence previously shown by in vitro experiments to be the target of PKA mediated-phosphorylation: —S756SRRASS762— [125,126]. The bacterial surface protease SopA (IcsP), a member of the OmpT/OmpP family of serine proteases, is involved in cleavage of IcsA [127, 128]. The processing of IcsA by SopA(IcsP) at the bacterial surface leads to secretion of IcsAa into the culture medium. A AsopA mutant is unable to polarize IcsA and unable to induce comet tail formation, suggesting that polar distribution of IcsA is important for bacterial motility [127]. The IcsAa domain is exposed at the bacterial surface. Expression of IcsAa at the surface of a AompTE. coli K12 mutant induces actin comet tail formation and bacterial motility in cytoplasmic extracts of cell-free Xenopus eggs [129, 130]. Expression of IcsAa at the inner face of the eukaryotic plasma membrane induces subcortical actin polymerization and membrane ruffles [130a]. Thus, membrane presentation of IcsAa is necessary and sufficient to induce actin polymerization.
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IcsAa
IcsAp
surface exposed domain (53-758)
membrane anchoring domain (759-1102)
SP ,
N-WASP binding site (103-433)
Autotransporter (623-1102)
Vinculin binding site (103-506)
B 1-Actln Nucleation
3-Capping and Cross-ilnking
2-Elongation
Actin niaments
Capping protein
Actin bundling protein
Proniin Actin
VASP
Vinculin
N-WASP
Fig. 9 (A) Map of the functional domains of the 120-kDa IcsA protein. SP = signal peptide. (B) Scheme summarizing the three major steps of IcsA-mediated actin polymerization leading to intracellular motility of Shigella.
Functional (domains of IcsAa involve(J in actin polymerization have been identified by expressing in-frame truncates on the surface of A/cM bacteria [131, 132]. Expression of the Rio3-Ala433 domain of IcsA, which encompasses the GRR domain, is sufficient to elicit F-actin accumulation [132]. The carboxy-terminal portion of IcsAa (residues I509-T720) seems to be involved in establishment of the asymmetric distribution of IcsA on the bacterial surface and in formation of the actin tail [131].
8. SHIGELLOSIS 3.
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CELLULAR PARTNERS INVOLVED IN ACTIN-BASED MOTILITY
The mechanism of actin polymerization induced by IcsA is still unclear. As the IcsA protein does not interact directly with actin and has no actin-nucleating properties, recruitment of a cytosolic protein complex to the bacterial vicinity is likely to create the proper conditions for actin polymerization and formation of the comet tail [133]. The following steps are currendy considered: (1) actin nucleation, (2) elongation of actin filaments, and (3) capping of filament pointed ends and bundling of filaments, achieving comet stabilization. Several cytoskeletal proteins found in lamellipodial structures have been detected in Shigella actin tails by immunofluorescence analysis. These include T-plastin, a-actinin [134], VASP [135], vinculin [131, 136], Mena, Arp2/3 (C. Egile and P. J. Sansonetti, unpublished results, 1999), and neural Wiskott-Aldrich syndrome protein (N-WASP) [132]. However, the contribution of these proteins to actin-based motility has been demonstrated for only a few of them. Among them, vinculin and N-WASP are the only IcsA ligands so far identified [131, 132]. The role of cellular proteins involved in Listeria actin-based motility (Arp2/Arp3 complex, profilin, VASP, and ADF/Cofilin) [113] in IcsA-mediated motility is currently unknown. Vinculin is a structural protein involved in the crosslinking of actin cytoskeleton to the cell membrane via focal adhesion complexes [137]. Vinculin is present in the entire Shigella actin comet. The globular amino-terminal domain of vinculin interacts in vitro with the amino-terminal portion of IcsA (residues 53-506) [131]. However, the relevance of IcsA-vinculin interaction for Shigella actin-based motility is still debated. Microinjection of an FEFPPPPTDE peptide of ActA or of a (GPPPPP)3 peptide of VASP inhibits Shigella motility [116], and microinjection of the vinculin head portion in Shigella-infccitd cells leads to a threefold increase of bacterial rate of movement [136]. Based on these results, it was proposed that IcsA binding to vinculin may unmask a VASP interaction motif present in the vinculin head portion leading to recruitment of the VASP-profilinactin complex, which could then serve as a molecular scaffold for Shigella actin polymerization [136]. Listeria ActA is also able to recruit VASP at the bacterial surface for formation of the actin comet tail [135, 138]. These observations suggest that Listeria and Shigella have evolved a similar motility mechanism involving VASP recruitment directly in the case of Listeria and indirectly by an ActA analog, such as vinculin, in the case of Shigella. An alternative model was proposed in which vinculin head interaction with IcsA and tail interaction with actin filaments may serve as a link between the bacterial surface and the actin comet [131]. However, Shigella motility appears to be unaffected in a vinculindeficient murine cell line [139], raising the possibility that a functional homolog of vinculin is recruited by IcsA in this cell line. Vinculin does not seem to be involved in the nucleation step, but rather in elongation and stabilization of actin filaments (Fig. 9). Further studies are required in order to identify the precise role of vinculin in Shigella motility.
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N-WASP belongs to the WASP-related proteins family [140-142], which includes WASP, the protein deficient in patients suffering of the Wiskott-Aldrich syndrome [143], the Dictyostelium SCAR protein [144], and its human homolog WAVE [145]. Both WASP and N-WASP interact with Cdc42Hs, but ectopic expression of these proteins generates different actin structures: WASP induces formation of cytoplasmic F-actin clusters [146], while N-WASP generates cortical actin polymerization [140]. In addition, N-WASP enhances Cdc42HsV12-induced formation of filopodia, while WASP antagonizes it [145]. WASP and N-WASP share several functional domains, including a verprolin-homology domain, a cofilin-homology domain, and an acidic domain. N-WASP may act as a severing or depolymerization factor of actin filaments. The acidic verprolin-cofilin (VCA) domain generates uncapped small actin filaments, and subsequent profilin-based elongation may induce polymerization of new actin filaments. The VCA domain is probably buried inside N-WASP, thus requiring the Cdc42Hs-N-WASP interaction to unmask it. Several lines of evidence suggest a role for N-WASP during the nucleation step of Shigella actin comet tail formation [132]. First, during Shigella infection, N-WASP is recruited to the bacterial pole. Recruitment of N-WASP requires interaction of the verprolin region of N-WASP with the glycin-rich repeats of IcsA (residues Rio3-Ala433). Second, Shigella motility is impaired in cells expressing an N-WASP form lacking four conserved residues of the cofilin-homology region, suggesting that this domain is required for comet tail formation. Third, immunodepletion of N-WASP in Xenopus extracts abolished bacterial motility, and add-back of recombinant N-WASP could only restore the nucleation step. Unexpectedly, N-WASP-mediated nucleation during Shigella motility does not seem to require Cdc42. Inactivation of Rho GTPases by TcdB toxin in infected cells or in Xenopus extracts has no effect on bacterial motility [99a]. Thus, it is likely that IcsA binding to the verprolin domain of N-WASP could itself unmask the VCA domain, thus bypassing the requirement for Cdc42Hs.
4.
CELL-TO-CELL SPREAD
When contact occurs between a moving organism and the inner face of the cytoplasmic membrane, a protrusion is formed that is phagocytosed by the adjacent cell [51, 147]. This process involves interaction with components of the cellular junction, allowing bacterial passage via an actin-driven protrusion into the adjacent cell. Expression of cadherins is a prerequisite to allowing phagocytosis of these protrusions by the adjacent cells [51]. Once absorbed by the adjacent cell, the bacteria are trapped inside a pocket surrounded by a double membrane, which is subsequently lysed, a process that needs the intervention of IcsB, a 57-kDa protein encoded by a gene located upstream from the ipa genes in the entry locus [148]. Because icsB mutants remain trapped inside large vacuoles, the
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IcsB protein is likely to be required for membrane degradation, although this has not been direcdy established. In summary, in the context of an epithelial lining, the invasive phenotype of Shigella leads to an efficient process of intracellular colonization. It encompasses entry into nonphagocytic cells, escape to the cytoplasm, intracellular growth, intracellular motility, and cell-to-cell spread. This multifactorial process is a spectacular example of integradon of several defined steps leading to progression of infecdon in a sanctuary that is relatively protected from humoral and cellular effectors of the innate and adapdve immune response (Fig. 10). F.
Apoptotic Killing of Macrophages and Induction of the Release of Mature IL-ip by Shigella
Shigella are phagocytosed by macrophages, but, unlike their noninvasive isogenic mutants, the wild-type strains escape from the phagosome and induce apoptodc death of the host macrophage (Fig. 11), causing typical DNA fragmentadon after 2-3 hours of incubadon [55]. This observadon has been confirmed in vivo in infected Peyer's patches in the rabbit ligated-loop model. A background level of apoptosis is detected in this area when infection is carried out with a noninvasive mutant, whereas numerous apoptodc cells can be seen (Fig. 12) only if an invasive Shigella is used [149]. All invasive clinical isolates belonging to the different species of Shigella induce macrophage apoptosis [150], and the number of
Ipa^/Mxi-Spa
IcsB Ipa^/Mxi-Spa?
Fig. 10 Scheme of Shigella entry, escape into the cytoplasm, and intracellular and cell-to-cell spread, with reference to the effectors required for each step of invasion of a polarized epithelium.
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Fig. 11 Shigella-induced apoptosis of J774 macrophages, (a) Invasive Shigella escaping into the cytoplasm and causing a typical process of apoptosis to its host macrophage after 2 hr of incubation. Note the shrinking nucleus with peripheral condensed chromatin and the formation of pits by the cytoplasmic membrane, preceding cellular fragmentation, (b) Noninvasive Shigella mutant phagocytosed by a J774 macrophage showing an intact host cell in spite of numerous intracellular bacteria and extended incubation period. Bacteria remain inside individual vacuoles. Reprinted through the courtesy of M. C. Prevost, A. Zychlinsky, and P. Sansonetti, Station Centrale de Microscopic Electronique, Institut Pasteur. Bar = 1 )im.
apoptotic cells appears significantly higher than the background level in the rectal mucosa of patients experiencing bacillary dysentery [151]. Apoptosis remains restricted to monocytes/macrophages and does not occur during epithelial cell infection [152].
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Fig. 12 Evidence for apoptotic cells in the dome of lymphoid follicles infected by Shigella, (a) Infected macrophage at early stage of apoptosis. (b) Tingible bodies corresponding to phagocytosis of several apoptotic cells by a macrophage. Reprinted through the courtesy of M. C. Prevost, P. Gounon, and P. Sansonetti, Station Centrale de Microscopic Electronique, Institut Pasteur. Bars = I |Lim.
IpaB is required for apoptotic killing of macrophages, provided that the bacteria are phagocytosed and the vacuoles disrupted. The cytotoxicity of IpaB has been demonstrated both by a genetic approach [153] and, more direcdy, by microinjecting the purified protein into macrophages [154]. The mode of action of IpaB appears to reside essentially in its capacity to bind to Caspase-1 (ICE), the IL-ip-converting enzyme [155]. It belongs to the growing family of cysteine
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proteases that act in a proteolytic cascade involving initiator and effector caspases, the latter achieving degradation of various substrates, eventually causing programmed cell death. All caspases are made as proenzymes and subsequently cleaved into a prodomain and two polypeptides that dimerize or tetramerize to form the active enzyme. Activation of these proteolytic cascades often responds to activation of cell surface receptors by their specific ligands (e.g., binding of Fas to FasL, TNF-a to TNF-Rl, or Apo3L to Apo3). This leads to activation of the initiating Caspase-8. Activation of caspases can also respond to the occurrence of internal damage, particularly in mitochondria, thus causing release of cytochrome c and activation of the initiating Caspase-9. However, Caspase-1 belongs to a group of proinflammatory caspases whose actual role appears to be cleavage of pro-IL-lp and IL-18. Its role in the induction of cell death is so far unknown, despite its homology with the Caenorhabditis elegans CED3 cell death protein [156]. Caspase-1 knockout mice develop normally and do not show any defect in the various processes that require apoptosis [157]. It is likely that the binding of IpaB on the Caspase-1 molecule [154, 158] activates its cryptic proapoptotic potential. The fact that Caspase-1 mediates IpaB-induced macrophage death is supported by the observation that the YVAD-CHO tetrapeptide, which inhibits Caspase-1 and closely related caspases, protects macrophages against Shigellainduced apoptosis [154]. Moreover, peritoneal macrophages from Caspase-1 knockout mice are not killed by invasive Shigella [158]. When activated by LPS, macrophages express cytokines such as TNF-a, IL-6, and IL-1. However, IL-ip remains intracellular as a promolecule until these macrophages are challenged by invasive Shigella. Shigella-infecied macrophages start producing and releasing large quantities of this strongly proinflammatory cytokine under its mature 17-kDa form [56, 158] before cell death occurs. Therefore, Shigella has evolved a remarkable strategy combining two potentials: (1) efficient killing of its front line predator, the macrophage, and (2) the use of this process to initiate inflammation, thereby destabilizing the intestinal tissue. This original strategy, compared to other pathways of programmed cell death, is summarized in Figure 13.
G. Activation of PMNs Also Reflects the Shigella Invasive Phenotype Unlike monocytes/macrophages, PMNs do not undergo apoptotic killing in the presence of Shigella. Moreover, in vitro, shigellae are unable to lyse the endocytic vacuole and are consequently killed [159]. A major effector of bacterial death is the bactericidal/permeability-increasing protein (BPI) [160]. This suggests that PMNs are the major phagocytic cells involved in the killing of Shigella. However, it has also been shown that infection of PMNs by invasive shigellae enhances their expression of adherence molecules and release of bactericidal granules [161]. Even though the outcome of the interaction is bacterial death, activation of
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I
Fas/FasL
EC
ic
Alteration of Intracellular membranes and/or Mitochondria
C^^^
Apoptosis
^r
InilammatioiT Fig. 13 Molecular bases of IpaB induction of macrophage apoptosis and IL-1 [3-mediated initiation of inflammation. IpaB-mediated activation of the Caspase-1 pathway is compared to other established pathways of induction of apoptosis in cells.
PMNs is likely to enhance to the proinflammatory potential characteristic of the Shigella invasive phenotype, thus causing further tissue destruction.
X. Pathogenic Mechanisms: In Vivo Expression of the Invasive Phenotype After oral ingestion, Shigella progresses along the intestinal tract until it reaches the mucosal surface of the colon and rectum, which it eventually invades, causing inflammation and tissue destruction.
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A. Does Shigella Express Organ-Specific Adhesins? S.flexneri carrying the virulence plasmid are more hydrophobic and adhere 10 times better to eukaryotic cells than plasmid-less mutants [162]. This observation cannot account for organ specificity, and the basis for colonic and rectal specificity of Shigella is currently unknown. Like almost all Enterobacteriaceae, Shigella harbors mannose-specific type I fimbriae [163], the expression of which can be induced in vitro by serial passages in aerobic conditions in liquid medium [164]. Type 1-piliated strains bind to isolated human colonic epithelial cells in a mannose-dependent manner [163]. Although no role has been ascribed to type 1 pili, one can speculate that they help survival in the environment, or may be involved in adherence to the colonic and rectal surfaces. Recent electron microscopic studies have shown that fresh clinical isolates oi Shigella can express at least two additional types of fimbriae [165]. Another study has shown that the binding of S. flexneri to colonic epithelial cells of guinea pigs could be inhibited by fucose, A^-acetyl neuraminic acid, and N-acetyl mannosamine [166]. Shigella may therefore express various carbohydrate-specific adhesins on its surface. Identification of an organ-specific adhesin has become a priority, and a strategy to identify such candidate molecule(s) needs to be defined. As described below, M cells, which are part of the follicular-associated epithelium, seem to represent the major entry route of Shigella. Lectins specific for M cells are known [167]. Previous work has also shown that lectins belonging to the guinea pig mucus could achieve bridging adherence between the 5. flexneri LPS and the glycosylated motives of mucus glycoproteins [168]. Finally, it is possible that this putative adhesin is expressed only in the intestinal lumen. In this case, methods such as signature-tagged transposon mutagenesis may be required in order to identify, in the in vivo context, mutants that have lost their colonizing capacities [169]. The tropism of Shigella for primates makes the experimental approach potentially extremely difficult.
B.
Crossing the Epithelial Lining by Shigella: Which Route is Best?
In vitro. Shigella is inefficient at invading an epithelial lining apically [54]. A similar situation is likely to prevail in vivo. There are potentially two ways for bacteria to reach the basolateral pole of the epithelium: (1) to cross the M cells of the follicular-associated epithelium (FAE) covering the mucosal lymph nodes associated with the mucosa, and (2) to take advantage of the inflammation, which disrupts epithelial integrity, to translocate and reach subepithelial tissues. Based on a combination of in vitro and in vivo models of Shigella infection, both mechanisms may occur, although the former may dominate during the early stages of invasion.
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TRANSLOCATION THROUGH THE FOLLICULAR-ASSOCIATED EPITHELIUM
Experiments carried out in macaque monkeys and in the rabbit ligated-intestinal-loop model have indicated that Shigella primarily translocated through the M cells of the FAE (Fig. 14) [48, 49, 119]. Once established in the dome region of the FAE, bacteria are phagocytosed by numerous macrophages present in this area. These macrophages quickly die (Fig. 12), many of them showing a typical pattern of apoptosis [49, 149]. A current hypothesis is that those macrophages that are permanently exposed to bacterial material from the intestinal flora may be in a chronic state of subactivation. In the presence of the cell death message they receive from invading shigellae, these macrophages release large amounts of IL-lp, which initiates inflammation [170]. In addition, during the first 4 hours of infection, the balance between the tissue concentration of IL-1 receptor antagonist (IL-lra) and IL-1 is tipped toward a very low ratio, reflecting poor expression of IL-lra [170a]. This enhances the proinflammatory capacity of Shigella. Infusion of IL-1 ra to rabbits prior to and during Shigella infection of ligated intestinal loops
Fig. 14 Entry of Shigella through M cells of the follicular-associated epithelium in a rabbit Peyer's patch. M and arrow point to the ruffle that has been elicited by the entering shigellae. E points to adjacent enterocytes. Reprinted through the courtesy of M. C. Prevost, J. Perdomo, and P. Sansonetti, Station Centrale de Microscopic Electronique, Institut Pasteur. Bar = 1 |Lim.
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causes dramatic reduction of inflammation-mediated tissue destruction, thus confirming the major role played by IL-1 in pathogenesis of the disease [171]. 2.
INITIAL INFLAMMATION INCREASES PERMEABILITY OF THE EPITHELIAL BARRIER TO SHIGELLA
Transmigration of polymorphonuclear leukocytes through an epithelial lining loosens cellular junctions, thus facilitating Shigella entry. This has been shown both in vitro on human colonic epithelial cells cultured on filters and in vivo in the rabbit ligated-loop model of infection by Shigella [58, 172]. Inflammation may be initiated exclusively at the level of the follicular structures. It is also possible that the presence of Shigella on the apical side of the epithelium elicits transepithelial signaling sufficient to induce local transmigration of PMNs, thus causing the formation of multiple zones of bacterial invasion away from the follicular-associated epithelium. The vicious circle that involves crossing of the epithelial barrier and inflammation is summarized in Figure 15. More recent evidence indicates that expression of virulence plasmid genes is required to induce trafficking of neutrophils across polarized monolayers of the intestinal epithelium [173]. SepA, a 110-kDa surface protein, is encoded by the virulence plasmid. It is the protein that is most abundantly secreted by Shigella flexneri [ 174]. This protein has a signal peptide and is secreted in a way similar to IgA protease. Its secretion does not require the Mxi-Spa type III system. Even though one portion of this protein is very similar to the N-terminal portion of the IgA protease oi Haemophilus influenzae, SepA does not express an IgA-protease activity [175]. An sepPs. mutant produces weaker inflammation in the rabbit ligated-loop infection assay. While this is indicating a proinflammatory potential for this protease, no specific target has been identified so far for SepA. B. Intestinal Inflammation during Human Infection The first study on increased levels of cytokines elicited by shigellosis was performed on children from Sri Lanka infected with S. dysenteriae 1 [176]. These children showed elevated levels of IL-6 and TNF-a in their serum. Expression of all the pro- and antiinflammatory cytokines was observed after immunostaining of rectal biopsy sections of Bangladeshi patients developing shigellosis. However, severe cases were associated with higher levels of IL-1 p, IL-6, TNF-a, and IFN-y [34]. In another study, the plasma and stool levels of these cytokines, except for IFN-y, appeared elevated during the first 2 weeks of infection [35]. How these observations fit with the recent demonstration that IFN-y is essential for elimination of Shigella in a murine model of pneumonia remains to be established. Downregulation of IFN-a production at the early stage of infection may be an efficient strategy for Shigella to successfully resist and survive the initial response of the innate immune system [177]. These in vivo data are in general in agreement with in vitro data, although factors other than expression of the invasive phenotype may participate in the development of inflammation.
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Shigella entry routes
J4
Fig. 15 General scheme summarizing the early steps of Shigella crossing of the follicular-associated epithelium and initiation of inflammation (i.e., mostly influx of PMNs), following induction of macrophage apoptosis, which destabilizes the architecture of the epithelial lining and enhances bacterial invasion, the process being subsequently amplified by the capacity of Shigella to spread from cell to cell.
XL Role of Chromosomally Encoded Genes in the Virulence of Shigella Aside from the armamentarium of genes encoded by the virulence plasmid, some chromosomal genes are also involved in virulence. They can be classified in regulatory genes such as virR, and structural gene-encoding factors such as LPS and toxins. A. Regulation of Plasmid Virulence Genes Shigella expresses an invasive phenotype when grown at 37°C, but not at 30°C [178]. Transcription of the ipa and mxi-spa genes is under the control of a dual
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regulation system: a positive regulatory cascade involving two plasmid products—VirF and VirB—and a negative regulatory cascade involving a chromosomal regulator—VirR. The virF gene encodes a 30-kDa regulatory protein belonging to the AraC family of transcriptional activators and is required for expression of genes of the entry region, as well as for expression of icsA [179]. VirF operates essentially by controlling the transcription of virB, a gene encoded on the virulence plasmid, directly downstream of the ipa operon [180], which activates transcription of the ipa, mxi, and spa operons of the entry region. The screening of transposon-induced mutants for expression of entry genes at 30°C has led to identification of virR [181], a chromosomal gene that encodes the H-NS (HI) protein [182]. H-NS is a major component of a family of histone-like proteins, which control DNA supercoiling, thereby regulating expression of numerous genes [183]. Current evidence indicates that H-NS binds to the virB promoter at 30°C, thereby preventing its activation by VirF [184, 185]. Another chromosomal gene, vacB, regulates the invasive properties of Shigella [186]. A vacB mutant showed decreased expression of both icsA and ipaB gene products, despite unaltered transcription of these genes, indicating that vacB-mediated regulation may occur at the posttranscriptional level. In addition to temperature, osmolarity also modulates expression of invasion genes. Transcription of an mxi-lac gene fusion was enhanced in conditions of high osmolarity and reduced in t^envL and AenvZ-ompK mutants. These mutants are less invasive than their isogenic wild-type strain [187]. More recent evidence indicates that other regulatory systems exist. A set of plasmid-encoded proteins comprising VirA [188] and IpaH9.8 [189], which are secreted through the Mxi-Spa apparatus, are encoded by genes whose transcription depends on activation of this type III system, unlike transcription of the ipa genes, which does not depend on activation of protein secretion [190].
B.
Lipopolysaccharide
Smooth Shigella strains are considerably more virulent than rough mutants, thus indicating an essential role for LPS 0-sidechains in virulence. When the virulence plasmid and chromosomal sequences from S. flexneri were introduced into an E. coli K12 strain by conjugation, expression of a smooth LPS appeared essential for the exconjugants {E. coli K\2IShigella hybrids) to become virulent in in vivo conditions such as in the rabbit ligated-loop-infection assay and in the Sereny test [191]. This work confirmed previous data showing that rough Shigella mutants were consistently negative in the keratoconjunctivitis assay in guinea pigs and rabbits [192]. Although rough strains can invade epithelial cells in vitro, they are still impaired in their capacity to spread from cell to cell. This could be related to an indirect effect where the lack of 0-sidechains alters surface localization of
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IcsA, thereby preventing its polar localization, an important factor for optimal actin-driven motility [193]. Both mislocalization and insufficient proteolytic cleavage of IcsA may be taking place in the mutants lacking 0-sidechains. Aside from suffering alterations in their motility, rough mutants of Shigella may also be avirulent in in vivo assays due to their hypersensitivity to killing by complement [194]. The complement system protects the host against invading bacterial pathogens by generating opsonic, chemotactic, and lytic factors [195]. Its protein components are present in the serum, but also in inflammatory exudates, as it occurs in the mucosal tissues in the course of shigellosis. A recent study describes S. flexneri mutants with short 0-sidechains expressing a normal motiUty phenotype, but exhibiting susceptibility to serum-mediated kilUng [13].
C. Toxins Shigella produces various toxins, among which Shiga toxin is the best characterized [30]. Among the numerous species and serotypes of Shigella, only S. dysenteriae 1 produce this toxin. It is a holotoxin composed of five B subunits of 7 kDa each, and one A subunit of 32 kDa. The B subunit binds to the disaccharide Galal-4galp found in glycolipids such as the globotriaosylceramide Gbs. The A subunit is the toxic part of Shiga toxin that becomes enzymatically active on proteolytic cleavage, releasing a 27-kDa A1 fragment corresponding to the toxic moiety and a 4-kDa carboxy-terminal A2 portion [30]. Shiga toxin is an A^-glycosidase that cleaves adenine off one specific adenosine of the 28 S component of the 60 S ribosomal subunit, thus irreversibly destroying ribosomal functions [196]. Therefore, Shiga toxin is a potent inhibitor of protein biosynthesis, in both eukaryotic and prokaryotic systems [197, 198]. The function of Shiga toxin in eliciting diarrhea in the course of shigellosis due to S. dysenteriae 1 has not been convincingly demonstrated in humans, although its enterotoxicity in the rabbit ligated-loop model would indicate that it has this function [24]. As Gb3 is mainly detected in the nonepithelial fraction of colonic sections, it is not clear how the toxin could interact with the lumenal pole of epithelial cells to cause diarrhea. On the other hand, Shiga toxin is cytotoxic to intestinally derived epithelial cells in vitro [199] and could thereby participate in the cytotoxic effect to the epithelium. This could occur either by release of the toxin by extracellular or intracellular bacteria. In any event, considering the localization of Gb3, it is likely that the toxin has subepithelial targets. Macaque monkeys inoculated intragastrically either with a wild-type or with a stxA (knockout mutant lacking expression of the catalytic A subunit of Shiga toxin) mutant of 5. dysenteriae 1 developed dysentery. However, animals infected with the wild-type strain consistently showed the presence of blood in their dysenteric stools. In support of this difference in clinical symptoms, the histopathological analysis showed severe alterations of the capillaries in the lamina propria of
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colonic tissues from animals infected with the toxin-producing strains [200]. These observations indicate that Shiga toxin may primarily behave as a toxin for the vascular endothelium, thereby adding a component of ischemic and hemorrhagic colitis to the dysentery caused by the invasive phenotype. Epidemiological observations confirm this finding, showing that the patients infected with S. dysenteriae 1 have more blood in their stools, compared to patients infected with other Shigella species [17]. The classical neurotoxic effect of Shiga toxin [201] is also likely to be due to brain capillary damage. Another severe condition, primarily observed in the context of S. dysenteriae 1 infection, is the hemolytic uremic syndrome (HUS). Although the pathogenesis of this acute (often fatal) renal failure is not fully understood, evidence points to Shiga toxin penetrating the blood vessels of the intestinal mucosa and reaching the kidneys via the bloodstream as a major etiological factor. The glycolipid Gb3 is found in high concentrations on human kidney endothelial cells, which are exquisitely sensitive to Shiga toxin [202]. Histopathological analysis shows thrombosis and destruction of the glomerular capillaries, and sometimes renal cortical necrosis [203]. It is possible that LPS acts in synergy with Shiga toxin to destroy renal blood vessels [204], Shigella produces toxins other than Shiga toxin. Two enterotoxins have recently been described that may account for the early diarrheal phase often observed during shigellosis. Shigella enterotoxin 1 is a chromosomally encoded, iron-dependent toxin of 55 kDa, mainly expressed by S.flexneri 2a [205]. Shigella enterotoxin 2 is a plasmid-encoded protein of 63 kDa [206].
D.
Other Virulence Factors
Other virulence factors encoded by the chromosome include the OmpC porin [207]. Also, all Shigella strains express siderophores and their corresponding outer membrane receptors. Siderophores are low-molecular-weight molecules with high affinity for ferric iron that they can extract from their physiological carriers, transferrin and lactoferrin. Most isolates of S. flexneri and S. sonnei express aerobactin siderophores, whereas 5". dysenteriae and 5". sonnei produce enterochelin siderophores. Some isolates can express both types [208]. Aerobactin-negative mutants are impaired in their growth in tissues but not inside cells [209]. Last, but not least, the interesting concept of "black holes" was presented [9] in 1998, according to which portions of the bacterial chromosome encoding enzyme that may affect pathogenicity have been deleted as the pathogenic species evolves toward maximum fit with the host. For example, lysine decarboxylase (LDC), which is present in more than 90% of E. coli isolates, is absent in Shigella and enteroinvasive E. coli. When cadA, the gene encoding lysine decarboxylase.
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was introduced into Shigella, invasiveness and enterotoxicity were both affected. Cadaverin, a byproduct of lysine decarboxylase, was identified as the inhibitor. Moreover, the chromosomal region encompassing cadA was shown to be deleted in Shigella and entero-invasive E. coli. Similarly, the E. coli ompTgQne attenuates virulence in Shigella, which normally lacks ompT [210].
XIL Conclusions Thanks to a combination of in vitro and in vivo approaches, the pathogenesis of shigellosis is progressively unraveling its secrets. The major contributions in this area over the last two decades have been: (1) the establishment of the genetic basis for cell invasion and its regulation by environmental cues such as temperature, (2) the description of the infectious cycle of the bacteria in epithelial cells and the recognition of the molecular crosstalks accounting for entry, intracellular movement, and cell-to-cell spread, (3) the description of apoptotic killing of macrophages with its dual implications for bacterial survival and elicitation of inflammation, and (4) the description of the role of inflammation in facilitating mucosal invasion. These concepts have also led to the development of a series of promising live attenuated vaccine candidates against shigellosis. Several questions remain open, such as: (1) understanding the colonic specificity of Shigella; (2) understanding the dramatically high infectiousness of the pathogen (i.e., 100 cfu absorbed orally); (3) deciphering the signaling pathways that, on expression of the invasive phenotype, lead either to entry into epithelial cells or to programmed cell death of macrophages; (4) confirming that translocation through the epithelium occurs essentially via M cells, since inflammation occurring at distant sites may also facilitate entry; (5) identifying the specificities of the signaling cascades that cause the particularly severe inflammation observed during shigellosis; and (6) understanding the bases of immune protection against the disease and developing live oral or subunit parenteral vaccines.
Acknowledgments We wish to thank Colette Jacquemin for outstanding editorial work on this manuscript. We also wish to express our gratitude to all past and present members of Unite de Pathogenie Microbienne Moleculaire whose work made a significant part of this manuscript possible.
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179. Sakai, T., Sasakawa, C , and Yoshikawa, M. (1986). Expression of the four virulence antigens oi Shigella flexneri is positively regulated at the transcriptional level by the 30 kD VirF protein. Mol Microbiol 2, 589-597. 180. Adler, B., Sasakawa, C , Tobe, T., Makino, S., Komatsu, K., and Yoshikawa, M. (1989). A dual transcriptional activation system for the 230-kb plasmid genes coding for virulence associated antigens oi Shigella flexneri. Mol. Microbiol. 3, 627-635. 181. Maurelli, A. T., and Sansonetti, P. J. (1988). Identification of a chromosomal gene controlling temperature regulated expression of Shigella virulence. Proc. Natl. Acad. Sci. U.S.A. 85, 2820-2824. 182. Dorman, C. J., Ni Bhriain, N., and Higgins, C. F. (1990). DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri. Nature 344, 789-792. 183. Hulton, C. S. J., Seiraf, A., Hinton, J. C. D., Sidebotham, J. M., Waddel, L. Pavitt, G. D., Owen-Hughes, T., Spassky, A., Buc, H., and Higgins, C. F. (1990). Histone-like protein JHl (H-NS), DNA supercoiling and genes expression in bacteria. Cell 63, 631-642. 184. Tobe, N., Nagai, S., Okada, N., Adler, B., Yoshikawa, M., Sasakawa, C. (1991). Temperatureregulated expression of invasion genes in Shigella flexneri is controlled through the transcriptional activation of the virB gene on the large plasmid. Mol. Microbiol. 5, 887-893. 185. Tobe, T, Yoshikawa, M., Mizumo, T, and Sasakawa, C. (1993). Transcriptional control of the invasion regulatory gene virB of Shigella flexneri: Activation by virF and repression by H-NS. J. Bacteriol. 175, 6142-6149. 186. Sasakawa, C. (1995). Molecular basis of pathogenicity of Shigella. Rev. Med. Microbiol. 6, 257-266. 187. Bemardini, M. L., Fontaine, A., and Sansonetti, P.J. (1990). The two-component, osmo-dependent, regulatory system (ompR-ompZ) controls the virulence of Shigella flexneri. J. Bacteriol. 172,6274-6281. 188. Uchiya, K., Tobe, T, Komatsu, K., Suzuki, T, Watarai, M., Fukuda, I., Yoshikama, M., and Sasakawa, C. (1995). Identification of a novel virulence gene, virA, on the large plasmid of Shigella, involved in invasion and intercellular spreading. Mol. Microbiol. 17, 241-250. 189. Venkatesan, M. M., Buysse, J. M., and Hartman, A. B. (1991). Sequence variation in two ipaH genes of Shigella flexneri 5 and homology to the LRG-like family of proteins. Mol. Microbiol. 5, 2435-2445. 190. Demers, B., Sansonetti, P. J., and Parsot, C. (1998). Induction of type III secretion in Shigella flexneri is associated with differential control of transcription of genes encoding secreted proteins. EMBO J. 17(10), 2894-2903. 191. Sansonetti, P J., Hale, T L., Dammin, G. J., Kapfer, C., Colins, H., and Formal, S. B. (1983). Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri. Infect. Immiin. 39, 1392-1402. 192. Okamura, N., and Nakaya, R. (1977). Rough mutant of Shigella flexneri 2a that penetrates tissue culture cells but does not evoke keratoconjunctivitis in guinea pigs. Infect. Immun. 17, 4-8. 193. Sandlin, R. C , Lampel, K. A., Keasler, S. P, Goldberg, M. B., Stolzer, A. L., and Maurelli, A. T (1995). Avirulence of rough mutants of Shigella flexneri: Requirement of O antigen for correct unipolar localization of IcsA in the bacterial outer membrane. Infect. Immun. 63, 229-237. 194. Rowley, D. (1968). Sensitivity of rough Gram-negative bacteria to the bactericidal action of serum. J. Bacteriol. 95, 1647-1650. 195. Joiner, K. A. (1988). Complement evasion by bacteria and parasites. Annu. Rev. Microbiol. 42, 201-230. 196. Endo, Y, Tsurugi, K., Yutsudo, K., Takeda, T, Ogasawara, T, and Igararshi, K. (1988). Site of action of verotoxin (VT2) from Escherichia coli 0157:H7 and of Shiga toxin on eukaryotic ribosomes: RNA N-glycosidase activity of the toxins. Eur J. Biochem. 171, 45-50.
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197. Thompson, M. R., Steinberg, M. S., Gemski, P., Formal, S. B., and Doctor, B. P. (1976). Inhibition of in vitro protein synthesis by Shigella dysenteriae 1 toxin. Biochem. Biophys. Res. Commun. 71, 783-788. 198. Olenick, J. G., and Wolfe, A. D. (1980). Shigella toxin inhibition of binding and translation of polyuridylic acid by Escherichia coli ribosomes. J. Bacteriol. 141, 1246-1250. 199. Moyer, M. P, Dixon, P S., Rothman, S. W., and Brown, J. E. (1987). Cytotoxicity of Shiga toxin for primary cultures of human colonic and ileal epithelial cells. Infect. Immun. 55, 1533-1535. 200. Fontaine, A., Arondel, J., and Sansonetti, P. J. (1988). Role of Shiga toxin in the pathogenesis of bacillary dysentery as studied with a Tox-mutant of Shigella dysenteriae 1. Infect. Immun. 56,3099-3109. 201. Bridgwater, F. A. J., Morgan, R. S., Rowson, E. K. E. K., and Payhing Wright, G. (1955). The neurotoxin of Shigella shigae: Morphological and functional lesions produced in the central nervous system of rabbits. Br J. Exp. Pathol. 36, 447^53. 202. Obrig, T. G., Louise, C. B., Lingwood, C. A., Boyd, B., Barley-Maloney, L., and Daniel, T. O. (1993). Endothelial heterogeneity in Shiga toxin receptors and responses. J. Biol. Chem. 268, 15484-15488. 203. Koster, F., Levin, J., Walker, L., Tung, K. S., Gilman, R. H., Rahaman, M. M., Majid, M. A., Islam, S., and WilUiams Jr., R. C. (1978). Hemolytic-uremic syndrome after shigellosis: Relation to endotoxemia and circulating immune complexes. New Engl. J. Med. 298, 927-933. 204. Heyderman, R. S., Fitzpatrick, M. M., and Robin Barclay, G. (1994). Haemolytic-uraemic syndrome. L««c^r 343, 1042. 205. Noriega, F. R., Liao, F. M., Formal, S. B., Fasano, A., and Levine, M. M. (1995). Prevalence of Shigella enterotoxin 1 among Shigella clinical isolates of diverse serotypes. J. Infect. Dis. Ill, 1408-1410. 206. Nataro, J. P., Seriwatana, J., Fasano, A., Maneval, D. R., Guers, L. D., Noriega, F., Dubovsky, F., Levine, M. M., and Morris Jr., J. G. (1995). Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella. Infect. Immun. 63, 4721-4728. 207. Bernardini, M. L., Sanna, M. G., Fontaine, A., and Sansonetti, P. J. (1993). OmpC is involved in invasion of epithelial cells by Shigella flexneri. Infect. Immun. 61, 3625-3635. 208. Lawlor, K. M., Daskaleros, P. A., Robinson, R. E., and Payne, S. M. (1987). Virulence of iron transport mutants oi Shigella flexneri and utilization of host iron compounds. Infect. Immun. 55, 594-599. 209. Nassif, X., Mazert, M. C , Mounier, J., and Sansonetti, P. J. (1987). Evaluation with an iuc::TnlO mutant of the role of aerobactin production in the virulence of Shigella flexneri. Infect. Immun. 55, 1963-1967. 210. Pal, T, Newland, J. W, Tall, B. D., Formal, S. B., and Hale, T. L. (1989). Intracellular spread of Shigella flexneri associated with the kcpA locus and a 140 kDa protein. Infect. Immun. 18, 94-98.
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CHAPTER 9
Pathogenic Escherichia
coli
JOSE L. PUENTE B. BRETT FINLAY
I. Introduction 11. Enterotoxigenic E. coli (ETEC) A. Disease B. Virulence Factors C. Virulence Gene Regulation III. Enteroinvasive E. coli (EIEC) A. Disease B. Virulence Factors C. Virulence Gene Regulation IV. Enteropathogenic E. coli (EPEC) A. Disease B. Virulence Factors C. Virulence Gene Regulation V. Enterohemorrhagic E. coli (EHEC) A. Disease B. Virulence Factors C. Virulence Gene Regulation VI. Enteroaggregative E. coli (EAEC) A. Disease B. Virulence Factors VII. Diffusely Adhering E. coli (DAEC) A. Disease B. Virulence Factors VIII. Uropathogenic £. co// A. Disease B. Virulence Factors IX. E. coli That Cause Sepsis and Meningitis A. Disease B. Virulence Factors X. Conclusions References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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/- Introduction Escherichia coli is the most extensively studied microorganism. It has been a model system for the study of bacterial metabolism, the cell division process, cell wall biosynthesis, chemotaxis, bacterial genetics, and the physiological role of enteric bacteria as part of the normal fecal flora [1]. Despite the vast knowledge that has been accumulated over the years, the recent release of its full genomic composition has made it obvious that there are still many things to learn about this microorganism [2]. Analysis of the E. coli K-12 genome sequence also shows that about 2% of its DNA consists of mobile genetic elements, including phages, plasmids, and transposons [2]. These elements are responsible for the continuous evolution of the bacterial genomic repertoire, providing significant diversity in E. coli strains. In this regard, pathogenic E. coli appears to have evolved from nonpathogenic strains by acquiring new virulence factors by the horizontal transfer of accessory DNA, which is often organized in clusters (pathogenicity islands) in the chromosome or on plasmids [3]. The high genetic diversity of the E. coli genome is also reflected by the large variation in DNA content between different strains [4-6] and by the distribution or genomic location (insertion site) of different virulence determinants [7, 8]. In this context, it seems that most pathogenic E. coli strains do not have a single evolutionary origin, but instead have emerged as a result of different events of DNA transfer, and that even strains capable of causing the same disease do not constitute a monophyletic group [9]. E. coli pathogenic variants are represented by strains of specific serogroups possessing a particular set of virulence factors, which are responsible for the different clinical manifestations that characterize E. coli infections. Pathogenic E. coli cause various diseases in humans, including several types of diarrhea, urinary tract infections, sepsis, and meningitis (Table I). E. coli strains that cause human diarrhea of varying severity have been divided into six major categories: enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enteroaggregative E. coli (EAEC), and diffuse adhering E. coli (DAEC). E. coli strains causing urinary tract infections are known as uropathogenic E. coli (UPEC), while E. coli Kl are often responsible for cases of meningitis or sepsis (Table I). Different bacterial virulence attributes appear to dictate the types of interactions that occur between the pathogenic organism and its host cells, and where in the body these interactions occur. Tissue tropism plays an important role in disease— for example, UPEC infects the urinary tract and kidneys, EPEC the small bowel, and EHEC the large bowel. For nearly all pathogenic E. coli, colonization of a particular host surface is mediated by fimbriae or pili, which are often called colonization factors [10-12]. Common adhesins are frequently found within several E. coli types. For example, type I pili are found in most of the different pathogenic E. coli, making it difficult to assign a specific role for this adhesin in disease, although it has been suggested to be important for spreading and
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Table I
E. coli That Are Pathogenic for Humans
Type of E. coli
Disease
Virulence factors
Enterotoxigenic (ETEC)
Watery to cholera-like diarrhea
Heat-labile toxin (LT), heat-stable toxin (ST), colonization factors (CFs)
Enteroinvasive (EIEC)
Watery diarrhea to dysentery
Ipas, type III secretion (Mxi and Spa), VirG/IcsA
Enteropathogenic (EPEC)
Watery diarrhea
Esps, type III secretion (Sep and Esc), intimin, Tir, and BFP
Enterohemorrhagic (EHEC)
Hemorrhagic colitis, hemolytic uremic syndrome (HUS)
Above EPEC factors and Shiga toxin, hemolysin
Enteroaggregative (EAEC)
Watery to mucoid diarrhea
AAF adhesins, EAST-1, Pet, Pic, hemolysin
Diffusely adhering (DAEC)
Watery diarrhea
F1845 and AIDA-I fimbriae
Uropathogenic (UPEC)
Urinary tract infections
Type I pili, P pili, Afimbrial adhesins (Afa), hemolysin, CNF-1
Septic (SEC)
Neonatal sepsis, meningitis
Capsule, type I pili, S-fimbrial adhesin, IbeA and IbeB (invasion proteins)
colonization by commensal E. coli [13, 14] or colonization of the urinary tract [15, 16]. Once localized to a particular tissue, the molecular interactions that occur between pathogenic E. coli and their host cells follow specific steps, and are quite different between different pathogenic types. Some strains adhere to mucosal surfaces and secrete specific toxins that either intoxicate localized epithelial cells or spread systemically to affect distant host cells. Other strains interact more intimately with host cell surfaces, and this intimate interaction results in disease. Finally, other strains actually enter host cells and live as intracellular pathogens, or penetrate host barriers and live systemically within the human host, resulting in septic disease [17, 18]. The wide diversity of virulence factors identified and characterized in different pathogenic E. coli resemble many of the virulence mechanisms found in other pathogens [17, 19]. ETEC utilizes a cholera-like toxin to cause cholera-like disease [20]. EIEC behaves as Shigella, in that it contains the same virulence factors (e.g., type III secretion system, invasins, and intracellular spread mechanism) that are responsible for producing a dysentery-like disease [21]. EHEC produces a Shiga-like toxin (similar to that found in Shigella dysenteriae) that seems to be involved in causing the hemolytic uremic syndrome in a proportion of cases [22]. EHEC and EPEC utilize a type III secretion system, similar to those seen in Salmonella, Shigella, Yersinia, and other Gram-negative pathogens, to inject E. C(9//-specific factors into the host cell. These factors induce actin
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rearrangements and activation of particular signal transduction pathways that result in disease [23]. As with other pathogens that cause systemic disease and meningitis, E. coli Kl also produces a polysaccharide capsule that prevents clearance by phagocytic cells [24]. Expression of the genes encoding this variety of virulence factors is often modulated in response to a series of environmental cues such as temperature, ion concentrations, osmolarity, iron levels, pH, carbon source availability, growth phase, and oxygen levels [25]. Virulence gene expression is determined by a consensus response to a mixture of these different biochemical and physical parameters that allows the bacterial cell to identify and exploit a particular extracellular or intracellular niche. Mechanistically, it often involves the interplay of regulatory proteins acting independently or as a cascade; these proteins share similarity with members of different families of regulatory proteins [17, 26]. The aim of this chapter is to examine our present understanding of the molecular basis of E. coli pathogenesis and the function and regulation of the various virulence determinants that distinguish each category in the context of their contribution to disease. An excellent comprehensive review was published in 1998 that examines the epidemiology, clinical symptoms, detection, diagnosis, and virulence of the diarrheagenic E. coli [18].
//. Enterotoxigenic E. coli (ETEC) A.
Disease
ETEC causes a watery diarrhea, ranging in severity from mild and self-limiting to a severe cholera-like profuse diarrhea. Diarrhea is usually without mucus, blood, pus, fever, or vomiting, consistent with it being an intoxication (i.e., toxin mediated), rather than a systemic infection. ETEC can affect adults and is often seen in travelers in developing countries (thus its name "traveler's diarrhea"), being contracted through contaminated food or water [27]. Serious (life-threatening) disease is seen in infants in developing countries. ETEC is responsible for more than 650 million cases of diarrhea and between 700,000 and 800,000 deaths in children under the age of 5 years. Antibiotics decrease the severity and duration of diarrhea, but antibiotic-resistant strains of ETEC are increasingly common. Like cholera, therapy is mainly rehydration (usually oral). With proper hydration, the disease is usually self-limiting [18]. B. Virulence Factors ETEC strains cause diarrhea through the action of thermolabile and thermostable enterotoxins (reviewed in [20]). Approximately 30% of the ETEC strains express a heat-labile toxin (LT), 35% produce a heat-stable toxin (ST), and the rest express both. In addition, ETEC strains produce one or more colonization factors
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(adhesins) that mediate attachment to intestinal mucosal surfaces [28-30]. Figure 1 summarizes the current proposed action mechanisms of ETEC toxins.
-CF
ETEC
GSa
Fig. I ETEC interactions with intestinal epithelial cells. ETEC adherence to intestinal cells is mediated by different colonization factors (CFs). Once established in the proximal small intestine, ETEC strains produce the heat-labile toxin (LT) and/or the heat-stable toxin (ST). The LT holotoxin, consisting of an A subunit and a pentamer of B subunits, is internalized by endocytosis on binding to its ganglioside GM] receptor. The A subunit is proteolitically cleaved into the A] and A2 subunits, which remain linked by a disulfide bond. The Ai subunit ADP-ribosylates the alpha subunit of the GTP-binding protein, Gs, inhibiting its intrinsic GTPase activity, resulting in constitutive activation of adenylate cyclase at the basolateral membrane. This activation leads to increased levels of intracellular cyclic AMP, activation of a cAMP-dependent A kinase, and supranormal phosphorylation of intestinal epithelial cell chloride channels, such as CFTR. These events result in inhibition of NaCl absorption and stimulation of chloride secretion. ST acts by binding to guanylate cyclase type C (GC-C), localized in the brush-border membrane of intestinal epithelial cells. Activation of GC-C results in increased levels of intracellular cyclic GMP that stimulates chloride secretion and/or inhibits NaCl absorption, resulting in net intestinal fluid secretion. In vivo chloride secretion may occur through activation of a cGMP-dependent protein kinase (G-kinase), which ultimately activates the chloride channel CFTR.
392 1.
JOSE L. PUENTE AND B . BRETT FINLAY HEAT-LABILE TOXIN (LT)
There are two forms of LT: LT-I and LT-II [31]. LT-I, the predominant form, is quite similar to CT at the sequence level (above 80% identity) [32], and is thought to act mechanistically in an identical fashion (see [33] and Chapter 10 for details on Vibrio cholerae, its toxin, and mechanism of action). LT-I is oligomeric in structure with one enzymatic A subunit and five identical B subunits [34]. The five B subunits are arranged symmetrically in a ring-like structure that binds the ganglioside GMi and weakly to ganglioside GDlb [35]. The A subunit is proteolytically cleaved into two domains—A] and A2—that remain linked by a disulfide bond and span the center of the ring [36, 37]. The toxin is endocytosed, and the A subunit reaches the basolateral (bottom) surface of the epithelial cell after escaping the endocytic vesicle (reviewed in [20, 33]). The Al peptide transfers an ADP-ribosyl group from NAD to the a-subunit of the GTP binding protein G^. This modification of G^a inhibits its intrinsic GTPase activity, which results in permanent activation of adenylate cyclase, leading to accumulation of intracellular levels of cyclic AMP (cAMP). As cAMP accumulates inside intestinal cells, it is thought that a cAMP-dependent kinase (A kinase) is activated, which then results in phosphorylation of apically located chloride channel proteins such as CFTR (the channel affected in cystic fibrosis patients). This causes channel opening, and chloride ion efflux out of cells along with a block in ion and fluid absorption into cells, resulting in a net osmotic imbalance (Fig. 1). The result is watery diarrhea. It has been proposed that the reason for the high prevalence of cystic fibrosis in the Caucasian population is that a defective CFTR provides a protective mechanism against CT- (and LT-) mediated disease [38, 39], though other data suggest it confers protection to S. ry/?/2/-mediated typhoid fever. Although the above explanation is logical, several other more complex mechanisms have also been implicated in LT- and CT-mediated disease (reviewed in [18]). These include promotion of the production of prostaglandins, stimulation of a mild inflammatory response, and activation of the enteric nervous system. It is likely that the watery diarrhea resulting from ETEC infection is a combination of the classic mechanism described above along with one or more of these other events. The LT-II form shows less identity to LT-I and CT (ca. 57%) and basically no identity to the B subunit [40, 41]. These differences are also reflected in their specificity for binding to gangliosides, since LT-II binds best to gangliosides GDlb or GDI a [35] and is found associated mainly with animal, but not human, disease [42]. Like ST and the colonization factors, LTs are usually encoded on a plasmid. 2.
HEAT-STABLE TOXIN (ST)
As mentioned above, about one-third of ETEC strains expresses ST, and another third expresses ST and LT Thus, ST alone is capable of causing watery diarrhea, without LT being present. In contrast to LT, ST is heat stable (thus its
9. PATHOGENIC ESCHERICHIA COLI
393
name), a property provided by its intramolecular disulfide bonds. There are two major STs: STa and STb (reviewed in [20, 43]). STa has been studied in more detail and provides an excellent bacterial example of a hormone-like peptide that affects normal host cell function [44]. STa enterotoxins are small, cysteine-rich molecules (18-19 amino acids) that can form three intramolecular disulfide bonds [45]. These toxins are made from larger precursors (72 amino acids) that are cleaved as the molecule transits out of the bacterium [46]. The eukaryotic membrane receptor for STa is guanylate cyclase C (GCC), which is located in the apical membrane of intestinal cells [47]. Because of the apical location of guanylate cyclase, STa-mediated cell activation is quite rapid. It has been suggested that STa receptors are more abundant on enterocytes of infants or young animals, but that their numbers decrease in older individuals. This variation in receptor abundance may determine the severity of the secretory response, and give a plausible explanation for the high susceptibility of human infants and newborn animals to STa-mediated diarrhea [48-50]. GCC activation triggers a cascade of events (reviewed in [18, 20]) (Fig. 1), including the accumulation of intracellular cGMP levels, the cGMP-dependent activation of protein kinase A (PKA), and the PKA-dependent phosphorylation and activation of the cystic fibrosis transmembrane conductance regulator (CFTR), which finally leads to increased chloride secretion and blockage of sodium chloride uptake, resulting in diarrhea [38, 51] (Fig. 1). GCC-null mice were protected against an infection with ETEC, further evidencing the importance of this receptor in Sta-mediated diarrhea [52]. Several other intracellular signals are activated in response to STa, but the contribution of these signals to diarrhea has not been fully defined. STa behaves like guanylin, the endogenous intestinal peptide hormone that binds to guanylate cyclase [53, 54]. Guanylin regulates ion and fluid levels by modulating cGMP levels, thereby mediating intestinal homeostasis. It is a 15-aa peptide that contains four cysteines (two disulfide bonds) and, ironically, is less efficient than STa in activating guanylate cyclase. The discovery of guanylin was one of the first demonstrations that bacteria have evolved the ability to mimic eukaryotic endogenous functions to take advantage of host cells and, also, an indication of how the study of microbial pathogens is teaching us important aspects of the cell biology of eukaryotes. Although STb is primarily found in animal pathogens, it has also been isolated from human ETEC isolates. STb bears no sequence similarity to STa, although it does have four cysteine residues that form disulfide bridges [55]. It is also initially synthesized as a larger precursor of 71 aa, which is then proteolitically processed to a 48-aa mature protein [56]. Sulfatide has been suggested as a functional receptor for STb [57, 58]. STb affects neither cAMP or cGMP levels nor stimulates chloride secretion [59]; instead, it seems to elicit an intestinal response characterized by the secretion of bicarbonate, to which human cell lines seem to be insensitive [60].
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ADHESINS
ETEC attachment to an intestinal surface is mediated by colonization factor antigens (CFAs), coli surface antigens (CSs), and putative colonization factors (PCFs), which are generally referred to as colonization factors (CFs) (reviewed in [11, 30]. These structures are essential for ETEC to colonize the small intestine, a central step in ETEC's virulence. At least 20 different and antigenically distinct CFs have been described in human ETEC, and these are found in varying combinations along with LT, ST, or both [30]. The main CFs associated with human ETEC strains include CFA/I, constituted by a single fimbrial structure, and CFA/II and CFA/IV, which can be a combination of a particular set of CSs. CFA/II strains can express CS3 alone or in combination with CSl or CS2 [61, 62], while CFA/IV strains can express CS6 alone or mixed with CS4 or CS5 [63]. It is thought that CFs dictate host and tissue specificity, since animal ETEC CFs are not found in human ETEC isolates. Morphologically, CFs can be subdivided into four major groups: rigid rods (e.g., CFA/I), bundle-forming (longus), fibrillar, and nonfimbrial adhesins [11, 30]. The genetics of ETEC CFs have been studied extensively, and most are encoded within standard fimbrial operons, much like that seen with type I and Pap pili (see below). These operons usually encode 4-8 proteins whose functions include regulation, the major subunit that forms the adhesin, and accessory factors that include a periplasmic chaperone and an outer membrane molecular usher (Fig. 2) [61, 62, 64]. One of the best-studied systems is CSl, which has served as a prototype to study the mechanisms of assembly of CF [65]. The coo operon is composed of the cooB, cooA, cooC, and cooD genes, which are required for expression of functional CS1. CooB has been shown to act as a chaperone-like protein, which is required for pilus assembly but is not present in the final pili structure [66]. CooA is the major structural subunit [67]. CooC is an outer membrane protein likely to be involved in transmembrane transport of CSl [68]. CooD, which is a located at the tip of the pilus and potentially involved in adherence, determines the initiation of pilus assembly and modulates the number of CSl pili on bacterial cells [69, 70]. Unlike Pap and type I pili, which produce minor tip adhesins, the major structural subunit (the stalk protein) also functions as the major adhesin. Operons encoding CFs have a low G+C content, are usually flanked by transposons, and are mainly contained on plasmids that also contain LT and/or ST, suggesting that horizontal transfer and transposition events have been responsible for generating the combination of virulence factors found in ETEC strains [30]. The intestinal receptors that CFs bind include sialoglycolipids such as GM2, sialic acid containing glycoconjugates, asialogangliosides, and several other glycoconjugates (glycolipids and glycoproteins) found on the cell surface (reviewed in [11]). The oligosaccharides expressed on mammalian cell surfaces vary widely, providing an extensive range of options that might contribute to host and tissue specificity for the ETEC CFs.
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Rns
/ /
/ .
oo-.o«
Fig. 2 Genetic organization and regulation of the ETEC CSl fimbrial operon. The plasmid-encoded coo operon contains four genes that are co-expressed from a promoter located upstream from the cooB gene. Transcriptional activation of this operon requires the product of the plasmid-encoded rns gene, which exhibits sequence similarity to the AraC family of regulatory proteins. Rns seems to positively activate its own transcription. Transcription of the coo operon and rns is negatively regulated by H-NS, a small histone-like protein that potentially binds transcriptional silencer sequences present in both the cooB and rns upstream regulatory regions and open reading frames. Mutations in H-NS abolish negative regulation at low temperatures, suggesting that H-NS antagonizes Rns-mediated activation.
4.
OTHER VIRULENCE FACTORS
Some ETEC strains are capable of invading epithelial cell lines, although the role of invasion in ETEC pathogenesis remains undefined [71]. Two separate chromosomally encoded invasion loci, designated da and tib (toxigenic invasion loci A and B), direct noninvasive E. coli to adhere to and invade intestinal epitheUal cell lines [71,72]. The tib locus directs the synthesis of Tib A, a 104-kDa outer membrane protein that has been correlated with the adherence and invasion phenotypes [73]. Tib A is synthesized as a 100-kDa precursor (preTibA) that is subsequently glycosylated to render the active form [74]. Tib A shares similarity with members of the autotransporter family of outer membrane proteins (afimbrial adhesins) that play an important role in the virulence of different Gram-negative bacteria [74]. C. Virulence Gene Regulation Expression of the CSl and CS2 adhesins requires the product of the plasmid-encoded regulatory gene rns (Fig. 2) [75]. Rns belongs to a family of transcriptional regulators known as the AraC family and is considered the prototype of a number of AraC-like proteins involved in regulation of virulence determinants [76]. The
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binding sites identified by Rns by footprinting analysis are neither palindromic nor repeated sequences, as has been observed for other AraC-like proteins [77]. Likewise, CFA/I expression requires CfaR/CfaD, a close homolog of Rns [78, 79]. The expression of these adhesins is negatively regulated at low temperatures. Interestingly, mutations in the gene coding for the histone-like protein H-NS abolish this negative regulation, suggesting that Rns and CfaR are required to overcome the repression mediated by H-NS on the expression of these adhesins [80, 81]. It is likely that, in the same way, rns positively regulates its own transcription [82]. Transcriptional silencer regions have been described for the coo operon and for rns [80, 82]. Some of these silencer regions, which are potential DNA-binding sites for H-NS, overlap the cooB and rns open reading frames, but act in conjunction with upstream sequences [80, 82].
///. Enteroinvasive E. coli (EIEC) A.
Disease
EIEC causes a watery diarrhea that often resembles that caused by ETEC. However, some patients do experience a dysentery-like disease, with mucus, blood, and pus in the stool, along with fever. Since the virulence factors in EIEC are virtually identical to those in Shigella species (see below and Chapter 8 herein), disease symptoms are often similar to those caused by S.flexneri (significant inflammation, ulcer formation, and clinical dysentery) [21, 83]. The infectious dose of EIEC is much larger than that of Shigella. Unlike S. dysenteriae, EIEC does not contain a Shiga toxin and so does not cause hemolytic uremic syndrome. The incidence of EIEC is low in developed countries, but foodbome outbreaks have been reported [84]. B. Virulence Factors Both EIEC and Shigella species invade the colonic epithelium. To achieve this, they follow a series of steps as they interact with the intestinal mucosa: invasion of colonic epithelial cells, lysis of the endocytic vacuole, bacterial multiplication, spread to adjacent cells, and host cell killing by apoptosis if the host cell is a macrophage [21, 83]. The virulence factors required for these multiple steps are encoded on a 140-MDa plasmid (pinv), although some other factors, such as regulators, are encoded on the chromosome [21, 85]. Nearly all we know about EIEC virulence mechanisms is based on extrapolation from the Shigella systems, so only an overview of ElEC/Shigella invasion and the necessary bacterial factors is provided here (Fig. 3). The homologous invasion mechanism of Shigella species is described in detail in Chapter 8 in this volume.
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Fig. 3 EIEC interactions with intestinal epithelial cells. EIEC strains secrete Ipa proteins via a type III secretion system onto and into host cells, causing actin rearrangements and membrane ruffling, resulting in bacterial internalization. Once inside a vacuole in the host cell, the IpaB protein degrades the vacuole, releasing the bacterium into the cytosol, where IcsA polymerizes actin. This action propels the organism through the cell and into neighboring cells.
1.
INVASINS (IPAS)
Like Shigella, EIEC secretes Ipas (invasion plasmid antigens) A-D into the host cell. Ipas mediate invasion by triggering several events in host cells resulting in membrane ruffling, macropinocytosis, actin rearrangements, and bacterial engulfment (reviewed in [83]). Once accomplished, internalized bacteria are surrounded by a membrane-bound inclusion in the cytoplasm of host cells. Unlike phagocytosis, this process can occur in nonphagocytic (e.g., epithelial) cells. Like most Gram-negative pathogens, EIEC and Shigella utilize a specialized secretion system, designated the type III system, to export the invasion plasmid antigens (Ipa proteins) out of the bacteria and into their host cells [23]. This system is encoded by more than 20 mxi and spa genes located on the large plasmid [21]. 2.
VIRG(ICSA)
Once free in the cytoplasm, EIEC and Shigella trigger an event that is responsible for their cell-to-cell spreading. Analogous to the process described for Listeria monocytogenes (see Chapter 16), these bacteria trigger nucleation of actin
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at one pole of the bacterial cell, which propels the bacterium through the cytoplasm at the head of this "comet tail" [86]. The VirG protein (also called IcsA) is critical and sufficient for triggering of this actin-mediated motion [87], probably by binding cytoskeletal components such as N-WASP [88]. When the moving bacteria encounter the cytoplasmic face of the host cell membrane, they propel outward, pushing into adjacent cells, thereby avoiding extracellular exposure. C. Virulence Gene Regulation Shigella, and presumably EIEC, coordinately regulate their virulence factors in response to certain environmental conditions, including temperature, osmolarity, and DNA supercoiling. In addition, secretion of Ipas is increased in the presence of mammalian cells or serum [89]. VirR is a chromosomally encoded protein that regulates the various virulence factors in concert with the plasmid-encoded AraC-like transcriptional activator VirR VirR is a histone-like protein, functioning like H-NS (see Chapter 3 for details), that modulates the expression of virulence genes by repressing transcription at 30°C or in low osmolarity [90-93]. Interestingly, mutations in the hns gene derepress transcription of invasion genes at 30°C only in the presence of VirF [90, 91, 93], indicating that the role of VirF is not simply to overcome the negative influence of H-NS, as has been suggested for CfaD in ETEC (see above). In addition, H-NS also seems to control expression of virF in a temperature-dependent manner [94, 95].
IV. Enteropathogenic Escherichia coli (EPEC) A.
Disease
Enteropathogenic E. coli (EPEC) is the predominant cause of infant diarrhea worldwide and represents a major endemic health threat to children under 6 months of age living in developing countries [96]. In addition, isolated outbreaks in day care centers, nurseries, and pediatric wards, as well as among adults in developed countries, have also been reported. It has been estimated that EPEC kills several hundred thousand children each year worldwide. EPEC disease is characterized by prolonged watery diarrhea of varying severity, with vomiting and low fever often accompanying fluid loss (reviewed in [18, 96, 97]). Despite our increasing knowledge of the bacterial factors and host molecules that mediate EPEC interactions with epithelial cells, the actual molecular mechanisms that cause diarrhea remain undefined [98]. Unlike other diarrheas caused by pathogenic E. coli strains such as ETEC (see above), no toxin has been implicated in EPEC-mediated diarrhea. Instead, EPEC binds to intestinal surfaces of the small bowel, causing a characteristic histological lesion called the attaching and effacing (A/E) lesion [99-101]. A/E lesions are marked by dissolution of the intestinal brush-border surface and loss of epithelial microvilli (effacement) at the sites of bacterial attachment. Once bound, EPEC reside on cup-like projections
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or pedestals formed by cytoskeletal rearrangements in host actin. EPEC-associated diarrhea has been attributed to the loss of absorptive surface area due to effacement of epithelial cell microvilli [102]. Although this mechanism remains to be proven, a series of recent in vitro studies have indicated that a more complex, yet not well-defined, multifactorial mechanism might be involved. Active ion secretion has been involved in the development of infectious diarrheal diseases [20]. It has been reported that EPEC causes significant depolarization of cultured HeLa and Caco-2 epithelial cells, altering the distribution of ions across the cell membrane [103]. In addition, EPEC infection of Caco-2 cell monolayers induced rapid but transient increases in short-circuit current (Isc) and CI" secretion [104]. Occurrence of such a process in the gut would reduce the electrochemical gradient available for sodium ion absorption from the gut lumen, thereby contributing to ionic imbalance, fluid loss, and diarrhea. However, other reports suggest that this may not be the case by demonstrating that EPEC-infected epithelial cell monolayers show a diminution in net ion transport, reflected as a decrease in short-circuit current (Isc), without revealing any difference in Cl~ channel activity [105, 106]. Early studies have also indicated that EPEC infection of HEp-2 cells increased the intracellular concentration of free calcium ([Ca^^in) [107-109]. It has been hypothesized that this rise in [Ca^"^]in leads to disruption of the microvillus actin cytoskeleton by activating a calcium-dependent actin-severing protein, providing a plausible explanation for microvilli effacement [107]. However, in contrast to these data, a significant increase in [Ca^'^]in was not observed on EPEC infection in HEp-2 cells, using calcium-imaging fluorescence microscopy, which allows spatial and temporal measurements of [Ca^"^]in in live cells [110]. Furthermore, chelation of intracellular calcium with BAPTA did not inhibit pedestal formation, as previously reported [107, 109], suggesting that calcium fluxes are probably due to EPEC-mediated cytotoxicity [110]. In addition to morphological rearrangements that occur on the apical surface of cells, EPEC causes a large decrease in transepithelial electrical resistance in polarized epithelial cell monolayers [111]. EPEC infection induces phosphorylation of myosin light chain [112], an event that also leads to a decrease in transepithehal electrical resistance by disrupting tight junction integrity [113, 114]. EPEC infection also causes transmigration of polymorphonuclear leukocytes across the epithelial cell monolayers, which may contribute to disruption of epithelial barrier function [115]. Such transepithelial disruptions may occur in vivo, and this could lead to ionic imbalances, an increase of intestinal permeability, and possibly diarrhea. By using EPEC mutant strains, it has been shown that some of the virulence factors required for the formation of A/E lesions in vitro are also required to induce some of the events described above and are also needed to cause disease in human volunteers or animal models [103, 116-118]. What is not yet clear is how these virulence factors actually cause disease. Although EPEC can enter (invade) tissue culture cells [119, 120], it does not normally cause invasive disease and rarely penetrates the intestinal barrier.
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EPEC belongs to a family of related pathogenic organisms that form A/E lesions, including enterohemorrhagic E. coli (EHEC), several EPEC-like animal pathogens that cause disease (e.g., REPEC in rabbits and PEPEC in pigs), Citrobacter rodentium, and Hafnia alvei [98, 121]. These organisms all cause cytoskeletal rearrangement and pedestal formation on host epithelial cells, and encode conserved proteins that mediate these effects.
B. Virulence Factors
The development of an A/E lesion has been divided in three stages (Fig. 4) (reviewed in [18, 98, 122, 123]). The first stage is characterized by the initial nonintimate attachment of EPEC to the epithelial cell surface in a pattern termed localized adherence (LA) [124-127]. LA is associated with the production of a type IV fimbriae known as bundle-forming pili (BFP) [128]. During the second stage, a set of EPEC-secreted proteins (Esps) triggers the activation of signal transduction pathways leading to a complex response by the epithelial cell and cytoskeletal rearrangements [129-132]. Finally, during stage three, an outer membrane protein called intimin allows EPEC to attach intimately to the host cell membrane on interaction with its translocated intimin receptor call Tir [133, 134]. Like many other pathogens, EPEC's virulence factors are found in clusters or pathogenicity islands. EPEC possess a large 69-kb plasmid that encodes two major virulence attributes—a bundle-forming pilus (BFP) needed for initial localized adherence to host cells and bacterium-bacterium interactions, and the regulatory locus Per (BfpTVW), which regulates expression of EPEC virulence factors (see below) [135]. All the factors necessary to form A/E lesions are clustered in a 35.5-kb region in the EPEC chromosome, inserted at the selenocysteine {selQ tRNA gene [136, 137]. This region of DNA (called the locus for enterocyte effacement, or LEE region) encodes >40 open reading frames, and has a low G-i-C content (38% versus 51% for E. coli), indicating it was acquired by horizontal transfer [138]. The LEE can be divided into three functional regions: one encoding a type III secretion system, a region needed for intimate binding to host cells (encodes Tir, CesT and intimin), and the one harboring the genes for E. coli effector proteins (Esps) that are secreted by the type III system and their chaperones (Fig. 4). The Esps trigger signals and actin rearrangements in host cells and are needed for A/E lesions (see below). In addition to the Esps, EPEC also insert another molecule (Tir) into host cell membranes [134, 139]. Upon insertion, Tir is tyrosine phosphorylated and serves as the receptor for intimin, an EPEC surface protein [133, 134]. Tir-intimin binding leads to underlying cytoskeletal organization and resultant pedestal formation (Fig. 4). Thus, this enteric pathogen uses a very specialized sequence of events to establish an intimate interaction with host cells, which then results in disease.
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tight junction^ disruption
PMN
Fig. 4 EPEC interactions with epithelial cells. The initial nonintimate attachment of EPEC microcolonies to the epithelial cell surface (the localized adherence phenotype) is mediated by type IV fimbriae known as BFP. After this initial interaction, secretion and translocation of the EspA, EspB, EspD, and Tir proteins onto and into the host cell cytosol is mediated by the type III secretion apparatus. EspA is assembled into a large filamentous organelle that is essential for translocation of EspB. Upon translocation, Tir is phosphorylated and inserted into the plasma membrane, where it binds to the bacterial OM protein intimin. At this stage, Tir nucleates cytoskeletal components including actin, alpha-actinin, talin, and ezrin. These cytoskeletal rearrangements form actin-rich pedestals beneath the adhering EPEC. Translocated proteins trigger the activation of signal transduction pathways that lead to a complex cellular response. Enhancement of membrane-associated protein kinase C (PKC) activity might stimulate CI" secretion. Tyrosine phosphorylated phospholipase Cy converts phosphatidyinositol-4,5-biphosphate (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG), which might stimulate the release of intracellular calcium ([Ca^"^]i from IP3-sensitive stores. Activation of the transcription factor NF-KB initiates interleukin 8 (IL-8) transcription, which in turn stimulates transmigration of polymorphonuclear cells (PMNs). Myosin light-chain kinase (MLK) activation leads to tight junction phosphorylation and increased intestinal permeability.
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BUNDLE-FORMING PILUS ( B F P )
EPEC initial adherence to host cells involves the generalized nonintimate interaction of bacterial microcolonies in a pattern known as localized adherence (LA) [124]. This pattern of attachment requires the 69-kb EPEC adherence factor (EAF) plasmid [125-127], which is considered a common property of the classic EPEC serotypes [140]. The EAF plasmid contains a cluster of 14 genes that is sufficient to direct synthesis of BFP, a 7-nm-in-diameter type IV fimbriae associated with microcolony formation and interbacterial interactions, that tends to aggregate into bundles (Fig. 5) [128, 141-143]. Several of the proteins encoded by the bfp genes exhibit similarity to other proteins involved in the biogenesis of
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PerC t, (BfpW)
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Fig. 5 EPEC virulence genes: organization and transcriptional regulation. Two major groups of genes are involved in EPEC virulence. The EAF plasmid contains the bfp operon, consisting of 14 genes that encode the functions required for BFP biosynthesis. Transcription of the bfp operon requires the product of the perA (bfpT) gene. PerA (BfpT) belongs to the AraC family of transcriptional activators and is required for autoactivation of the per (bfpTVW) operon. The genes encoding the secreted proteins, the type III secretion system, and the proteins involved in intimate attachment are located in the LEE region organized in at least five operons, denominated LEEI-LEE5. Expression of these operons, except LEEI, requires the product of ler, the first gene of the LEEI operon, to overcome the negative effect of H-NS. Interestingly, Ler shares significant similarity with H-NS. In addition, PerC (BfpW) seems to positively influence the expression of ler.
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Other type IV pilus such as TCP in Vibrio cholerae and the Pseudomonas aeruginosa piH [142, 143]. In addition to the proteins encoded by the bfp cluster, BFP biosynthesis requires DsbA, an enzyme that mediates disulfide bond formation [144]. EPEC can encode at least two other types of fimbriae, although the role of these in pedestal formation and disease has not been characterized [145]. The role of BFP in the development of EPEC-mediated diarrhea has not been clearly established. Experiments with pediatric small intestinal tissue in in vitro organ culture have suggested that BFP are not involved in initial adherence but mediate interbacterial interactions that allow formation of three-dimensional bacterial aggregates at a later stage of infection [146]. However, it has been demonstrated that a functional bfpA gene, the first gene of the bfp operon that encodes the major structural subunit of BFP [ 147, 148], is required for production of BFP and full virulence in human volunteers [149]. A mutation in bfpF that encodes one of the putative nucleotide binding proteins that provide energy for pilus biosynthesis significandy affected the capacity of EPEC to cause diarrhea in humans [149]. Mutations in BfpF increased piliation, enhanced the localized adherence phenotype, and abolished twitching motility, affecting the dispersal phase of microcolony formation, which is associated with dramatic alterations in the structure of BFP bundles [ 149-151]. Other A/E pathogens do not contain BFP, but instead encode other adhesins that mediate adherence to host tissues [152]. For example, AF/Rl and AF/R2 fimbriae mediate the initial adherence of rabbit EPEC strains RDEC-1 and REPEC O103, respectively [153-156]. These differences in initial adherence might relate to their host and tissue specificity, as EPEC is a human-specific pathogen and does not infect animals [12]. 2.
EPEC-SECRETED PROTEINS (ESPS)
When EPEC interact with cultured epithelial cells, several signal transduction pathways are activated in the epithelial cells, including release of the eukaryotic secondary messenger IP3 [109, 129, 157], activation of phospholipase Cy [158], protein kinase C [159], and NF-KB [160], and phosphorylation of host proteins [161] (Fig. 4). In addition, tyrosine dephosphorylation of several host proteins, which is observed following EPEC infection, correlates with inhibition of bacterial uptake by macrophage-like cell lines [162]. EPEC binding to cultured epithelial cells also causes tyrosine phosphorylation of Tir (formerly Hp90) [133, 134]. The key to EPEC activation of eukaryotic signal transduction pathways is effector proteins secreted by the type III secretion system (described below). Strains containing mutations in genes encoding the EPEC-secreted proteins EspA {E. coli secreted protein A), EspB (formerly EaeB), or EspD do not stimulate signal transduction, cytoskeletal rearrangements, or the antiphagocytic phenotype [129, 131, 132, 162]. As for other type III effectors in bacterial pathogens, the Esps lack an amino-terminal signal sequence characteristic of sec-dependent secreted proteins [23] and require a chaperone for proper secretion [163].
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EspA is a secreted protein with an apparent molecular weight of 25 kDa [131] that constitutes a major component of a bacterial surface organelle [164]. It was proposed that this structure serves as a delivery system for other Esps and Tir, as mutants lacking EspA cannot deliver these molecules into host cells [134, 164]. EspA does not appear to be injected into host cells. These filaments provide an essential first step in the molecular crosstalk between the bacterium and the host cell. Furthermore, the role of EspA is critical for virulence, as mutations in the espA gene render rabbit EPEC avirulent [118]. EspB (38 kDa, formerly EaeB) is translocated directly into host cells, where it is distributed between the mammalian cell membranes and cytosol, where it presumably mediates its effects [164-166]. Strains lacking EspB are unable to activate host signals, rearrange actin, or form A/E lesions [165, 167]. This molecule is central for EPEC pathogenesis, as mutants in EspB are not virulent, and are unable to form A/E lesions in an animal model [118]. How EspB mediates its effects has not been clearly defined, but experiments with HeLa cell clones transfected with the espB gene have suggested that EspB may act as a cytoskeletal toxin disrupting filamentous actin distribution and function [168]. Alternatively, it may form a pore in the host membrane to allow other EPEC effectors such as Tir to pass into the host cell [130]. EspB does not seem to be a component of the EspA filament, since this filament is produced in an espB mutant strain and anti-EspB antiserum does not stain the filaments [164]. EspD (40 kDa) is the third secreted protein involved in A/E lesion formation [132]. Upon secretion, EspD is inserted into the host cell membrane, but apparently not translocated into the cytoplasm [169]. By analogy with the Yersinia YopB protein, it has been suggested that EspD is part of the putative EPEC translocation apparatus [169]. Its role in virulence has not been established, but, given the results with EspA and EspB, it is likely to be required for disease. Another EPEC-secreted protein (EspF) was identified in 1998 [170]. This protein requires the type III secretion system for its secretion; however, EPEC mutants lacking EspF still formed pedestals on cultured cells that were indistinguishable from those seen with the parental strain [170]. Although no role for EspF could be identified using tissue culture cells, it is possible that it may play a role in vivo. Another major EPEC protein, EspC (110 kDa), is still secreted in strains lacking the type III secretion system, and instead mediates its own secretion using an autotransporter mechanism [171]. It is quite homologous to several other members of the autotransporter family, including EspP in EHEC [172], Pet in EAEC [173], and Sep A in S.flexneri [174], and shares some similarity to Neisseria gonorrhoeae'^ IgA protease and Haemophilus influenzae^ high-molecular-weight adhesins [175]. Autotransporters utilize a sec-dependent secretion system to cleave a signal peptide off as it passes to the periplasm. These proteins then insert their C terminus into the outer membrane in a (J-barrel, through which the N terminus passes out of the bacterium [176] (see Chapter 2 in this volume). Several
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members of this family, including EspC, cleave themselves, releasing a smaller secreted protein [177]. The role of EspC in EPEC pathogenesis is unclear, as mutants in EspC still form pedestals in tissue culture [171]. An EspC mutation has not been tested for virulence in a relevant animal model, and the rabbit EPEC strains do not produce an EspC homolog. EspC is not encoded within the LEE region. 3.
THE TYPE III SECRETION SYSTEM
As with many other Gram-negative pathogens, EPEC use a type III system to secrete effector proteins out of the bacteria and translocate these proteins into host cells [23] (see chapter by Silhavy and Harper for details). This type III system is encoded by >20 esc {E. coli secretion) and sep (secretion of E. coli proteins) genes, conforming at least three potential operons (Fig. 5) [138]. Work with mutant strains has shown that some of the genes are important for secretion [178, 179]; however, the specific role and function of all the predicted components of EPEC's type III secretion system remains undetermined. As with other type III secretion systems, there are also predicted chaperones encoded within the LEE to assist in secretion of the Esp proteins. For example, a chaperone for EspD, CesD (chaperone for E. coli secretion D), has been described [163], and CesT has been shown to be a chaperone for Tir [180, 181]. The main function of the type III system is to transport Tir and the Esps (other than EspC) out of the bacteria and into the host cell, although some of the Esps (such as EspA) also play a role in establishing the process of translocation into the host cell (see above). 4.
INTIMIN
Intimin, the product of the eae locus within the LEE, is a 94-kDa outer membrane protein needed for intimate adherence [182] and full virulence in both human and animal models [117, 183-186]. Intimin mutants form immature A/E lesions, characterized by diffuse actin accumulated near adherent bacteria, that is not focused and reorganized into defined pedestals beneath adherent organisms; however, epithelial signals are still activated, since the Esp effectors are still delivered to host cells [103, 133, 161]. Intimin molecules direct the final condensation and reorganization of the underlying host cytoskeleton by binding to Tir [133, 134]. The amino-terminal region of intimin is needed for export to the outer membrane and shares homology with Yersinia invasin (see Chapter 6 by Boyd and Comelis), suggesting that invasin and intimin share secretion and membrane insertion mechanisms. In contrast, its cell binding domain is located at the highly divergent C-terminal 280 amino acids (Int280) [187], which are sufficient for adherence to the EPEC protein Tir [134]. Sequence comparison of the different intimin types, which have been designated alpha, beta, delta, and gamma, has
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revealed a nonrandom clustering of polymorphic sites mainly in the C-terminal domain, suggesting that protein divergence has been accelerated by recombination and diversifying selection [188, 189]. It has also been reported that, like invasin, intimin binds P-1 integrins [190, 191]; however, the significance of these findings remains unclear, since a more recent report indicates that P-1 integrins are not essential for intimin-mediated cell attachment and A/E lesion formation [192]. To date, a Tir-independent cell-binding activity for intimin cannot be ruled out [102]. Immunological and amino-acid sequence analysis of the cell-binding domain of different intimins has revealed the existence of at least five different intimin subtypes that may be responsible for tissue tropism [188, 193]. The global fold of Int280 has been determined by nuclear magnetic resonance, revealing that it is structured in three domains: two immunoglobulin-like domains and a C-type lectin-like module [194].
5.
TRANSLOCATED INTIMIN RECEPTOR (TIR)
The A/E lesion (or pedestal) formed by EPEC on interaction with epithelial cells is associated with the assembly of highly organized cytoskeletal structures in the epithelial cells immediately beneath adherent bacteria [98]. These structures contain actin, a-actinin, talin, ezrin, myosin light chain, and other molecules associated with polymerized actin structures (Fig. 4) [112, 195, 196]. Although this pedestal usually raises the bacterium slightly above the epithelial cell surface, EPEC can trigger extended pseudopod formation, with projections extending up to 10 microns above the epithelial cell surface, with the bacteria located extracellularly at the tip of these extensions [196]. Extended pedestals are not seen when strains containing mutations in eae, espB, tir, or type III loci are used, reinforcing the linkage between signal transduction events and cytoskeletal rearrangement. Interestingly, in contrast with other enteric pathogens that trigger cytoskeletal rearrangements, small GTP-binding proteins such as Rac, Rho, and Cdc42 do not seem to be involved in pedestal formation [197, 198]. At the tip of these structures in the epithelial membrane is Tir, where it interacts with intimin on the bacterial surface, linking the bacterium intimately to the host cell. Tir (formerly Hp90, also called EspE) is a bacterial protein that is inserted into host membranes, requiring the type III secretion system and Esps for its delivery to host cells [133, 134, 199]. Tir is predicted to be an integral membrane protein with two transmembrane domains, both N- and C-terminal domains probably facing toward the cell cytosol and a central extracellular loop [122, 134, 200]. Tir is secreted out of EPEC as a 78-kDa unphosphorylated protein. Once inserted into the host membrane, it is tyrosine phosphorylated and its apparent molecular weight shifts to 90 kDa. Alkaline phosphatase treatment reduces the 90-kDa form to 78 kDa. Unlike the Esp proteins and other type III secreted proteins, its amino-terminal methionine is missing from the bacterial secreted protein [134].
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The role of tyrosine phosphorylation of Tir in the host cell is unclear. The bacterial secreted form of Tir (unphosphorylated, 78 kDa) binds intimin in a dose-dependent, saturatable, and competitive manner. Additionally, EHEC Tir is not tyrosine phosphorylated (see later), yet it binds intimin and is interchangeable with EPEC Tir [200]. The phosphorylation of tyrosine 474 of Tir seems to be essential for actin nucleation activity, but not for the increase in apparent molecular mass observed in target cells, suggesting additional modifications [201]. Deletion analysis of Tir has led to identification of the intimin-binding domain, an extracellular loop that resides between the two previously predicted membrane-spanning regions [191, 201, 202]. Tir binding to intimin triggers additional signals in host cells, including activation of phospholipase C-y. Thus, it seems that Tir has several functions: to bind intimin, to focus the cytoskeletal rearrangements induced by EPEC, and to potentially send additional signals to the host cell [122, 134].
C. Virulence Gene Regulation
Despite the current knowledge about EPEC pathogenesis, little is known about the regulation of virulence gene expression in this microorganism. Transcriptional regulation of BFP expression occurs selectively during the exponential phase of growth in tissue culture media at 37°C, where it is modulated by ammonium concentration and temperature [203]. The coordinate regulation of the genes contained in the bfp operon is controlled by a promoter located upstream of bfpA [141, 203, 204]. Activation of the bfpA promoter requires the product of the bfpT gene (BfpT), which is the first gene of the bfpTVW operon (previously identified as the perABC locus), localized 18 kb downstream of bfpA on the EAF plasmid [205, 206]. BfpT (PerA) belongs to the AraC/XylS family of transcriptional factors [76, 205, 206], and its expression is autoregulated and modulated by the same environmental signals that regulate bfpA expression [207] (Fig. 5). The genes encoding the type III secretion apparatus {esc and sep), the EPEC-secreted effectors {esp) and the proteins involved in intimate attachment {tir, cesT, and eae) are organized in at least five different polycistronic units or operons {LEEl to LEE5) (Fig. 5) [208, 209]. Except for LEEl, expression of these operons requires the product of the ler gene, which is located at the beginning of the LEEl operon [208-210]. The Ler protein exhibits amino acid similarity with H-NS, a histone-like DNA binding protein that has been involved in negative regulation of virulence factors [138]. Ler is required to overcome the repression carried out by H-NS on expression of the promoters located upstream of the LEE2, LEE3, and LEE4 operons. These promoters are Ler independent in the absence of H-NS or on removal of negative regulatory sequences located upstream of the putative -35 boxes [210].
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The bJpTVW (per) operon is also involved in the regulation of genes contained within the LEE region [205, 208, 211], and in the production and/or secretion of EspB and other secreted proteins, which also respond to conditions similar to those found in the gastrointestinal tract [130, 205, 212]. However, in contrast to its direct role in bfpA and Z?^7 activation [204, 206, 207], it is still not clear how this locus participates in expression of LEE-encoded genes, since transcriptional activation of LEE2 to LEE5 promoters is similar in both EPEC wild-type and EPEC strains lacking the EAF plasmid [209, 210]. In addition, it has also been suggested that initial contact with HeLa cells induces de novo protein synthesis by EPEC and activation of its type III secretion system [165, 213].
V. Enterohemorrhagic E. coli (EHEC) A.
Disease
Over the last 20 years, EHEC strains have emerged as the cause of a major health problem, particularly in developed countries [22, 214, 215]. Following ingestion of EHEC-contaminated food or water and transit to the large bowel, there is an incubation period of 3 to 4 days. The onset of disease is a nonbloody watery diarrhea, abdominal pain, and fever. Vomiting may also occur at this stage. As the disease progresses, abdominal pain increases and bloody diarrhea commences. In the majority of cases, the bloody diarrhea subsides and symptoms resolve. However, in 10-20% of cases (especially in the pediatric and geriatric populations), EHEC infections can lead to the development of serious life-threatening complications such as hemolytic uremic syndrome (HUS) (hemolytic anemia), thrombotic thrombocytopenic purpura (TTP) (decreased platelets in the blood), and renal failure [216]. It has a fatality rate of 5%, while about 25% of patients will have permanent kidney damage. The typing of EHEC strains is often based on their O (LPS) and H (flagella) antigens (e.g., 0157:H7). However, there is no evidence that either of these antigens is involved in disease. Despite the broad variety of EHEC serotypes found in the gastrointestinal tract of domestic animals, only a limited number of them (particularly 0157, 0111, and 026) are associated with the serious clinical manifestations seen during human EHEC infections [22, 216]. EHEC is acid resistant, and it is thought that, like Shigella species, acid resistance accounts for its low infectious dose (10-100 organisms) [217-219]. EHEC outbreaks have been caused by contamination of various foodstuffs, including beef, radishes, lettuce, sprouts, apple juice, salami, yogurt, and even chlorinated water [220-224]. Cattle can be asymptomatic EHEC carriers, and contamination with beef products or manure can often be traced as the source of EHEC [225-228].
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Virulence Factors
The disease is currently associated with three major virulence attributes: the capacity to cause formation of A/E lesions, mediated by the genes encoded within the LEE; the expression of Shiga toxin (Stx); and the presence of a 60-MDa plasmid that encodes a hemolysin (see below) [18, 22, 229]. However, it has been observed that other pathogenic serotypes (such as 026, 0103, and 0111) have a distinctive pattern of virulence factors, with respect to that of E. coli 0157:H7 [230]. In addition, there are "atypical EHEC" strains that express Stx but do not produce A/E lesions nor possess the plasmid. It is thought that the capacity to attach tighdy to enterocytes and elicit the formation of A/E lesions contributes to the nonwatery diarrhea; this process appears necessary for intestinal colonization. Once established, EHEC secrete the Stx, which has systemic effects, causing the bloody diarrhea and HUS. 1.
SHIGA TOXIN
Also known as Shiga-like toxins (SLTs) or verotoxins (VTs), the Shiga toxins produced by EHEC strains possess high similarity to the cytotoxins produced by 5. dysenteriae [231]. The production of Stx constitutes a key element to EHEC pathogenesis and a distinctive characteristic that distinguishes EHEC strains from EPEC. Because of this, EHEC is also called STEC (for Stx-producing E. coli) or VTEC (for verotoxigenic E. coli). Two subgroups of serologically distinguishable toxins, Stxl and Stx2, have been recognized in EHEC. While Stxl is identical to S. dysenteriae Stx, Stx2 is 56% identical to the other toxins and presents a number of variant forms, such as Stx2c, Stx2d, and Stx2e [232-235]. The sequence variability, which is mainly observed in the B subunit, is reflected not only antigenically, but also in receptor binding and toxicity for tissue culture cells or in animal models [236-239]. Stxl and Stx2 toxins (except Stx2e) are encoded by bacteriophages (i.e., toxin-converting bacteriophages), which are able to spread stx genes among enteric E. coli strains [244]. Interestingly, some antimicrobial agents have been shown to cause prophage induction, which could increase the copy number and transcription of the stx genes and cell lysis-mediated toxin release [245, 246]. This process is mediated by the Rec A protein, because recA mutants showed a significant reduction in toxin synthesis and were deficient of specific phage production [246,247]. Sequence and genetic analysis has shown that the stx genes are part of an apparent Q-dependent late transcript, suggesting that toxin production and phage release would be regulated by the Q gene product, rendering maximal expression during the lytic growth of the phage [246,248,249]. Despite the common elements found for Stxl and Stx2 prophages, they show sequence and morphological differences (Stx 1 phages are more closely related to lambda), as well as different phage immunity and receptor affinity [248-250]. As mentioned above, the Stx produced by EHEC is identical to that produced by S. dysenteriae and uses the same mechanism (reviewed in [20]). Like LT, CT, and several other toxins, Stx (Stx) is an AB5 toxin, with one 32-kDa catalytic
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JOSE L. PUENTE AND B . BRETT FINLAY
subunit (A) and five 7.7-kDa binding subunits (B) [251]. The A subunit is nicked into two products (Ai and A2) that are Hnked by a disulfide bond. Toxin binding to its specific glycolipid receptor, globotriaosylceramide, or GB3, on cell surfaces, occurs by the B subunits [252]. Following binding, toxin uptake occurs by endocytosis, followed by retrograde transport to the Golgi apparatus and endoplasmic reticulum (litde is known about this unconventional endocytic routing in cells) [253]. The A subunit enters the cytoplasm, where the Al peptide acts as a specific ^-glycosidase that cleaves an adenine from the 28S ribosomal RNA [254, 255], inhibiting elongation factor 1 (EF-1 )-dependent aminoacyl tRNA binding [256]. This action blocks protein synthesis, resulting in death of intoxicated cells (Fig. 6). Intestinal cell death may result in hemorrhagic colitis (bloody diarrhea) due to a breach in the intestinal barrier, while the pathogenesis of HUS and TTP is characterized by Stx-mediated destruction of endothelial cells in venules and Stx hoioloxln
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Fig. 6 Structure and mode of action of Stx. The Stx holoenzyme is composed of a single catalytic subunit (A) that is associated with a pentameric ring formed by B subunits. The B subunit binds the toxin to a specific glycolipid receptor, globotriaosylceramide (Gb3). Once bound to the cell membrane, the toxin molecules seem to be internalized by receptor-mediated endocytosis, through clathrin-coated pits. Vesicles containing toxin-receptor complexes are transported to the Golgi apparatus and then to the endoplasmic reticulum, before being translocated to the cytosol (retrograde transport). The A subunit is proteolitically nicked during this process, generating the catalytically active A] amino-terminal portion, which remains linked to the C terminus by a disulfide bond. The A| subunit has an A^-glycosidase activity that depurinates the 28S rRNA of 60S, resulting in inhibition of protein synthesis and cell death.
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arterioles, which results in a thrombotic microangiopathy [257]. This damage is also believed to be due to circulating host-derived cytokines such as tumor necrosis factor alpha (TNF-a), TNF-P, interleukin-ip (IL-IP) and IL-6, which sensitize endothelial cells (ECs) to the cytotoxic action of the toxins [258-260]. This enhanced sensitivity to the toxins is probably achieved by inducing or increasing the expression of the glycolipid Gb3 receptor, thus increasing Stx binding [257, 261-266]. Animals differ in their susceptibility to Stx probably due to varying expression of Gb3. For example, rabbits infected with Stx-expressing RDEC-1 (a rabbit EPEC that encodes an LEE) showed more serious histological changes, including edema and inflammation (much like hemorrhagic colitis) than those infected with RDEC-1 alone [267]. This study suggested that Stx is responsible for the bloody diarrhea and HUS seen in EHEC outbreaks, presumably due to intoxication of intestinal and renal cells. In contrast, Stx is not an essential factor to produce disease in piglets [268]. In addition, experiments with T- or B-cell lines from different origins have led to the conclusion that lymphocytes are also susceptible to Stx and that these toxins contribute to the pathogenesis of EHEC-associated diarrhea by suppressing the mucosa-associated immune response [269, 270]. It has been suggested that Stx2 expressing strains are more likely to be associated with the development of HUS [271], although this is not always the case. Using human intestinal microvascular endothelial cells (HIMECs), it was observed that the binding affinity of Stxl was 50-fold greater than that of Stx2. Nonetheless, Stx2 was more toxic to HIMECs than an equivalent amount of Stxl, a feature that may explain the higher association of Stx2-producing STEC with cases of hemorrhagic colitis and its systemic complications [272]. 2. LEE EHEC possesses an LEE that is functionally and structurally similar to the LEE found in EPEC [136]. Interestingly, a recent characterization of the EHEC 0157:H7 LEE revealed that the molecules that are thought to be on the bacterial surface or interact with host cells (i.e., those that would be exposed to the host immune system)—such as the Esps, Tir, and intimin—are much more diverse than the others when compared to the EPEC products [273]. In addition, the EHEC LEE also encodes a cryptic prophage of the P4 family [273]. The LEE region in 0157:H7 is inserted at the same position as EPEC within the selenocysteine tRNA gene. However, other EHEC serotypes contain an LEE inserted at different positions such as the pheU locus and other undefined insertion sites [274]. Intriguingly, in contrast to what was observed with the EPEC LEE [137], the LEE from EHEC 0157:H7 was unable to confer on an E. coli K-12 strain the capacity to form A/E lesions or to secrete Esp proteins [275]. Like EPEC, EHEC 026:H~ produces a Tir (also called EspE) protein that becomes tyrosine phosphorylated on translocation into host cells, serves as the
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intimin receptor, and focuses cytoskeletal components beneath the adherence site [199]. In contrast, EHEC 0157:H7 Tir is not tyrosine phosphorylated and acts as the primary determinant of bacterial adherence to epithelial cells [200, 276]. As for EPEC and EHEC 026:H-, EHEC 0157:H7 Tir binds intimin and focus cytoskeletal rearrangements, suggesting that tyrosine phosphorylation is not needed for pedestal formation. Despite the remarkable heterogeneity found in the amino-acid sequence of Tir proteins from different EHEC serotypes [277], as well as between the C-terminal domain of different intimins [278], it has been shown that EHEC and EPEC intimins are functionally interchangeable; although EHEC Tir shows a much greater affinity for EHEC intimin than for EPEC intimin [200]. In vivo studies have shown that E. coli 0157:H7 requires intimin to efficiendy colonize the intestinal tract, to cause diarrhea and A/E lesions in neonatal calves, and to cause colonic edema and A/E lesions in piglets [184, 186, 279], as well as for determining the site of intestinal colonization [185]. As mentioned above, the LEE region of EHEC also encodes proteins homologous to EspA, EspB, and EspD [273], which are secreted through the type III apparatus [280-282]. As for EPEC, EHEC EspA is essential for bacterial attachment and is a part of filamentous appendages that appear during the early stages of the attachment process and are necessary for protein translocation of other effector proteins [283]. Similarly, EHEC EspD is required to obtain efficient bacterial attachment to target cells and to establish a direct link between bacteria and eukaryotic cells via EspA-containing surface appendages [284]. EspD is transferred to the cytoplasm and is also found as an integral protein of the cytoplasmic membrane of infected cells. By interacting with EspB to form a pore in the cytoplasmic membranes of the target cells, it may also facilitate translocation of the effector proteins required for A/E lesion and intimate attachment, resembling the interaction between Yersinia YopB and YopD proteins [284].
3.
THE 6 0 - M D A PLASMID
In addition to the Stx phages and the LEE region (see above), EHEC also possess a 60-MDa plasmid termed p0157 that is not found in EPEC. The contribution of this plasmid to EHEC adherence or colonization has not been clearly established. While some authors have described a plasmid-dependent adherence to epithelial cells in vitro or intestinal colonization in vivo [285-287], others have been unable to confirm these observations [268, 288, 289]. Despite the lack of consistent experimental evidence about the role of p0157 in disease, the determination of its full nucleotide sequence confirmed that it encodes several potential virulence factors, including a hemolysin (HlyA), a catalase-peroxidase (KatP), a serine protease (EspP) and a type II secretion system, as well as a protein containing a putative active site shared with the large clostridial toxin (LCT) [290, 291].
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a. The hemolysin. The EHEC hemolysin encoded within the p0157 plasmid is highly conserved between many EHEC strains of different serotypes isolated from outbreaks [292-296]. Although its role in pathogenesis has not been clearly established, it has been suggested that it may stimulate bacterial growth in the gut by releasing hemoglobin from red blood cells, thus providing a source of iron [297]. Alternatively, one can speculate that erythrocyte lysis would lead to cellular debris that could affect renal function. The EHEC-hlyCABD (also called ehxCABD) operon codes for the EHEC hemolysin (HlyA), a protein required for HlyA activation (HlyC) [298], and the proteins that constitute the transport mechanism (HlyB and HlyD); this operon shares around 60% of identity with the alpha-hemolysin operon of uropathogenic E. coli [299-301]. EHEC hemolysin and alpha-hemolysin (discussed below) belong to the RTX family of pore-forming cytolysin toxins [299], which are widely distributed among Gram-negative bacteria [302]. b. The serine protease (EspP). EspP is a 104-kDa extracellular protein that shares significant similarity with a group of surface-associated or secreted bacterial proteins that are also known as autotransporters, which includes the IgAl protease of Neisseria gonorrhoeae, the Pet protein of EAEC (see below), and the EspC secreted protein of EPEC [172]. In contrast to the hlyA gene (see above), the gene coding for EspP is less widely conserved between EHEC isolates. Although present in EHEC 0157:H7 and 026:H~ strains, it was not detected in a significant number of EHEC isolates belonging to different serotypes (e.g., 0157:H-) [296]. This serine protease cleaves the coagulation factor V and is cytotoxic for Vero cells [172, 303]. These features have led to the suggestion that it might have a synergistic effect during the development of the hemorrhagic disease [172], a role that is supported by the presence of EspP antibodies in patients with EHEC infections [172].
4.
SIGNAL TRANSDUCTION
EHEC 0157:H7 induces rearrangements of cytoskeletal proteins such as F-actin and alpha-actinin independently of detectable tyrosine phosphorylation of Tir or host cell proteins. This suggests that EHEC (in contrast to EPEC) uses different signal transduction mechanisms to produce A/E lesions or that tyrosine phosphorylation is not important for this phenomena [200, 304]. EHEC infection of T84 cells decreases transmonolayer resistance and increases intercellular permeability, events that seem to be a consequence of the EHEC-mediated disruption of ZO-1 distribution in the tight junctions [305]. The increased epithelial permeability could lead to an alteration of the electrochemical gradients in the intestinal epithelium, resulting in diarrhea or initiation of inflammatory
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responses [305]. As for EPEC (see above), EHEC infection also results in the release of host second messenger molecules, including Ca^"^ and inositol triphosphate [276]. Signaling responses also include PKC and MLCK activation, which seem to be involved in altering tight junction permeability in T84 cells [305]. A possible additional mechanism involved in diarrheal disease has been suggested through the study of C57BL/6J mice infected with EHEC, which showed a progressive increase in both NF-KB activation and galanin-1 receptor (Gall-R) expression by epithelial cells lining the colon. On activation, Gall-R causes Cl~ secretion, promoting fluid secretion [306]. This observation was reproduced in vitro, where galanin also increases short-circuit current (Isc) in EHEC-infected T84 cells, in contrast to uninfected cells [306].
C. Virulence Gene Regulation As in EPEC, production and secretion of secreted proteins, such as EspA and EspB, is enhanced in tissue culture media at 37°C [280]. Likewise, Tir synthesis is stimulated in bacteria grown in defined media M9 or tissue culture media, but not in complex media LB [200]. Activation of esp operon transcription is favored at high osmolarities, modulated by temperature, and influenced by the degree of DNA supercoiling [307]. As for other virulence factors, the H-NS protein and sigma S factor participate in its regulated expression, which also seems to be switched off in tighdy attached bacteria [307]. EspP secretion is reduced in tissue culture media [280] but is optimally produced in nutrient broth at 37°C and pH 7, showing a dramatic reduction at 20°C and pH 5 [172].
Vl. Enteroaggregative E. coli (EAEC) A.
Disease
EAEC is associated with persistent pediatric diarrhea in developing countries and is characterized by its aggregative adherence pattern (for recent reviews see [308, 309]). EAEC causes a watery secretory diarrhea, often mucoid in nature. There is often low-grade fever, but no vomiting [310, 311]. Grossly bloody stools can occur, although the majority of cases do not involve bloody diarrhea [312]. Formation of a thick mucus gel on the intestinal mucosa and mucosal damage probably mediated by a mucosa damaging toxin are pathogenic features of EAEC histopathology [313, 314]. EAEC often affects children [310, 312], although adults cases have also been reported [315, 316].
9.
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B. Virulence Factors EAEC strains are a heterogeneous collection of pathogenic E. coli that share certain chromosomal and plasmid-bome genes [18, 308, 317]. Their defining feature is that they adhere to cultured HEp-2 cells in small clumps or aggregates ("aggregative adherence," or AA), resembling a stacked-brick configuration [318] (Fig. 7). As expected, adherence to host cells and neighboring bacteria is mediated by fimbrial adhesins encoded on a large plasmid. EAEC can increase mucus secretion, leading to a blanket of adherent bacteria trapped in a layer of mucus. Some EAEC can cause tissue damage, resulting in villus atrophy and other cytotoxic effects that are probably mediated by toxins, although the contribution of each toxin to disease has not been defined (see below). Invasiveness of tissue culture cells by some EAEC strains has been suggested [319]. However, although EAEC seem to be able to colonize many regions of the gastrointestinal tract, as studied in human intestinal explants, these explants do not show bacterial intemaHzation [313].
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V, Fig. 7 EAEC interactions with intestinal cells. AAF fimbriae mediate the initial adherence of EAEC strains to the intestinal mucosa in a stacked-brick configuration (aggregative adherence). Colonization enhances mucus production, leading to accumulation of a thick mucus layer where bacterial cells remain embedded. During this process, EAEC delivers different toxins that damage the mucosa and promote intestinal secretion. The heat-stable enterotoxin (EASTl) is related to ETEC ST and may act similarly (see Fig. 1). Pet and Pic belong to the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily.
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1.
ADHERENCE FACTORS
EAEC produce at least two fimbrial adhesins: aggregative adherence fimbriae I and 11 (AAF/I and AAF/II, respectively). AAF/I mediates adherence to tissue culture cells and human erythrocytes and is encoded by two regions on the 60-MDa plasmid [320, 321]. One region encodes the fimbrial structural gene and assembly genes (such as chaperones) [322], while the other is a regulatory region encoding an AraC-like regulator named AggR [323]. AAF/I is a member of the Dr family of adhesins, which adhere to the Dr blood group antigen. AAF/I are flexible fimbriae 2-3 nm in diameter and are capable of forming bundles, although they do not share homology with the type IV bundle-forming pilus of EPEC [320]. AAF/I fimbriae are produced by a small number of EAEC strains. AAF/II are a second fimbriae produced by some EAEC strains [324]. Like AAF/I, they are encoded by two regions (separated by 12 kb), although, unlike AAF/I, the structural subunit and the assembly genes are encoded in separate regions (a characteristic of the Dr family of adhesins) [325]. The AAF/I and AAF/II subunits are 25% identical. AAF/II fimbriae are filaments 5 nm in diameter arranged in semirigid bundles that may play an important role in adherence, as strains lacking AAF/II are no longer capable of adhering to human intestinal tissue [324]. 2. EAST-1 Many EAEC strains produce a plasmid-encoded heat-stable toxin designated enteroaggregative E. coli heat-stable toxin 1 or FASTI [326]. This toxin shares identity with STa (see above) and probably works in a similar manner by activating guanylate cyclase, leading to secretory diarrhea. It is composed of 38 amino acids (4.1 kDa) and contains four cysteine residues (like guanylin, but differing from STa, which has 6) that form disulfide bonds to stabilize the toxin [327, 328]. However, the contribution to disease by FASTI has not been established. Many E. coli strains produce FASTI, including 0157, several ETEC and even EPEC [326] strains; however, clinical isolates of EAEC that do not produce FASTI are common, and even nonpathogenic E. coli produce FASTI, indicating that it alone is not sufficient to cause disease [326]. Much like ETEC, it is likely that EAEC possess multiple adhesins and toxins that, in various combinations, contribute to disease.
3.
PET
AND P I C
EAEC also produce a 108-kDa plasmid-encoded enterotoxin (Pet) that may contribute to disease [173]. Purified Pet is capable of causing rises in short-circuit current (Isc) and falls in tissue resistance, characteristic of toxins that causes secretory diarrhea [329]. In addition, EAEC causes increased mucus release and exfoliation of cells, processes seen in EAEC disease [329], and is also able to
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elicit cytoskeletal changes in epithelial cells [330]. The cytopathic and enterotoxic effects induced by Pet are ascribed to its protease activity [330]. However, as with EASTl, not all EAEC strains produce this toxin [317], although it may contribute to the pathogenicity of some strains during infection [315]. Some EAEC strains also produce and secrete a chromosomally encoded 110-kDa protein denominated Pic (protein involved in intestinal colonization), which is synthesized as a 146.5-kDa precursor molecule that is processed at the N and C termini during secretion. Pic is presumably involved in mucinase activity, serum resistance, and hemagglutination [331]. Pet and Pic belong to the autotransporter class of bacterial proteins that mediate their own secretion, exhibiting most sequence similarity to a subgroup termed the SPATE subfamily (serine protease autotransporters of the Enterobacteriaceae) that includes EspC from EPEC and EspP from EHEC (see above) [175, 331].
VII. Diffusely Adhering E. coli (DAEC) A.
Disease
E. coli strains showing a diffuse adherence pattern in tissue culture cell assays are known as DAEC [125, 318, 332]. With the characterization of EAEC, DAEC is now recognized as a separate class of E. coli, although little is known about their virulence mechanism and their association with diarrheal disease remains controversial. While some studies clearly establish a link between DAEC strains and diarrheal disease [333-335], others suggest that there is no such association [312, 336]. DAEC strains are also considered a heterogeneous group that comprises strains with different pathogenic potential due to the presence of variable virulence factors (see below). DAEC strains have been more frequently associated with persistent watery diarrhea in children between the ages of 2 and 5 years [337, 338]. B. Virulence Factors Diffuse adherence has been associated with four different adhesins, while toxins have not been described in any detail. DAEC strain CI845 expresses the F1845 fimbria [339], a member of the Dr family of adhesins [340]. This fimbrial adhesin uses the membrane-associated decay-accelerating factor (DAF) as receptor [341]. Interaction of F1845-expressing strains with DAF induces elongation and nucleation of microvilli in differentiated Caco-2 cells [341] and formation of long thin membrane projections in Hep2 cells [342, 343], and promotes F-acting rearrangements in INT407 cells, a process that involves recruitment of signal transduction molecules [344]. It has also been shown that some daaC positive strains (daaC codes for a molecular usher of F1845 fimbriae [345]) secrete Esp homologs, which are necessary to induce signal transduction events and the A/E phenotype in EPEC and EHEC [346]. The expression mechanism of the F1845 fimbrial genes, which may be
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JOSE L. PUENTE AND B . BRETT FINLAY
encoded on a plasmid or the chromosome, has been a model system for the study of fimbrial-regulated expression by mRNA processing [339, 345, 347]. According to some studies, F1845 positive strains seem to be widely distributed around the world [18]; however, others suggest that this fimbria is rare among DAEC strains [348]. DAEC strain 2787 of serotype 0126:H27 expresses a 100-kDa outer membrane afimbrial adhesin denominated AIDA-I [349, 350], which belongs to the family of outer membrane autotransporters [351]. This plasmid-encoded adhesin is synthesized as a 132-kDa precursor molecule that is processed at the C terminus by an autocatalytic cleavage mechanism, and seems to require a 45-kDa cytoplasmic protein for its correct maturation [352, 353]. In addition, a 57-kDa mannose-resistant hemagglutinin [354] and two major surface proteins of 16 and 29 kDa [333] have been associated with diffuse adherence.
VIIL Uropathogenic E. coli A.
Disease
Uropathogenic E. coli (UPEC) strains are responsible for approximately 80% of community-acquired and 30% of nosocomial-acquired urinary tract infections (UTIs). Females under 10 years of age, or between 18 and 40, are at the highest risk for community-acquired infections. Infections in children are often due to blockages in the urinary tract, resulting in pools of stagnant urine. Similarly, nosocomial infections are usually associated with indwelling urinary catheters, resulting in loss of flushing action of urine, which, in some cases, can proceed to a systemic infection. Aside from the distal tip of the urethra, the urinary tract is usually sterile. UPEC can reside in the colon and then be introduced into the urethra. UTIs result from ascending colonization of the urinary tract by these strains. Infections can occur in the urethra (urethritis), bladder (cystitis), and kidneys (pyelonephritis), and, under some conditions, the microorganisms enter the blood stream. Disruptions of the normal flora (such as the use of vaginal spermicides) or direct inoculation (e.g., during intercourse) can cause infections. The clinical symptoms of UTIs vary depending on the region of the urinary tract that is colonized. Painful or difficult urination (dysuria) and sometimes suprapubic pain characterize cystitis. Pyelonephritis includes the above symptoms and fever, flank pain, shivers, and sometimes vomiting. Treatment includes antibiotic therapy, although there is increased incidence of antibiotic-resistant organisms, especially in nosocomial infections. Catheter removal facilitates treatment in most cases. B. Virulence Factors In order to successfully colonize and establish a UTI, UPEC strains take advantage of an assortment of virulence properties [355, 356]. Urine flow is the
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largest obstacle facing E. coli attempting to colonize the urinary tract. Not surprisingly, UPEC encode several adhesins, both fimbrial and afimbrial in nature, which facilitate adherence to uroepithelial cells. However, like most adhesins, it has been difficult to precisely define the role of any particular adhesin due to overlapping function (redundancy). Type I and P fimbriae, the most common fimbriae found in UPEC strains, enhance virulence and are involved in initial urethral colonization (see below) (Fig. 8). In addition, many UPEC produce hemolysin, which may be involved in kidney disease, and the CNF-1 toxin. Certain UPEC strains possess iron sequestration systems to assist in growth, whereas others produce a capsule (usually Kl or K5) that may help avoid clearance from the urinary tract. Lipopolysaccharide also
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Uroi^lakiii
Mr^M^!*?iMi5^H R0l«ase of cytokines
Fig. 8 UPEC interaction with uroepithelial cells. Most UPEC strains express P or type I pili. P pili bind to a glycolipid Gala(l,4)Gal, while type I pili bind to mannose-containing glycoprotein receptors, known as uroplakins. The components of both pili share functional similarities, although the tip fibrillum of P pili is more complex. Coupling of the P pili PapG adhesin with its surface receptor activates expression of a bacterial iron-acquisition system by inducing the expression of AirS, a sensor-regulator protein. In addition, this interaction also stimulates the intracellular release of ceramides, which leads to cytokine production after activation of protein kinases. In addition, the toxic activity of hemolysin and CNF-1 may contribute to the kidney damage seen in pyelonephritis. When hemolysin binds calcium, it can be inserted into host membranes to form a pore, which eventually lyses the host cell. CFN-1 induces profound changes on the cytoskeleton of epithelial cells, such as actin reorganization and membrane ruffling, by modifying the GTPase activity of Rho.
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appears to be involved in cytokine induction [357]. In general, it appears that the more virulence factors a uropathogenic E. coli strain possesses, the more severe the disease symptoms. 1.
TYPEIPILI
Type I pili, originally identified by their ability to mediate mannose-sensitive agglutination, are expressed by the majority of E. coli strains derived from patients with cystitis and pyelonephritis. Despite the extensive knowledge on type I pili, their role in disease is controversial. It is unlikely that type I pili play a role in gastrointestinal disease, although many enteric E. coli pathogens and nonpathogenic fecal isolates produce this type of pili. However, there is good evidence that it is involved in colonization of the oropharynx that contributes to invasive diseases (sepsis and meningitis) in neonates. There is also good evidence that type I pili contribute to infections of the lower urinary tract, such as cystitis, by mediating colonization of the bladder mucosa, promoting bacterial persistence, and enhancing the inflammatory response to infection, thus increasing UPEC virulence [15]. Type I pili have a composite structure of 7 nm in width and 1-2 microns in length and consist of a long rigid rod and a distal thin fibrillar structure [358]. Expression of type I pili requires at least nine genes in ihtfim cluster, encoding products that are assembled in a manner very similar to P pili (see below). FimH is the adhesin protein responsible for binding to mannosylated glycoproteins [359, 360] and is located at the distal tip of the heteropolymeric type-1 pilus rod, which is predominandy constituted by FimA subunits [358]. FimG and FimF are minor components that are probably needed as adaptors, initiators, or terminators, FimC is the chaperone and FimD the outer membrane usher [358, 361, 362]. FimH mediates E. coli binding to mannose-containing glycoprotein receptors, known as uroplakins, that are located on the luminal surface of the bladder epithelial cells [16]. Antibodies to FimH reduce colonization of the bladder mucosa and disease in a murine cystitis model, suggesting that a FimH-based vaccine may provide a means of preventing these infections [363-365]. FimH may have another role in promoting bacterial survival in the host by mediating binding to macrophages. This interaction, probably through CD48 receptors located on the macrophage surface, triggers a different endocytic pathway that appears to increase the ability of E. coli to survive within them, possibly by affecting phagolysosomal fusion with vacuoles containing UPEC [366]. 2.
PAP
One of the best-studied examples of pili and its associated assembly machinery is P pili (pyelonephritis-associated pili), which are encoded by pap genes found within pathogenicity islands (see below). As its name suggests, this adhesin is critical for upper UTI infections (pyelonephritis). The pap operon is a useful
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421
example of pilus assembly since it contains many conserved features that are found among various pilus operons, including type I pili (reviewed in [367-369]). Two molecules guide newly synthesized pilus components to the bacterial surface (reviewed in [370, 371]). PapD, a conserved chaperone molecule with an immunoglobulin-like domain, is necessary to transport several pilus subunits from the cytoplasmic membrane to the outer membrane [372, 373]. PapD-subunit complexes are targeted to the PapC outer membrane (OM) usher, which forms a pore through which the pili are translocated across the OM [374]. The major subunit of the pilus is PapA, which is assembled into a 6.8-nm thick helical rod that is anchored in the outer membrane by PapH [375-377]. At the distal end of the pilus rod is a 2-nm linear tip fibrillum composed of PapE [378], which is adapted to the PapA rod by PapK [379]. The actual molecule that mediates adherence (i.e., tip adhesin), PapG, is joined to the PapE tip fibrillum by the adapter protein PapE [379]. PapG mediates binding to the a-D-galactopyranosyl-(l-4)-P-D-galactopyranoside (Gala(l,4)Gal) moiety present in a globoseries of glycolipids found on host cells lining the upper urinary tract and erythrocytes [380-382]. There are three adhesin variants of PapG—G-I, G-II, and G-III—which recognize three different but related Gala(l,4)Gal receptors. It is thought that the distribution of these receptors differs among hosts and tissues, and differential expression of the PapG adhesins at the pilus tip could determine tissue and host specificity. Although the host receptor varies for different bacterial pili, the general features of the P pilus operon are conserved in many other pilus systems, and components are often interchangeable. For example, the PapD chaperone can modulate the assembly of type I pili, which mediate binding to mannose-containing molecules on the host cell surface. The gene organization of related pilus operons is also usually conserved. Thus, while type I and P pili are encoded by similar operons and functionally analogous sequences that can be aligned, they bind to quite different carbohydrates on the cell surface (reviewed in [367, 370]). One of the most interesting features of PapG-mediated interactions with its Gala(l,4)Gal-containing glycolipid receptor is the ability to activate specific responses in the bacteria and in the epithelial cell that promote virulence [383]. For example, AirS is a sensor-regulator protein that is expressed only when PapG binds to its receptor, thereby allowing activation of the UPEC iron-acquisition system. Efficient iron acquisition allows UPEC to grow in urine, an otherwise growth-limiting environment [384]. In addition, this interaction also triggers the intracellular release from receptor glycolipids of ceramide, an important second messenger that can activate cytokine production, through the activation of serine/threonine protein kinases and phosphatases [385-387]. 3.
AFIMBRIAL ADHESINS
E. coli produce a family of adhesins that bind to the same mammalian receptor. Members of this family include F1845 and Dr, which are fimbrial adhesins found
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JOSE L. PUENTE AND B . BRETT FINLAY
in gastrointestinal pathogens, and two nonfimbrial adhesins (afimbrial adhesins Afa-I and Afa-III) expressed by many uropathogenic E. coli [339, 388, 389]. All four of these adhesins use as receptor the Dr^ blood group antigen present on decay accelerating factor (DAF) on erythrocytes and other cell types, although they appear to recognize different epitopes of the Dr antigen [340]. Much like other fimbrial adhesins, expression and production of these adhesins requires 5-6 gene products. These include a periplasmic chaperone, an outer membrane anchor protein, 1 to 2 transcriptional regulators, and the adhesin [388, 390, 391]. At least for Afa-III, there appear to be two adhesins (AfaD and AfaE) encoded within this operon [392]. The AfaE and AfaD confer upon an E. coli laboratory strain the ability to bind to HeLa cells and to mediate internalization of the adherent bacteria, respectively [393]. Some adhesins of this family (F1845 and DR) form fimbriae, whereas others form nonfimbrial adhesins on the bacterial surface (Afa-I and Afa-III). This appears to be dictated by the sequence of the adhesin molecule because switching the genes encoding these adhesins switches the adhesin type [392]. It is possible that the E. coli afimbrial adhesins have evolved from the related fimbrial adhesins, but have been altered such that the properties needed to polymerize a pilus are missing, yet the adhesin domain remains anchored on the bacterial surface. 4.
HEMOLYSIN
Approximately half of UPEC strains that cause upper UTIs, about a third of those that cause lower UTIs, and only about 10% of fecal isolates produce a hemolysin (HlyA) that belongs to the RTX (repeats in toxin) family and shares similarity with the hemolysin described in EHEC (see above) [299, 302]. Four genes (hlyABCD) are required for the production and export of the hemolysin: hlyA encodes the hemolysin structural gene; HlyC acylates HlyA posttranslationally, adding fatty acids to two internal lysine residues via amide linkages [394]. It is thought these acyl chains anchor HlyA to host cell lipid bilayers or assist in HlyA oligomerization in host bilayers. The HlyB and HlyD proteins are needed for HlyA secretion out of E. coli, as is the TolC protein. When calcium binds to HlyA, this toxin is capable of inserting into host membranes to form a pore, which eventually lyses the host cell. Such toxic activity may contribute to the kidney damage seen in pyelonephritis. 5.
CYTOTOXIC NECROTIZING FACTOR
1 (CNF-1)
About one-third of UPEC strains produce cytotoxic necrotizing factor 1 (CNF-1), a 1014-aa cytotoxin that is also produced by some gastrointestinal E. coli (see above) [395, 396]. CFN-1 induces profound changes in the cytoskeleton of epithelial cells, including actin reorganization and membrane ruffling [397399], and impairs migration and proliferation of bladder cells that could interfere with repair of the bladder epithelium [400]. It has been demonstrated that CFN-1
9. PATHOGENIC ESCHERICHIA COLI
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interacts with Rho, a GTP-binding protein whose GTPase activity becomes constitutive on CFN-1-mediated deamidation of glutamine 63 [401^04]. However, a role for this toxin in urinary tract infection has not been defined. 6.
PATHOGENICITY ISLANDS
Like the LEE region seen in EPEC and EHEC, uropathogenic E. coli contain pathogenicity islands, which provides an excellent example of the role these regions play in acquisition of specific virulence factors [3, 405, 406]. The uropathogenic strain 536 contains two large unstable pathogenicity islands: PAI-I (70 kb) and PAI-II (190 kb) (Table II) [407]. Both pathogenicity islands are flanked by short (16-18 bp) direct repeats, which are likely responsible for their deletion due to recombination at a frequency of 10~^ [408]. PAI-I is inserted at the same site as LEE in EPEC and 0157:H7 EHEC (immediately downstream of selC at 82 min on the chromosome) and encodes, among other genes, the hly hemolysin operon. PAI-II is inserted at the leuX tRNA locus at 97 min on the chromosome, and encodes another hly operon and the prf (P-related fimbriae) pilus operon [3]. Like the LEE region, the G-HC content of these regions is lower (41%) than that of E. coli K-12 (51%). Similarly, another uropathogenic strain of E. coli (J96) contains two pathogenicity islands inserted in two different tRNA genes (pheV at 64 min for PAI-IV and pheR at 94 min for PAI-V) [409]. Each of these islands encodes an hly hemolysin operon; this is in addition to single copies of a pap operon (PAI-IV) and prs (P-related sequence) and cnfl for PAI-V. In addition, E. coli CFT073 possess the smallest of the five PAIs described (50 kb), which is inserted in the vicinity of the metV gene and carries an hly operon [410]. 7.
VIRULENCE GENE REGULATION
Like most virulence factors. Pap pili expression is tightly regulated in response to several environmental and nutritional factors, including temperature, amino Table II
Pathogenicity Islands of Uropathogenic E. coli
Strain
Pathogenicity island
536 536 J96 J96
Pail Paill PailV Pai V
CFT073
Pai VI
Genes hly hly, prf hly, pap hly, prs, cnfl hly
Size (kb)
Map location (min)
tRNA
70 190 170 110
82 97 64 94
selC leuX pheV pheR
50
26/63
metV
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JOSE L. PUENTE AND B . BRETT FINLAY
acids, and glucose, and modulated by different regulatory factors such as the leucine-responsive regulatory protein (Lrp), CRP (cAMP-binding protein), H-NS, Papl, and PapB. Pap pili expression is also controlled by a methylation-dependent phase variation mechanism (on/off switching) in response to these conditions [412, 413]. The promoter region of the pap operon contains two GATC sites that can be methylated by deoxyadenosine methylase (Dam) (Fig. 9B). GATC boxes I and II overlap with the DNA-binding sites for the leucine-responsive regulatory
Major i sutHintt I
Pap operon
I Milortip I comf>ofient
OM chaperone Periplasmic ch«{>«fOfi«
Rugulatton
RN«9e E
B
D»fti
Pha$# ON
Dam
papl Phase OFR
Vhkm
I RNA polymera^^e # l>apB HCIRP # Pa{>l
Fig. 9 Organization of the pap operon and regulation by phase variation. (A) Organization of the pap operon and posttranscriptional regulation. The papB and papA genes are cotranscribed, but the PapA protein is produced in a substantial molar excess over PapB. This difference is achieved through differential stability of the papB and papA segments, after mRNA processing by RNase E at a site located between the two genes. Higher stability of the papA transcript is in part due to a stem-loop structure that forms at its 5' untranslated region. (B) Phase variation is controlled at the transcriptional level by Dam methylation transition at two GATC sites within the papI-papB intergenic region. An increase in cAMP allows the CRP protein to interact with the papl upstream region, favoring its transcription. Papl then binds to Lrp, switching the protein complex from GATC-II to GATC-I, which then allows expression of papl and pap B (phase ON). Papl-Lrp inhibits methylation of GATC-I. When Lrp binds the nonmethylated GATC-II, transcription of the papB promoter is blocked and GATC-I remains methylated blocking transcription (phase OFF).
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protein (Lrp), which regulates the methylation state and, consequently, whether pili will be expressed (phase on) or not (phase off) [414, 415]. During phase on, the GATC-II site (nearest the main operon promoter) is methylated, while the distal GATC-I site is not, allowing the binding of an Lrp-Papl complex to this site, which protects it from methylation and promotes transcription by RNA polymerase. In contrast, during phase off, while the GATC-I site is methylated, the GATC-II site is unmethylated and bound by LRP alone (not associated with Papl), a configuration that blocks RNA polymerase binding and transcription [416, 417]. Transcription of the pa/?/gene, divergently located with respect to the papBA operon, is also modulated by PapB [418,419], CRP [420,421], and H-NS [422], in response to environmental cues. Thus, the phase switch frequency seems to be regulated by the varying concentrations of free Lrp and Lrp-Papl complex. Higher levels of the major fimbrial subunit Pap A are obtained by posttranscriptionally controlling the synthesis of other components through mRNA processing and differential RNA stability. Processing of the papBA transcript by RNAse E proceeds at an intercistronic-specific cleavage site, which consists of an mRNA conformation that exposes A/U-rich nonpaired regions. This step leaves a very unstable papB mRNA and a papA mRNA with a 10-fold longer half-life that is mainly stabilized by a stem-loop structure located at its 5' untranslated region [423-427]. Expression of type I pili is also regulated by a phase variation mechanism that controls the RecA-independent inversion of a 314-bp DNA fragment that carries ihtfimA promoter. This inversion is mediated by two recombinases encoded by the fimB Siud fimE genes, which are divergently transcribed upstream offimA, and occurs by a site-specific recombination at a 9-bp inverted repeat sequences [428, 429]. FimB functions independently of the orientation of the promoter fragment, while FimE prefers to switch from the on phase to the off phase [430, 431]. Expression of type I pili is also controlled at the translational level in strains lacking a functional leuX tRNA5^^" gene, which is disrupted as a result of deletion of the pathogenicity island known as Paill (see below). The fimB gene contains few TTG codons recognized by this tRNA, which is required for efficient translation of FimB, as well as other virulence factors and proteins influencing metabolic properties. In consequence, FimB synthesis is reduced in an leuX mutant strain, thus allowing FimE to switch the system off. This process can be alleviated by replacing the TTG codons in the fimB gene by CTG (the most abundant leucine-specific codon) or by expressing the leuX gene from a multicopy plasmid [432-434]. IHF is involved in the DNA inversion process, as well as in optimizing transcription of fimA [435, 436]. In addition, H-NS seems to be responsible for inhibiting the inversion event, while Lrp stimulates phase switching in both directions [437-439]. Differences in the expression of the fim recombinases or sequence changes around the fim switch affect regulation of type I pili in different UPEC isolates [440]. The infection of an animal model seems to favor selection of bacteria carrying the ON orientation [441].
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IX. E. coll That Cause Sepsis and Meningitis A.
Disease
In addition to gastrointestinal and urinary tract infections, some strains of E. coli can cause invasive (septic) diseases in newborns. The most common cause of neonatal sepsis is E. coli, accounting for about one-third of the cases. Neonatal sepsis is a serious disease, with an incidence of 1 in 1000 of all infants, climbing to about 65 per 1000 in live births in newborn intensive care infants. It has a case fatality rate of 20-75% and a significant incidence of neurological damage in survivors. Gram-negative organisms cause about one-quarter of the cases reported for neonatal meningitis, and about one-half of these organisms are E. coli [442-444]. E. coli can colonize the oropharynx at birth, and it is thought that this is the first step toward septic disease. Bacteremia can then occur, which may progress to septicemia. Circulating bacilli then colonize the meninges, resulting in meningitis. B. Virulence Factors Because of the different host environments encountered on mucosal surfaces or within the body, strains of E. coli causing systemic diseases have specialized virulence factors that are different from those seen in diarrheagenic or uropathogenic E, coli. These factors include a capsule that blocks complement activity and phagocytosis from circulating phagocytic cells. However, since colonization of the tract is an initial step in the infecting process, these strains, like other pathogenic E. coli, also possess adhesins. Few other virulence factors have been described for these strains, although they undoubtedly must have specialized mechanisms to acquire iron and other nutrients as they grow in body fluids. 1.
CAPSULE
E. coli that cause septic diseases produce a polysaccharide capsule that blocks complement and antibody deposition on the bacterial surface. If complement is deposited on the capsular surface, it is distal to the bacterial outer membrane, and the membrane attack complex is unable to insert into the membrane and lyse the bacteria. By blocking antibody deposition, capsules avoid opsonic uptake and clearance by antibody-mediated immune events. Capsules are also often poorly immunogenic, and this may be due to similar oligosaccharides in the capsule and on host cell surfaces. Collectively, these features make capsules a critical virulence factor for strains of £". coli causing invasive diseases [24]. Capsules are made of repeating carbohydrates loosely associated with the bacterial surface [445]. The dominant capsule type is Kl: approximately 40% of septicemic and 75-80% of meningitis disease caused by E. coli is due to strains
9.
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expressing the Kl capsular antigen [446]. The Kl capsule confers serum resistance and antiphagocytic properties, and thus contributes to the high level of bacteremia required for development of E. coli meningitis [447]. Although it is not required for invasion of brain endothelial cells, the Kl capsule protects encapsulated bacteria from killing during invasion [448]. The Kl polysaccharide is a homopolymer of sialic acid residues in a a2,8 linkage. Polysialic acid capsules are also found in other pathogens that cause invasive disease, such as Neisseria meningitidis group B. Fourteen genes encoded within a 17-kb region (the kps gene cluster) are needed for Kl capsule production in E. coli. This region can be functionally divided into three regions. There is a central region encoding the neu (for neuraminic acid, which is sialic acid) gene products necessary for polysialic acid synthesis, which are different for each capsular type containing a different carbohydrate base. The flanking regions (which are remarkably conserved in other encapsulated bacteria) are needed for polymer transport from the cytoplasm to the bacterial surface. One of them harbors the kpsM and kpsT genes, which encode ATP-binding cassette (ABC) transporter proteins involved in transport of polysialic acid through the inner membrane. At least two periplasmic kps products are found in the cytoplasm, which assist in further transport of polysialic acid. A functional porin is needed to move the complex through the outer membrane. Phosphatidic acid anchors polysialic acid to the bacterial surface (reviewed in [449]).
2.
TYPE I Piu AND S-FIMBRIAL ADHESIN
As with uropathogenic E. coli, strains that cause meningitis and sepsis often produce type I pili. Although the presence of type I pili does not seem to increase the virulence of an organism, it appears to help in initial colonization of the oropharynx of neonates, allowing these strains to be more prevalent than other E. coli strains in the oropharynx [450]. It is unlikely that type I pili are needed once the organism enters circulation, and they are not involved in bloodstream invasion [451]. Some E. coli strains associated with newborn meningitis, such as E. coli IHE3034 (018:K1:H7), express S fimbriae that mediate binding to glycoproteins containing terminal sialyl-a2-3-galactoside moieties [452^54]. S fimbriae are expressed in vivo in blood and cerebrospinal fluid, and mediate binding to cells lining the choroid plexuses and brain ventricles, and to cellular fibronectin, which is found at sites of tissue trauma or inflammation [455-458]. The sfa gene cluster encodes the proteins involved in biogenesis and regulation of the S fimbriae. SfaA is the major structural subunit, SfaS is the sialic acid-binding adhesin, and SfaG and SfaH constitute minor subunits that are associated with the fimbrial filaments [459, 460]. The sfaC and sfaB genes encode regulatory proteins that exhibit similarities with Papl and PapB of UPEC, respectively [460, 461]. Lrp is also a positive regulator of sfa expression, although not in response to aliphatic amino acids [462].
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INVASION
Certain strains of E. coli show a propensity to invade the central nervous system. Using in vitro and in vivo models, it has been possible to study the mechanisms that allow E. coli strains to cross the blood-brain barrier and cause meningitis [447, 458, 463, 464]. Two genes, ibeA and ibeB, required for invasion of brain endothelial cells, have been identified and shown to contribute to invasion both in vitro and in vivo [463, 465]. ibeA codes for an 8.2-kDa protein with multiple predicted transmembrane domains. The purified protein is also capable of inhibiting invasion of brain microvascular endothelial cells [463]. ibeB codes for a 50-kDa putative membrane protein that exhibits 97% identity with a hypothetical protein oi E. coli K-12 [465]. Likewise, it has also been shown that the outer membrane protein OmpA contributes to the invasion of brain microvascular endothelial cells by E. coli Kl [466, 467].
X. Conclusions Pathogenic E. coli are a heterogeneous lot when their virulence factors are compared. It is becoming clear that pathogenic strains have arisen by acquiring a collection of virulence factors on plasmids, transposons, phage, and pathogenicity islands. It is also clear that these organisms continue to move these and other genetic elements around, potentially causing the creation of new pathogens. Acquisition of a specific set of virulence factors dictates the potential to cause a specific disease. Some virulence factors, especially some toxins and adhesins, can be redundant, making it difficult to define specific roles for each of these factors and to classify the various heterogeneous strains that all may cause a similar disease. However, significant inroads have been made into characterizing pathogenic E. coli virulence factors, and many of these strains provide models for other pathogenic organisms. The future lies in exploiting our knowledge about these mechanisms to develop new therapeutics to control these infections.
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428. Abraham, J. M., Freitag, C. S., Clements, J. R., and Eisenstein, B. I. (1985). An invertible element of DNA controls phase variation of type 1 fimbriae of Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 82, 5724-5727. 429. Eisenstein, B. I. (1988). Type 1 fimbriae of Escherichia coli: Genetic regulation, morphogenesis, and role in pathogenesis. Rev. Infect. Dis. 10(Suppl. 2), S341-S344. 430. McClain, M. S., Blomfield, I. C , and Eisenstein, B. I. (1991). Roles of fimB and fimE in site-specific DNA inversion associated with phase variation of type 1 fimbriae in Escherichia coli. J. Bacteriol. 173, 5308-5314. 431. McClain, M. S., Blomfield, I. C , Eberhardt, K. J., and Eisenstein, B. I. (1993). Inversion-independent phase variation of type 1 fimbriae in Escherichia coli. J. Bacteriol. 175, 4335^344. 432. Susa, M., Kreft, B., Wasenauer, G., Ritter, A., Hacker, J., and Marre, R. (1996). Influence of cloned tRNA genes from a uropathogenic Escherichia coli strain on adherence to primary human renal tubular epithelial cells and nephropathogenicity in rats. Infect. Immun. 64, 5390-5394. 433. Ritter, A., Gaily, D. L., Olsen, R B., Dobrindt, U., Friedrich, A., Klemm, R, and Hacker, J. (1997). The Pai-associated leuX specific tRNA5(Leu) affects type 1 fimbriation in pathogenic Escherichia coli by control of FimB recombinase expression. Mol. Microbiol. 25, 871-882. 434. Ritter, A., Blum, G., Emody, L., Kerenyi, M., Bock, A., Neuhierl, B., Rabsch, W., Scheutz, R, and Hacker, J. (1995). tRNA genes and pathogenicity islands: Influence on virulence and metabolic properties of uropathogenic Escherichia coli. Mol. Microbiol. 17, 109-121. 435. Dorman, C. J., and Higgins, C. F. (1987). Fimbrial phase variation in Escherichia coli: Dependence on integration host factor and homologies with other site-specific recombinases. J. Bacteriol. 169, 3840-3843. 436. Eisenstein, B. I., Sweet, D. S., Vaughn, V., and Friedman, D. I. (1987). Integration host factor is required for the DNA inversion that controls phase variation in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 84, 6506-6510. 437. Higgins, C. F., Dorman, C. J., Stirling, D. A., Waddell, L., Booth, I. R., May, G., and Bremer, E. (1988). A physiological role for DNA supercoiling in the osmotic regulation of gene expression in 5". typhimurium and E. coli. Cell 52, 569-584. 438. Kawula, T. H., and Omdorff, P. E. (1991). Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS. J. Bacteriol. 173, 4116-4123. 439. Blomfield, I. C , Calie, P J., Eberhardt, K. J., McClain, M. S., and Eisenstein, B. I. (1993). Lrp stimulates phase variation of type 1 fimbriation in Escherichia coli K-12. / Bacteriol. 175, 27-36. 440. Leathart, J. B., and Gaily, D, L. (1998). Regulation of type 1 fimbrial expression in uropathogenic Escherichia coli: Heterogeneity of expression through sequence changes in the/Im switch region. Mol. Microbiol. 28, 371-381. 441. Struve, C, and Krogfelt, K. A. (1999). In vivo detection of Escherichia coli type 1 fimbrial expression and phase variation during experimental urinary tract infection. Microbiology 145, 2683-2690. 442. Unhanand, M., Mustafa, M. M., McCracken Jr., G. H., and Nelson, J. D. (1993). Gram-negative enteric bacillary meningitis: A twenty-one-year experience. J. Pediatr 122, 15-21. 443. Quagliarello, V., and Scheld, W. M. (1992). Bacterial meningitis: Pathogenesis, pathophysiology, and progress. New Engl. J. Med. 327, 864-872. 444. Tuomanen, E. (1996). Entry of pathogens into the central nervous system. FEMS Microbiol. Rev. 18, 289-299. 445. Bliss, J. M., and Silver, R. P. (1996). Coating the surface: A model for expression of capsular polysialic acid in Escherichia coli Kl. Mol. Microbiol. 21, 221-231.
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446. Robbins, J. B., McCracken Jr., G. H., Gotschlich, E. C , Orskov, K, Orskov, I., and Hanson, L. A. (1974). Escherichia coli Kl capsular polysaccharide associated with neonatal meningitis. New Engl. J. Med. 290, 1216-1220. 447. Kim, K. S., Itabashi, H., Gemski, P., Sadoff, J., Warren, R. L., and Cross, A. S. (1992). The Kl capsule is the critical determinant in the development of Escherichia coli meningitis in the rat. J. Clin. Invest. 90, 897-905. 448. Hoffman, J. A., Wass, C , Stins, M. F., and Kim, K. S. (1999). The capsule supports survival but not traversal of Escherichia coli Kl across the blood-brain barrier. Infect. Immun. 67, 3566-3570. 449. Whitfield, C., and Roberts, I. S. (1999). Structure, assembly and regulation of expression of capsules in Escherichia coli. Mol. Microbiol. 31, 1307-1319. 450. Guerina, N. G., Kessler, T. W., Guerina, V. J., Neutra, M. R., Clegg, H. W., Langermann, S., Scannapieco, F. A., and Goldmann, D. A. (1983). The role of pili and capsule in the pathogenesis of neonatal infection with Escherichia coli Kl. 7. Infect. Dis. 148, 395-405. 451. Omdorff, P E., and Bloch, C. A. (1990). The role of type 1 pili in the pathogenesis of Escherichia coli infections: A short review and some new ideas. Microb. Pathogen. 9, 75-79. 452. Hacker, J., Kesder, H., Hoschutzky, H., Jann, K., Lottspeich, E, and Korhonen, T. K. (1993). Cloning and characterization of the S fimbrial adhesin II complex of an Escherichia coli 018:K1 meningitis isolate. Infect. Immun. 61, 544-550. 453. Korhonen, T. K., Valtonen, M. V., Parkkinen, J., Vaisanen-Rhen, V., Finne, J., Orskov, E, Orskov, I., Svenson, S. B., and Makela, P. H. (1985). Serotypes, hemolysin production, and receptor recognition of Escherichia coli strains associated with neonatal sepsis and meningitis. Infect. Immun. 48, 486-491. 454. Korhonen, T. K., Vaisanen-Rhen, V., Rhen, M., Pere, A., Parkkinen, J., and Finne, J. (1984). Escherichia coli fimbriae recognizing sialyl galactosides. J. Bacteriol. 159, 762-766. 455. Saukkonen, K. M., Nowicki, B., and Leinonen, M. (1988). Role of type 1 and S fimbriae in the pathogenesis of Escherichia coli 018:K1 bacteremia and meningitis in the infant rat. Infect. Immun. 56, 892-897. 456. Parkkinen, J., Korhonen, T. K., Pere, A., Hacker, J., and Soinila, S. (1988). Binding sites in the rat brain for Escherichia coli S fimbriae associated with neonatal meningitis. J. Clin. Invest. 81, 860-865. 457. Saren, A., Virkola, R., Hacker, J., and Korhonen, T. K. (1999). The cellular form of human fibronectin as an adhesion target for the S fimbriae of meningitis-associated Escherichia coli. Infect. Immun. 67, 2671-2676. 458. Stins, M. E, Prasadarao, N. V., Ibric, L., Wass, C. A., Luckett, P, and Kim, K. S. (1994). Binding characteristics of S fimbriated Escherichia coli to isolated brain microvascular endothelial cells. Am. J. Pathol. 145, 1228-1236. 459. Schmoll, T, Hoschutzky, H., Morschhauser, J., Lottspeich, E, Jann, K., and Hacker, J. (1989). Analysis of genes coding for the sialic acid-binding adhesin and two other minor fimbrial subunits of the S-fimbrial adhesin determinant of Escherichia coli. Mol. Microbiol. 3, 1735-1744. 460. Morschhauser, J., Uhlin, B. E., and Hacker, J. (1993). Transcriptional analysis and regulation of the sfa determinant coding for S fimbriae of pathogenic Escherichia coli strains. Mol. Gen. Genet. 238, 97-105. 461. Schmoll, T, Ott, M., Oudega, B., and Hacker, J. (1990). Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen. J. Bacteriol. 112, 5103-5111.
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462. van der Woude, M. W., and Low, D. A. (1994). Leucine-responsive regulatory protein and deoxyadenosine methylase control the phase variation and expression of the sfa and daa pili operons in Escherichia coli. Mol. Microbiol. 11, 605-618. 463. Huang, S. H., Wass, C , Fu, Q., Prasadarao, N. V., Stins, M., and Kim, K. S. (1995). Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: Molecular cloning and characterization of invasion gene ibelO. Infect. Immun. 63, 4470-4475. 464. Meier, C , Oelschlaeger, T. A., Merkert, H., Korhonen, T. K., and Hacker, J. (1996). Ability of Escherichia coli isolates that cause meningitis in newborns to invade epithelial and endothelial cells. Infect. Immun. 64, 2391-2399. 465. Huang, S. H., Chen, Y. H., Fu, Q., Stins, M., Wang, Y., Wass, C , and Kim, K. S. (1999). Identification and characterization of an Escherichia coli invasion gene locus, ibeB, required for penetration of brain microvascular endothelial cells. Infect. Immun. 67, 2103-2109. 466. Prasadarao, N. V., Wass, C. A., and Kim, K. S. (1996). Endothelial cell GlcNAc beta l-4GlcNAc epitopes for outer membrane protein A enhance traversal of Escherichia coli across the blood-brain barrier. Infect. Immun. 64, 154-160. 467. Prasadarao, N. V., Wass, C. A., Weiser, J. N., Stins, M. F, Huang, S. H., and Kim, K. S. (1996). Outer membrane protein A of Escherichia coli contributes to invasion of brain microvascular endothelial cells. Infect. Immun. 64, 146-153.
CHAPTER 10
Molecular Basis of Vibrio cholerae Pathogenesis VICTOR J. DIRITA
I. Introduction II. Vibrio cholerae A. Bacteriology B. Serovars and Serotypes C. Biotypes D. Virulence Factors III. Cholera A. Aspects of the Disease IV. Molecular Mechanisms of Disease A. Surface Biology of Vibrio cholerae B. Cholera Toxin and Toxinogenesis C. Toxin-Coregulated Pilus (TCP) D. Regulation of Toxin and Pilus Production V. Natural and Induced Immunity against Vibrio cholerae Infection A. Immunity to Cholera B. Killed or Subunit Vaccines C Live Vaccines VI. Future Studies: The Past Is Prologue References
457 458 458 459 459 461 463 463 465 465 467 478 481 489 489 490 491 493 495
/. Introduction Infection with Vibrio cholerae leading to the disease cholera continues to threaten large portions of the world's population. The disease process is not very complicated, owing to the facts that the disease occurs at the mucosal surface, Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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with no invasion by the microbe into deeper tissue, and that disease symptoms are primarily due to the action of a single molecule, the cholera toxin. Within this simple system of mucosal pathogenesis, extremely interesting mechanisms have been uncovered at practically every level of study, and this chapter outlines many of those mechanisms. This chapter is developed as follows. Section II and III include a general introduction to the bacterium Vibrio cholerae and to the disease cholera, including a brief discussion of the major virulence factors. Section IV is devoted to analysis of the mechanistic aspects of cholera infection, with particular emphasis on the cholera toxin, the most important virulence factor in the pathophysiology of disease. Section V describes the ways that knowledge regarding the molecular basis of infection is being applied to control V. cholerae infection through vaccine efforts based on what is understood about the mechanisms of disease. In each section, I will try to point out areas where our knowledge about a particular mechanism is incomplete, to give the reader a sense of where future investigation into the molecular pathogenesis of V cholerae infections may be leading us.
//. Vibrio cholerae A.
Bacteriology
The genus Vibrio is part of the family Vibrionaceae, which also includes the genera Aeromonas and Plesiomonas. Vibrio cholerae is one of several medically important species within the Vibrionaceae, the others being V. parahaemolyticus and V. vulnificus. As with closely related Gram-negative bacteria in the family Enterobacteriaceae, Vibrios are facultatively anaerobic and capable of mixed acid fermentation. Vibrio spp. are predominantly oxidase positive, in contrast to members of the family Enterobacteriaceae. Growth of V. cholerae and of the closely related species V. mimicus is stimulated by addition of 1% NaCl to the media, although these species can grow in the absence of added salt. This is not the case for other Vibrio species, and a salt tolerance test using 1% (w/v) tryptone broth with or without added NaCl is used for purposes of identification [1,2]. Little by way of specialized media or facilities are used to culture pathogenic isolates of V. cholerae, as they grow well on a variety of standard laboratory media under a range of conditions. When grown in liquid culture in a rich medium such as Luria-Bertani broth, the organism can reach high optical densities in overnight cultures. One of the hallmark features of Vibrio spp. is their tolerance to alkaUne pH, and this feature is exploited in different enrichment media. Among these are alkaline peptone water (APW), thiosulfate-citrate-bile salt-sucrose agar (TCBS)
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[3] and Monsur's medium [4], which is alkahne taurocholate-tellurite-gelatin agar. APW is often used as an enrichment medium for V cholerae by inoculating samples followed by subculturing to a selective medium such as TCBS. On TCBS, V. cholerae produces yellow colonies, due to its ability to ferment sucrose, and this distinguishes its appearance from that of V. parahaemolyticus, which is a nonfermenter and grows as a green colony. V cholerae grown on Monsur's medium appears dark gray due to reduction of the tellurite. Production of a gelatinase by V. cholerae is also reported on Monsur's medium, as such colonies produce a halo in which the gelatin in the medium is digested. Given the highly selective nature of media such as TCBS, they are often very useful in the research laboratory. For example, TCBS provides an excellent medium to select plasmidcontaining V cholerae after electroporation with naked DNA or matings with E. coli. Yet, antibiotics in this medium tend to be less stable than in a standard laboratory medium, presumably due to its high pH.
B. Serovars and Serotypes As with other Gram-negative bacteria, the outermost sugars of the lipopolysaccharide in the V. cholerae outer membrane have been used in a classification scheme. There are 139 0-antigen groups (serogroups) known of V cholerae, and, until recently, only the 01 serogroup was associated with epidemic cholera. Within the 01 serogroup two serotypes associated with epidemic cholera predominate: Inaba and Ogawa. In 1992 and 1993, an outbreak of cholera occurred in India and near the Bay of Bengal, and strains isolated from this outbreak did not type with 01 antisera or with any of the other 137 extant typing sera, so, by definition, this organism was termed Y cholerae 0139 [5-7]. As it was the only O serogroup outside of 01 known to cause epidemic disease, it became the subject of intensive investigation around the world in the months following the outbreak. It was ultimately demonstrated that a nearly complete loss of 01-encoding genes and acquisition of 0139-encoding genes had taken place [8].
C.
Biotypes
In addition to serogroup and serotype designations, V cholerae has a biotype classification, which is based on the bacteriological tests shown in Table I. Two major disease-associated biotypes, classical and El Tor, have been characterized. El Tor strains are the cause of the seventh cholera pandemic, which began in Indonesia in 1961 and persists today [9]. The seventh pandemic may be the first of the recorded cholera pandemics dating to the early nineteenth century to be caused by El Tor and not classical strains (strain data on pandemics preceding the
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Table I
Laboratory Tests to Distinguish V. cholerae Biotypes
Test Hemagglutination of chicken erythrocytes Susceptibility to polymixin B Phage IV susceptibility Voges-Proskauer reaction Sheep blood hemolysis
Classical V. cholerae
El Tor V. cholerae +
+ +
-
-
+ +
fifth do not exist [10]). Based on epidemiological data, the El Tor biotype appears to be associated with less severe disease than the classical biotype, and often causes asymptomatic infections [11, 12]. It has been hypothesized that the El Tor biotype has evolved to a lower level of virulence than the classical biotype as a consequence of transmission mechanisms in human populations. According to this hypothesis, the level of virulence of a gastrointestinal pathogen like V. cholerae is directly related to the likelihood of waterbome transmission as opposed to transmission by some other route such as contaminated food. Retrospective analysis of the literature on cases of diarrheal disease supports the association between waterbome transmission and greater virulence, and also shows a link between waterbome transmission and infection with classical V cholerae. In this analysis. El Tor strains were more associated with outbreaks from contaminated food such as shellfish [13]. The usefulness of the biotype designation for classifying epidemic isolates of V. cholerae has been recently challenged [10]. Based on analysis of sequence variation in the asd gene, which encodes a housekeeping function likely to be present in all V. cholerae, it was suggested that isolates of the sixth and seventh pandemics, which are caused by classical and El Tor biotypes, respectively, represent independently derived clones of V. cholerae that arose from environmental strains [10]. In this hypothesis, V. cholerae became vimlent for humans by acquiring groups of genes encoding factors of pathogenicity, such as the cholera toxin and the toxin-coregulated pilus (see below). It was proposed that the differences between the sixth and seventh pandemic isolates could be accounted for by the sixth pandemic isolates having lost functions still expressed in isolates from the seventh pandemic, such as hemolytic activity, chicken erythrocyte hemagglutination, and the ability to produce acetylmethylcarbinol (the basis for a positive response in the Voges-Proskauer reaction) [10]. As these factors are not required for vimlence, the genes encoding them may have easily accumulated loss-of-function mutations. Given the fact that the sixth pandemic isolates have been associated with humans for a longer time than the seventh pandemic isolates.
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it is more likely that they would lose these functions before the seventh pandemic isolates would. Consistent with this hypothesis are reports describing El Tor isolates that are negative for both hemolysis and the Voges-Proskauer reaction [10, 14, 15].
D. Virulence Factors V. cholerae is an environmental organism, and many strains in nature are not capable of causing human disease. Disease from infection by pathogenic strains of V cholerae is due primarily to gene products encoded on the genomes of two filamentous phages that lysogenize virulent strains: CTX(|) and VPIcj) [16, 17]. CTX(t) encodes the major pathogenicity factor of V cholerae, the cholera toxin (CT). This is an ADP-ribosylating enzyme that modifies a component (G^a) of a heterotrimeric G protein complex that regulates adenylate cyclase function [18]. Modification of G^a by ADP-ribosylation results in constitutive adenylate cyclase activity and an associated elevation of intracellular cyclic AMP (cAMP) levels, which, through activation of a cAMP-dependent protein kinase, in turn causes a chloride-selective membrane channel to open. The result is that chloride ions leave the cell and enter the intestinal lumen, creating an osmotic gradient down which flow other ions and water. The outcome for the infected patient is excretion of water and ions as a diffuse, watery diarrhea, which, left unchecked, can lead to severe dehydration and death in a matter of hours. Other toxins have been identified in pathogenic V cholerae, but their roles in disease pathogenesis are not well understood. As noted above, some El Tor strains produce a hemolysin (HlyA) that can lyse sheep erythrocytes; the role of this molecule in pathogenesis is not completely established, although the purified molecule causes fluid accumulation when injected into rabbit ileal loops, a commonly used model for studying diarrhea-causing pathogens [11]. Two other molecules encoded by CTXcj) are accessory cholera enterotoxin (Ace) and zona occludens toxin (Zot). Both Ace and Zot have toxic activity when measured using in vitro models [19, 20]. However, there is little evidence supporting a direct role for either of these molecules in pathogenesis. Indeed, because zot and ace are phage encoded and exhibit sequence homology to coat protein genes of other filamentous bacteriophage, they are very likely to be the coat proteins of CTX(|) [16]; yet it has not been determined whether the CTXtj) particles per se exhibit Ace or Zot toxic activity. Genes encoding a toxin of the RTX (repeats-in-toxin) family have been identified immediately downstream from the inserted CTXcj) genome in El Tor V. cholerae [21]. The toxin encoded by this sequence, RtxA, is toxic to cultured epithelial cells, but its contribution to symptoms in cholera patients or animal models has not been determined. One question of particular interest regarding
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RtxA is whether it is responsible for the symptoms often reported after administration of vaccine strains to volunteers. These attenuated V. cholerae (see below) have deletions of the gene for the active moiety of the cholera toxin ctxA, yet they often cause adverse symptoms, a phenotype termed reactogenicity [11, 22]. The second bacteriophage playing a role in cholera pathogenesis is VPI(|), which encodes a variety of determinants whose roles are important for allowing the microbe to identify and colonize the proper host environment [17, 23, 24]. These include the toxin-coregulated pilus (TCP), and a surface organelle made up of a single protein called TcpA, which is required for colonizing humans [25, 26]. In addition to being a colonization factor, TcpA appears to be the coat protein of VPI(|) [17], suggesting the remarkable situation of one phage using another phage as its receptor where each phage contributes to the pathogenicity of the host bacterium [17, 27]. Another VPI(|)-encoded factor involved in colonization (based on an infant mouse model) is the accessory colonization factor (ACF) [28]. Four «c/genes have been identified and, like the tcp genes, they are regulated by the same system that controls expression of cholera toxin [17, 24, 29, 30]. Their exact role in pathogenesis is not worked out, but one of these, AcfB, exhibits sequence similarity with chemotaxis regulatory proteins found in other Gram-negative species [31]. A similar homology is found in one of the tcp gene products, Tcpl, and mutants lacking either AcfB or Tcpl display altered motility phenotypes [31, 32]. These observations and other work on motility mutants of V cholerae suggest that an inverse relationship exists between expression of motility and expression of toxin and pilus [33]. A hypothesis that accounts for this relationship in the pathogenesis of cholera is that V cholerae swim through the mucus gel covering the intestinal epithelium prior to colonizing the mucosal surface [33]. This hypothesis predicts that two populations of organisms exist during infection: one that is motile and does not produce toxin and another that is involved in colonization and produces toxin (Fig. 1). However, the role that motility and chemotaxis play in virulence is not completely understood [33-36]. Of potential importance to vaccine production is the observation that the reactogenicity associated with ingestion of some vaccine strains may be related to motility [33, 37-39]. Physical mapping of the genome from the classical Ogawa strain 395 showed that it has two chromosomes or megareplicons [40]. One of these carries known genes for essential functions such as ribosomal RNA, synthesis, which raises the question of whether the smaller replicon isn't simply a large plasmid. However, since this chromosome accounts for 40% of the genome, carries unique genes, and is found in a variety of isolates [40], it is likely to be an essential component of the natural Y cholerae genome. VPI(|) and CTXcj) and several virulence regulatory genes are on the larger replicon in 0395, although there is a second copy of CTXcj) on the smaller one [40]. In many El Tor strains, the ctxAB genes
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Fig. 1 Model for opposite regulation of motility and colonization. Vibrio cholerae cells are hypothesized to be in two distinct populations within the intestine. A motile population within the lumen swims through the mucus layer (hatched area) but lacks expression of colonization factors or toxin. Cells that migrate to the epithelial layer stop swimming, represented by lack of flagella, and produce cholera toxin (CT) and colonization factors such as the toxin-coregulated pilus. Signals such as bile within the lumen may contribute to downregulation of pilus and toxin production.
are in tandem duplication, and their copy number can therefore be ampUfied through a homologous recombination mechanism [41].
///. Cholera A. Aspects of the Disease 1.
PATHOPHYSIOLOGY
Cholera has earned a frightful reputation throughout history both for its epidemic nature and for the rapidity with which individuals can become gravely ill and even die. Disease is characterized by massive fluid loss, with rates as high as nearly a liter per hour [42]. If infection goes untreated, dehydration and shock may occur within half a day followed by death as soon as 24 hours to several days [42]. The symptoms of V. cholerae infection are consistent with the fact that the principal mechanism of pathogenesis is a toxin that causes fluid and electrolyte loss due to diarrhea: muscle cramps, dizziness, and low blood pressure [43]. The incubation period may vary from a few hours to several days and is dependent on both the dose of organisms ingested and the pH level of the stomach, as V. cholerae is sensitive to low pH, and therefore much of the inoculum does not survive passage through the stomach. Unlike diarrheas caused by invasive organisms, in which there is evidence of tissue damage such as the presence in stool of inflammatory cells and red blood cells, cholera stools are characterized by a clouded, milky white appearance termed "rice water stool" because it resembles water in which rice has been
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mixed. Resulting primarily from secretion from intestinal mucosal cells, the stool is isotonic with plasma concentrations of electrolytes such as Na"^, CI", HC03^, and K"^ [42, 43]. In addition to shock and fluid loss associated with this secretory diarrhea, another potentially dangerous aspect of cholera is a rise in the acidity of body fluids that can lead to pulmonary edema in severe cases of disease. This condition, known as metabolic acidosis, results partly from excretion of bicarbonate in stool, but also probably as a consequence of lactic acidemia from poor tissue perfusion and of other factors related to kidney failure and dehydration [42-44].
2.
HOST SUSCEPTIBILITY
Persons of the O blood group in the ABO grouping are at elevated risk for severe outcomes after infection by El Tor V cholerae [45-49]. This could be due to differential binding of CT to cells from the different blood groups. In order to enter cells, CT binds to a specific surface glycolipid called ganglioside GMi (discussed below). Cells from O blood group individuals lack an enzyme found in individuals of other blood groups that is involved in glycosylating surface antigens [50]. Decreased glycosylation may allow greater exposure of CT-binding sites and account for the greater severity of disease in this population. Experimental data supporting this is that the ganglioside repertoire in preparations of intestine from pigs of the A blood group is different than that of preparations from pigs of other blood groups [51]. The reason why El Tor organisms are associated with increased severity of disease in O blood group individuals is not necessarily explained by this hypothesis, but perhaps there are subtle differences in the binding of CT from the different biotypes that are exacerbated in the O blood group setting [52]. The association of certain populations with more severe disease has important ramifications for vaccine development against cholera. In fact, one study of vaccine efficacy demonstrated significantly less protection in persons of the O blood group [47]. And as live V cholerae are being developed as vaccines, determining their protective capacity will be difficult if individuals of different blood groups react differently to potentially protective epitopes on cholera toxin (discussed below). 3.
TREATMENT
Cholera treatment is simple and inexpensive. Fluids and electrolytes are replaced to prevent both the dehydration and the shock. Intravenous therapy may be used in cases of severe disease in which the patient is gravely dehydrated [43], but oral rehydration therapy is otherwise preferable because it is very effective as well as being cheaper and easier to administer. Although cholera toxin causes maladsorption by small intestinal tissue, glucose-coupled adsorption of Na^ is
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retained. This important observation led to the development of oral rehydration therapy (ORT) [53, 54]. Formulations for oral rehydration therapy vary, but that recommended by the World Health Organization (WHO-ORT) is 60-mM sodium chloride, 30-mM sodium bicarbonate, 20-mM potassium chloride, and 111-mM glucose [42, 43]. In order to achieve a maintainable balance of fluids, proper oral rehydration therapy must continue beyond the diarrheal phase of disease [43]. Intravenous solutions used to treat cholera have an electrolyte composition similar to WHO-ORT; one commonly available solution that can be administered intravenously is lactated Ringer's, lower in potassium (4 mM), but otherwise very similar to WHO-ORT. Except for extreme cases of potassium depletion, this lower amount of potassium is not problematic to therapy, as replacing fluid volume is judged to be more important in cholera treatment than the precise quantity of electrolyte solution because the kidneys can provide electrolyte balance once they are functioning properly [43]. Antimicrobial therapy against V cholerae may be used to limit the amount of ORT necessary, thereby limiting the extent of the diarrhea by about one-half and reducing the excretion of live organisms. Doxycycline or tetracycline are widely used, the former offering the advantage of requiring a single dose. The evolution of antibiotic resistance is a problem in cholera, as it is in so many other infectious diseases, based on the identification of multiply drug-resistant organisms involving R-factor plasmids and integrons encoding resistance genes reported over the past several years [55, 56].
IV. Molecular Mechanisms of Disease A. Surface Biology of Vibrio cholerae 1.
LlPOPOLYSACCHARIDE
Subdivision of pathogenic 01 strains of V cholerae into the serotypes Inaba and Ogawa is based on differences in the lipopolysaccharide O antigen. The predominant 0-antigen sugar in V. cholerae is perosamine (4-amino-l,6-dideoxyD-mannose), with quinovosamine (2-amino-2,6 dideoxy-D-glucose) present perhaps at the beginning or end of the O antigen [57, 58]. In addition to its normal role in the surface architecture of this Gram-negative pathogen, LPS may also be important in other ways for virulence and is a target for protective ^nix-Vibrio antibody production during infection [59, 60]. One potential role for LPS in pathogenicity is as an adhesin molecule, because purified LPS can inhibit attachment to the mucosa [61].
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2.
INTERCONVERSION BETWEEN O G A W A AND INABA S E R O G R O U P S
As O antigen is a major surface-exposed structure, it might be expected that alterations of it would arise from selection within hosts. This appears to be the case, and two predominant changes of 0-antigen structure in epidemic cholera are well characterized. The more subtle of these changes is interconversion between the two major 01 serogroups Inaba and Ogawa. Originally this conversion was observed during growth of Ogawa strains in the presence of anti-Ogawa serum, which results in outgrowth of Inaba isolates [62]. Conversion was also reported to occur during natural infection and in experimental infection of germfree animals; although the conversion from Inaba to Ogawa is rare (much less than the rate of 10"^ estimated for the Ogawa-to-Inaba conversion), it was demonstrated to occur in vivo, and conversion in both directions in vivo is the result of antibody-dependent selection [62-64]. The mechanism directing serotype conversion is mediated by the rfbT gene located at one end of the rfb gene cluster encoding the O antigen. The rfbT gene from an Ogawa isolate is sufficient to convert Inaba strains to Ogawa [65], and the dominance of the Ogawa rfbT allele over the Inaba allele suggests that the Inaba O antigen represents a loss-of-function mutation in rfbT. This was confirmed by allelic exchange in which a mutant rfbT gene was introduced into an Ogawa isolate, rendering a strain of the Inaba serotype [65]. Inaba isolates carry frameshift or missense mutations in the rfbT gents, leading to the conclusion that Inaba strains are rfbT mutants of Ogawa strains. This explains why the conversion rate from Ogawa to Inaba is so much higher than the reverse. Any mutation that results in loss of RfbT function would result in an Inaba O antigen, whereas conversion from Inaba to Ogawa requires specific reversion of the original mutation. This was demonstrated by molecular epidemiological analysis of strains from the 1991 cholera outbreak in South America. This epidemic began with Inaba strains, and sequence determination of the rfbT allele in these isolates showed them to carry a deletion of a single basepair that resulted in early termination of the open reading frame. Seven months into the outbreak, Ogawa isolates emerged, and sequence analysis of these showed that an insertion had arisen which restored the wild-type r/Z^r sequence [66]. RfbT is a 32-kDa cytoplasmic membrane protein [64, 65], but the exact modification of the Inaba LPS catalyzed by RfbT has not been reported. Given that rfbT is not essential for 0-antigen synthesis, it may function late in the synthesis or export of V cholerae LPS, analogous to the phage-encoded LPS modifications described in Shigella [64, 67]. 3.
EMERGENCE OF THE 0139
SEROVAR
A more radical alteration of LPS structure caused the emergence of a new serogroup, which came to be known as 0139 Bengal, in 1992 in India and Bangladesh [5, 7]. Most of the early cases of disease were in adults in areas
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endemic for 01 V. cholerae, suggesting that prior infection with 01 strains was not protective [5-7]. The 0139 strain was widely reported as being closely related to El Tor isolates [68-70] but having an O antigen of significantly different structure as well as the capacity for capsule production [8, 71, 72]. Analysis of the 0-antigen-encoding regions from 0139 V. cholerae showed that 22 kb of the 01 locus had been replaced by 35 kb of 0139-specific sequence [8, 73, 74]. This DNA harbors two new sets of genes involved in 0139 0-antigen and capsule synthesis separated by the insertion sequence IS 1358. The mechanism of acquisition of the 0139 O antigen/capsule locus has not been determined, although it likely occurred through horizontal transfer of DNA from a non-01 strain to an 01 strain. This conclusion rests on the observation that sequences from within the 0139 DNA are found in other non-01 isolates [74].
B.
Cholera Toxin and Toxinogenesis
The pathogenesis of cholera is due almost solely to the action of cholera toxin (CT), which causes elevation of cyclic AMP (cAMP) in affected cells through a pathway that includes activated protein kinase A, which leads to a number of events culminating in secretion of fluid and ions. What follows focuses on three important aspects of cholera pathogenesis related to CT: the basic structure and function of the enzyme, how it is excreted by V. cholerae, and how the toxin enters and moves within host cells. Much of what is known about the structure of CT and the mechanism of its secretion from V. cholerae is based on studies of a very closely related toxin from E. coli called heat-labile toxin (LT), although the CT structure has also been solved [75].
1.
ENZYMATIC ACTIVITY
CT causes severe disruption of intestinal cell function, leading to the watery, secretory diarrhea characteristic of cholera. CT acts by causing constitutive activation of adenylate cyclase, leading to elevated cAMP levels in intestinal epithelial cells. Cyclic AMP (cAMP) activates protein kinase A, which causes the opening of ion channels in the membrane, leading to chloride and bicarbonate secretion by intestinal crypt cells and disruption in absorption by villus cells [76] (Fig. 2). Modification of cAMP production by CT is a well-understood mechanism. CT is a heterodimeric protein made up of an active (A) subunit, and a receptor binding (B) subunit, in a ratio 1 A:5B [77]. Upon entry into host cells, the CT-A subunit is cleaved into two peptides, Al and A2, of 23 and 5 kDa, respectively, which are kept together in the holotoxin by means of a single disulfide bond [78]. In cholera pathogenesis, the CT-Al subunit catalyzes hydrolysis of NAD and subsequent
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'uUUuUU^
Fig. 2 Biochemical events leading to secretion caused by cholera toxin. The Al subunit of cholera toxin catalyzes ADP-ribosyl transfer from NAD to Gstt, a regulatory subunit of the adenylate cyclase complex. The ensuing constitutive adenylate cyclase activity leads to elevated cAMP levels, which activate protein kinase A (PKA), leading to opening of normally gated channels in the plasma membrane. ADP-ribosylation factors (ARFs), small GTP-binding proteins that stimulate ADP-ribosyltransferase activity by increasing the affinity of CT for both the substrate and the target, have not been shown to be required in vivo as yet. Chloride and other ions leave the cell, followed by water, leading to the profuse watery diarrhea characteristic of cholera.
transfer of the ADP-ribose group to the regulatory subunit (G^a) of a heterotrimeric G protein that controls adenylate cyclase function, thus leading to unregulated production of cAMP [79-81]. Although CT differs in primary sequence from other NAD-dependent ADP-ribosylating toxins such as pertussis toxin, diphtheria toxin, and exotoxin A from Pseudomonas aeruginosa, critical residues for these activities are conserved among these diverse toxins, and mutagenesis studies and structural modeling of these toxins suggest they may share a novel NAD binding fold [82, 83]. ADP-ribosylation activity of CT-Al is stimulated in vitro by host-derived Ras-like guanine nucleotide-binding proteins called ADP-ribosylation factors, or ARFs. This family of proteins, identified in both soluble and membrane fractions, acts by increasing the affinity of CT-Al for both the ADP-ribose donor and acceptor [84]. Whether ARFs play an important role in toxin activity during infection has not been demonstrated. 2.
INDUCTION OF PROSTAGLANDINS
An effect of CT that is unrelated to its role in stimulating cAMP production is its ability to stimulate prostaglandin synthesis, particularly prostaglandin E2 (PGE2) [85]. Prostaglandins can contribute to intestinal cell secretion and levels
10.
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MOLECULAR BASIS OF VIBRIO CHOLERAE PATHOGENESIS
of PGE2 produced during disease correlate with the volume of stool, suggesting that these molecules are at least theoretically associated with diarrhea production [86]. This effect occurs because CT activates phospholipase A2 (PLA2), which results in turnover of cellular phospholipids, causing the prostaglandin precursor arachidonic acid to be produced [87, 88]. The molecular basis for stimulation of PGE2 by cholera toxin is not well worked out. Administration of CT to a murine smooth muscle cell line results in upregulation of mRNA encoding the PLA2-activating protein (PLAP), which stimulates enzymatic activity of PLA2 and may therefore lead to elevated production of arachidonic acid from phospholipids [87, 89] (Fig. 3). Production of arachidonic acid does not require enzymatic activity or cyclic AMP production by target cells, because cells treated with either CT or a mutant CT lacking ADP-ribosyl transferase activity stimulate equivalent levels of arachidonic acid release from phospholipid in S49 cells, a murine lymphoma cell line. Also, a derivative of this cell line lacking the gene encoding G^a—and therefore insensitive to the action of CT-Al—releases arachidonic acid on treatment with CT [88]. CT-B alone could also stimulate arachidonic acid release from both cell lines. Taken together, these results suggest that receptor-mediated events caused by CT-B binding may cause PGE2 synthesis during cholera [88]. This would be consistent with observations that PLA2 activation can be caused by other receptor-binding events [90]. 3.
CT STRUCTURE
The overall picture arising from crystallographic determination of the LT structure is that of a ring of B subunits, the B pentamer, through which the A2 portion of the A subunit is inserted. The Al subunit sits above the B pentamer
« ^ ^
AA
- ^
lIMHi
iiiiili ^
'^^M Fig. 3 Induction of prostaglandin biosynthesis by cholera toxin. Cholera toxin (CT) induces expression of the phospholipase-activating protein (PLAP), which leads to a rise in phospholipase A2 (PLA2). This activity produces arachidonic acid (AA) from phospholipids; AA is the direct precursor of prostaglandin synthesis. Enzymatic activity is not required for prostaglandin synthesis by CT, suggesting that it may result from an event mediated by receptor binding.
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with the shape of an inverted triangle [83]. The CT structure, determined after that of LT, is identical, which is predicted from the high level of identity between LT and CT primary sequences [75]. This configuration is achieved because structural units from the B pentamer form a barrel surrounding a pore 30 A in length and between 11 and 15 A in diameter through which the carboxy terminus of the A2 peptide is inserted [83]. The B-helices that make up the sides of the barrel are heavily charged, with six basic residues (Lys-62, Lys-63, Arg-67, Lys-69, and Arg-73) and three acidic residues (Asp-59, Glu-66, and Asp-70). In contrast, the inserted sequence of the A2 chain has five acidic residues (Asp-225, Glu-229, Asp-231, Asp-238, and Glu-239 and two basic residues (Arg-235 and Arg-237). This charge complementarity is likely important for maintaining holotoxin structure because it is largely conserved in CT [91, 92].
4.
PERIPLASMIC ASSEMBLY
A significant aspect of V^ cholerae pathogenicity is that cholera toxin is secreted very efficiently into the surrounding milieu. In contrast, the very similar heat-labile toxin produced by enterotoxigenic E. coli (ETEC) is not exported beyond the periplasm, and diarrheal disease caused by ETEC is far less severe than cholera. As with the structural elements described above, much of the knowledge regarding the mechanism of assembly and secretion of CT has come from studying LT, which can be expressed from plasmids in V. cholerae [93-100]. In addition to defining this important aspect of cholera pathogenesis, study of toxin secretion has been a model for understanding the more general question of how a Gram-negative bacterium can transport a water-soluble protein across two membranes. The process takes place in two steps: secretion of subunits into the periplasmic space with rapid assembly followed by extracellular secretion of holotoxin using a dedicated transport system called Eps. Transport to the periplasmic space is accomplished by the Sec system (see Chapter 2). This system, which is responsible for delivering a variety of proteins to the periplasmic space, recognizes and cleaves a hydrophobic signal sequence encoded at the amino terminus of the target proteins, including CT-A and CT-B. Once the toxin gets into the periplasm, the mature A and B subunits assemble into the A:B5 holotoxin prior to being secreted across the outer membrane. The assembly process occurs very rapidly, as pulse chase experiments show that nearly half of the periplasmic B subunits are pentameric by 15 seconds, and nearly all (-85%) are pentamers by 2 minutes [95]. Monomers of the B subunit are not detectable in the supernatant, indicating that the pentamers remain intact during transport across the outer membrane. Transport from the periplasm across the outer membrane into the extracellular space is a much slower process than B subunit assembly, with a half-time of approximately 13 minutes [95].
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Interaction between the A and B subunits of LT, and by inference, of CT as well, are required for efficient assembly into the A:B5 holotoxin. First, the presence of the A subunit favors assembly of the holotoxin, as opposed to just B pentamers [99, 101]. In addition, mutations in the C terminus of the B subunit result in absolute dependence on the A subunit for pentamerization [99, 102]. Finally, consistent with the prediction from the crystal structure, removal of the last 14 amino acids of the A2 subunit causes accumulation of B pentamers at less than 1% the level seen in the presence of wild-type A subunit [103]. Given that the A2 subunit is embedded within the B-pentamer barrel in the crystal structure, it is not unexpected to find that these interacting domains appear to be involved in assembly of holotoxin.
5.
EXTRACELLULAR SECRETION: THE EPS SYSTEM
Cholera toxin (and several other proteins including chitinase, protease, and mucinase) is secreted from the periplasm of V cholerae using a complex of proteins designated as Eps proteins, for extracellular protein secretion [97, 104, 105]. The process of secretion through the Eps system (and similar systems from other organisms) is generally termed type II secretion or called the main terminal branch of the general secretory pathway (Gsp; see Chapter 2) [106, 107]. In the case of cholera toxin secretion and the Eps system, a large contiguous grouping of 12 open reading frames has been identified that share similarity with Gsp proteins from different genera [105]. Also important for the function of the Eps system is prepilin peptidase, an enzyme encoded by the vcpD gene that is not part of the eps gene cluster [108]. Prepilin peptidases are so named because of their role in an unusual amino-terminal processing step in the maturation of many bacterial pilin molecules, including the major subunit of TCP, TcpA (see below). But several of the type II secretion gene products, often called pseudopilins, require the same processing step for their undefined role in secretion [109]. The pseudopilins of the Eps system are the EpsG, H, I, and J proteins; VcpD, which is required for toxin secretion, is their likely processing enzyme [108]. In V. cholerae, the cytoplasmic protein EpsE was the first of the Eps proteins shown to be associated with toxin secretion [97]. Mutants lacking EpsE assemble toxin in the periplasmic space but do not secrete it extracellularly to any significant extent. The EpsE protein contains a Walker A box, a motif found in a variety of nucleotide-binding proteins [107]. The presence of an essential Walker A box in proteins within the EpsE family has led to the suggestion that these proteins may be ATPases that provide energy required for the secretion process [107], yet ATPase function of these proteins has not been conclusively demonstrated [107]. EpsE does have an autokinase activity that requires the Walker A box, and the possibility that kinase function may be a component of a signaling pathway important for Eps function or assembly has not been ruled out [98, 107].
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VICTOR J. DIRITA
In wild-type V cholerae, the majority of detectable EpsE fractionates with the membrane [98]. To associate with the membrane, EpsE requires the presence of EpsL, which has a putative transmembrane topology and fractionates to the cytoplasmic membrane, although it has an extensive cytoplasmic domain through which interaction with EpsE is likely mediated [100]. The first 93 residues of EpsE are sufficient to allow association with EpsL in the cytoplasmic membrane, leading to toxin secretion. This was demonstrated by fusing different amino-terminal portions of EpsE to the related ExeE, a homolog required for toxin secretion in Aeromonas hydrophila [98]. Although full-length ExeE does not suppress the secretion defect of an epsE mutant, an EpsE-ExeE fusion protein can substitute for EpsE. The limit of this requirement is the first 93 residues from EpsE: when these were used to replace the amino terminus of ExeE, the resulting chimera could restore secretion to an epsE mutant of V. cholerae. In addition, the fusion protein fractionated to the membrane in an EpsL-dependent manner [98]. Although it spans the membrane, much of the EpsL amino terminus is predicted to be in the cytoplasm, and it is with this domain that EpsE interacts [100]. In addition, coimmunoprecipation experiments suggest that another Eps protein, EpsM, which is also predicted to be membrane localized but to have an extensive periplasmic domain, forms a complex with EpsL. This complex appears to be quite avid, as it is resistant to Triton extraction [100]. Gel filtration of purified EpsL and EpsM suggests that both are dimers, although the significance of this observation is not clear [100]. A model accounting for the known interactions and activities in the Eps system is shown in Figure 4. In the multiprotein complex made up of Eps proteins, cytoplasmically localized EpsE protein, which has autokinase activity as well as potential ATPase activity, interacts with EpsL in the membrane. EpsL in turn forms an association with EpsM, which, by virtue of its periplasmic domain, is positioned for potential interaction with putative Eps outer membrane components. Although little has been done to characterize outer membrane components of the Eps system, the EpsD protein is homologous to outer membrane proteins called secretins in other Gsp systems that are very likely the pores through which the final step of secretion occurs [107]. To date, function has not been ascribed to the pseudopilin proteins of the Eps system (EpsG, EpsH, EpsI, EpsJ), but continuation of these studies should help us understand how they contribute to secretion and, ultimately, the precise mechanism by which the Eps complex delivers cholera toxin to the extracellular space.
6.
UPTAKE INTO HOST CELLS: A GMi-DEPENDENT PATHWAY
As noted above, the receptor for cholera toxin on host cells is the GMi glycolipid. Such molecules are ubiquitously distributed in the outer leaflet of the plasma membranes of eukaryotic cells, but their function is not clear. In animal cells, they are derived from ceramide as opposed to glycerol, which is the source
10.
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Fig. 4 Secretion of cholera toxin by the Eps system. Toxin assembles in the periplasm through intramolecular interactions between CT-A and CT-B after transport into the periplasmic space. Once assembled, it is acted on in an as-yet-uncharacterized fashion by the Eps gene products, which include components in all cellular compartments. EpsE, a cytoplasmic autokinase, associates with the inner membrane protein EpsL. EpsL and EpsM interact avidly with one another, and the latter protein is predicted to have an extensive periplasmic domain; both proteins are dimers in solution. The inner membrane pseudopilins Eps G, H, I, and J are processed at the amino termini by the VcpD protein, which is also the prepilin peptidase for the mannose-sensitive hemagglutinin, a type IV pilus. The final step in secretion is presumed to be delivery of the holotoxin to the extracellular space by moving through a pore formed by the oligomeric secretin EpsD.
of bacterial and plant glycolipids. GMi belongs to the family of complex glycolipids called gangliosides that contain A^-acetylneuraminic acid (NANA) and are found in cells throughout the body. As with all glycolipids, the sugar moieties in GMi are exposed on the cell surface, and it is to these that the cholera toxin binds through its B subunit. The structure of the B subunit of the closely related LT toxin of £. coli has been solved as a cocrystal with lactose, and this shows that binding is to the galactose molecule within lactose [110]. As galactose is present in the sugar component of GMi, this implies that galactose is the direct receptor for CT-B on host cell surfaces. The crystal structure shows identical binding of lactose to all subunits within the B pentamer, but it is unlikely that all of the B subunits need to bind to the receptor to initiate uptake of the cholera toxin [110]. Cholera toxin binds to target cells on the apical (lumenal) surface. However, the ultimate target of the toxin, adenylate cyclase, is on the cytoplasmic side of the basolateral membrane. Thus, after binding and entry, the toxin must traverse
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the cell. This fact is reflected in a lag phase that has been observed in vitro between the time that toxin binds and the time at which an effect can be measured [111, 112]. GMi binding by cholera toxin is required for activating the signal transduction pathway leading to chloride secretion in sensitive cells. This was shown by making chimeric toxin molecules using components from CT and the closely related £. coli toxin LTIIb [113] and testing their activity on cells that are sensitive to CT. The LTIIb toxin has the same ADP-ribosylating activity as CT (by virtue of very similar Al proteins), but its B subunit binds to a different ganglioside, GDia- The origin of the B subunit (either CT or LTIIb), and not the origin of the A subunit, dictated whether a particular chimeric protein induced chloride secretion; a chimeric protein with the CT-B subunit caused chloride secretion, but one with the LTIIb-B subunit did not, even though the cells used in the experiment have GD|a on their surface [113]. This sensitivity was inhibited by adding 10 |iM GMi to the assay, suggesting that the chimeric toxins are indeed binding to GMj. GMi binding appears to selectively sort CT into a pathway different from that of LTIIb bound to GDi^, because GMi-bound CT is associated with a Triton-insoluble membrane fraction in T84 cells, whereas GDi^-bound LTIIb is not [113]. That this pathway is required for toxin-promoted chloride secretion is suggested by the fact that the chimeric molecule with the LTIIb-A subunit and CT-B subunit also fractionates with Triton-insoluble membrane material in sensitive cells, whereas the ineffective toxin having the CT-A subunit and the LTIIb-B subunit does not [113]. In addition, when tested on cells that are sensitive to both CT and LTIIb, each toxin becomes associated with a Triton-insoluble membrane fraction on binding to its particular ganglioside receptor. These observations indicate that CT-GMi complexes are partitioning into caveolae, or caveolae-like structures [113, 114]. Caveolae are associated with endocytosis of different molecules through smooth invaginations (i.e., those lacking clathrin coating seen with a number of receptor-mediated uptake events [114]), and one of the hallmarks of caveolae-associated proteins is their resistance to extraction by Triton [113, 114]. That CT may be sorted into caveolae by binding to GMj is supported by the finding that caveolae are enriched for GMi [115]. In other systems there is evidence that the membrane constituents of caveolae are delivered to other vacuoles within the cell, similar to the way ligands are delivered to endosomal and postendosomal pathways through classical receptor-mediated endocytosis. Therefore, CT-GMi entering through caveolae may ultimately be trafficked through the cell to sites where enzymatic activity may lead to chloride secretion.
7.
GENERATION OF ENZYMATIC ALLY ACTIVE A1
The Al subunit is produced from the A polypeptide as a result of proteolytic cleavage at residue Arg-192 [116]. After this event, the Al and A2 peptides are kept together by a disulfide bond [117]. The role of A2 is not fully established.
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but, as noted above, crystallographic data suggest that it acts as a structural element to keep the Al and B subunits together [83]. Its C terminus contains the sequence Lys-Asp-Glu-Leu (KDEL), which is found in proteins that are retained in the endoplasmic reticulum (ER). Trafficking of CT appears to involve retrograde transport through the Golgi apparatus to the ER, and thus an ER retention signal such as KDEL is predicted to be required for toxin function. In fact, a mutant CT molecule lacking the KDEL sequence shows a lag phase that is nearly 10 minutes longer than that seen with wild-type CT and does not exhibit the same level of chloride secretion. The mutant CT also showed temperature sensitivity, with partial impairment at 27°C and complete impairment at 20°C [118]. Thus, it appears that the ER retention signal mediates efficient sorting and trafficking of CT, but that these processes may occur in the absence of such a signal, given that the toxin lacking the KDEL sequence was eventually able to induce chloride secretion after a longer lag period. Further experimental support for the role of the KDEL sequence in retrograde trafficking of CT is that antibodies against the KDEL receptor Erd2p can block transport of the toxin through the Golgi into the ER in unpolarized cells [119]. A proteolytic event processes the A subunit into A1 and A2 fragments. Although V. cholerae itself secretes a protease that can properly nick CT, mutant strains lacking this protein are unaffected in causing fluid accumulation in experimental animal models [11]. For many other toxin-mediated bacterial diseases, including anthrax and diphtheria, the host provides the nicking enzyme that activates the toxin [120]. This appears to be the case in cholera as well, because recombinant CT, not nicked during purification from E. coli, can be processed by T84 cells if administered to the apical surface. Administration of the recombinant molecule to the basolateral surface results in no A1 subunit production and a reduced (but detectable) amount of chloride secretion [121]. In addition, mutagenesis of the cleavage site by replacement of R192 with histidine (CTR192H) attenuates both the rate and the magnitude of chloride secretion when the toxin is applied to the apical surface of T84 cells. When applied to the basolateral surface, CTR192H causes chloride secretion with the same decreased kinetics as wild-type toxin applied to this surface. Cleavage appears not to be an absolute requirement for CT activity, as apically applied protease-resistant CTR192H ultimately causes chloride secretion even though the kinetics are altered significantly [121]. Alternatively, perhaps there is a small amount of nicking that occurs with CTR192H and basolaterally applied wild-type CT. These findings suggest that intestinal epithelial cells produce an apically localized protease, or perhaps an endosomal protease, that can cleave the CT-A subunit into Al and A2. The identity of this protease remains to be worked out, but its activity is blocked completely by DFP and 3,4-DCI, two inhibitors of serine proteases [121].
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8.
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VESICULAR MOVEMENT OF TOXIN
The lag period between the time of administration of the toxin and when chloride secretion is measurable is due (at least in part) to vesicular trafficking of CT from its receptor on the apical surface to its target on the basolateral surface. The lag period is 25-30% shorter, and the rates of chloride secretion are almost 70% faster if CT is applied to the basolateral surface [112]). There are equivalent numbers of GM| receptors on basolateral and apical surfaces [112], arguing against the hypothesis that the difference in lag periods following administration to different poles of the cell is due to more binding to the basolateral surface than to the apical surface. Support for the hypothesis that CT reaches its target through vesicular movement is the observation that chloride secretion in T84 cells is temperature dependent [112]. At 20°C chloride secretion is severely reduced relative to the levels observed at 37°C, and occurs only after a much exaggerated lag phase (200-300 minutes as opposed to roughly 30 minutes at 37°C) [112]. Although chloride secretion is diminished at 20°C whether toxin is applied apically or basolaterally, the attenuation of the response seen after apical administration at this temperature is far greater than that seen after basolateral administration [112]. Such temperature dependence has been observed for a range of intracellular trafficking processes [122-124], and the greater effect seen on apically applied toxin is consistent with a hypothesis that the CT is being trafficked through vesicles from the apical surface to the basolateral surface. When the toxin is applied apically at 4°C and then allowed to move into the cells at 37°C, it is observed within large cytosolic structures located beneath the apical surface. These structures are thought to be endosomes, lysosomes, and Golgi. At 20°C, entry of the toxin into this extensive network is not observed, although toxin is detected in smaller structures, perhaps endosomes, just beneath the apical surface [125]. Another temperature-sensitive step is seen after translocation of the Al subunit, and this step, which occurs late in the lag phase, is only observed if toxin is applied to cells on the apical surface, not the basolateral surface [112, 126]. Further insight into the cellular trafficking pathway for CT arises from studying the effects of various inhibitors of endosomal and vesicular processes [126, 127]. For example, the fungal toxin brefeldin A, which alters vesicular transport through a number of pathways in the cell [128], inhibits chloride secretion from T84 cells treated with CT [126]. Inhibition occurs at a step after endocytosis but prior to reduction of the A1/A2 complex, in contrast to the temperature-sensitive step noted above that occurs after A1/A2 reduction. In addition, administration of brefeldin A late in the lag phase does not appreciably alter chloride secretion [126]. Treatment of T84 cells with brefeldin A disrupts the Golgi structure, as determined by loss of staining with antibody directed against a Golgi-associated protein called p200 [126], indicating that toxin requires an intact Golgi apparatus to reach its target. As the ADP-ribosylating factors (ARFs) play a role in maturation of transport vesicles, the interaction between CT and ARFs is perhaps a component of this Golgi-dependent route [126].
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Taken together, the trafficking studies imply two distinct steps in the toxin trafficking pathway once toxin is brought into the endosomal network: an early brefeldin A-sensitive one preceding Al formation, and a later temperature-sensitive one occurring after A1 formation. It thus appears that CT traverses a pathway through the Golgi after endocytosis. From the fact that the putative ER retention signal KDEL present on CT-A2 is required for efficient trafficking of the toxin, a model develops in which cholera toxin undergoes retrograde transport through the Golgi cistemae to the ER during its movement through the cell, and that at some point in this trafficking route the Al subunit is translocated into the cytosol [112, 119, 126] (Fig. 5). The Al subunit must translocate into the host cell cytosol to reach the G^a protein target; however, the identity of the membrane crossed by the Al subunit remains unknown. In fact, both holotoxin and CT-B can transcytose T84 cells and be presented intact on the basolateral side of the monolayer [129]. Transcytosis of CT might explain how the toxin goes from the apical surface (after binding to the GMi receptor) to engage its basolateral target, and may also explain some of the systemic effects associated with cholera toxin, such as prostaglandin induction and stimulation of components of the intestinal autonomic nervous system and
GM|
GM| GM|
ARFs Fig. 5 Trafficking model of apically applied cholera toxin. The toxin must reach its target Gstt on the basolateral surface in order to cause chloride secretion, resulting in a lag period after administration of toxin before chloride secretion can be detected. Holotoxin enters the endosomal network of the cell through smooth membrane invaginations called caveolae after binding to its ganglioside receptor GM|. The toxin moves retrograde through the Golgi into the ER, a step that requires the ER retention signal KDEL. Eventually the Al subunit translocates an undefined membrane into the cytosol, where it can engage its target. ADP-ribosylation factors (ARFs), which stimulate ADP-ribosyltransferase activity in vitro, may also play a role in vesicle maturation. The fungal toxin brefeldin A inhibits a trafficking step early after endocytosis but before reduction of AI (I), whereas a later time in the lag phase, after Al reduction, is temperature sensitive (2).
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signaling events in subepithelial cells, as well as stimulation of the mucosal immune system [11, 85, 130]. Brefeldin A increases the rate of transcytosis and leads to a greater amount of the A subunit being delivered across the cells as opposed to the amount seen to transcytose in the absence of brefeldin A, suggesting that disruption of protein targeting resulting from brefeldin A treatment allows the transcytosis route to be more readily selected. It must be kept in mind that brefeldin A disrupts the signaling pathway induced by CT that leads to chloride secretion [112], so the greater amount of A subunit at the basolateral surface after brefeldin A treatment may be in a nonfunctional state.
C. Toxin-Coregulated Pilus (TCP) 1.
TYPEIVPILI
The major colonization factor for pathogenic V. cholerae is the toxin-coregulated pilus (TCP). This is a bundled surface structure named for the fact that it is expressed when CT is expressed and it is regulated by the same factors as CT, the ToxR/ToxT regulatory cascade (discussed below). The pilus structure consists of a single protein, TcpA, encoded on a pathogenicity island (VPI) that is itself a large portion of the genome from the single-stranded filamentous phage VPI(|) [17]. TcpA appears to be a coat protein for VPI(t) because anti-TcpA antibodies recognizes phage particles in immunogold electron microscopy [17]. Mutant strains of V. cholerae that do not express TCP are deficient for colonization in humans [25]. Volunteers given strains lacking TCP produce litde detectable anti-V7Z?no antibodies and are susceptible to subsequent challenge by wild-type Y cholerae, thus demonstrating that a key step in colonization in the human small intestine requires TCP [25]. Unlike many well-characterized pili of E. coli, which mediate tropism both for particular hosts and host tissues by binding to specific molecules [131], TCP has not been shown to require a host molecule as a receptor for binding. The question of whether direct adherence to a host structure is at all involved in colonization remains open, and another model for adherence will be discussed below. TCP is a member of a class of pili, termed type IV, that are frequendy associated with virulence in Gram-negative bacteria [107, 132-134]. A hallmark of the type IV pilus is that the major subunit is produced as a prepilin that is processed into mature pilin molecules by cleavage of a short signal sequence at a conserved glycine residue by an enzyme called prepilin peptidase [135]. The residue at the amino terminus of the processed molecule is then methylated by the prepilin peptidase. As described above, prepilin peptidases are also involved in type II secretion such as that controlled by Eps for delivery of cholera toxin, thus suggesting a functional association between type IV pilus assembly and type II secretion [107, 136]. Recall that the molecules processed by prepilin peptidases
10.
MOLECULAR BASIS OF VIBRIO CHOLERAE PATHOGENESIS
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in type II secretion systems have been termed pseudopilin molecules, although they are not thought to be displayed on the surface or to be involved in adherence the way that bona fide type IV pili are. The peptidase responsible for processing TcpA is called TcpJ [137, 138], and, in contrast to subunits of most type IV pili, processed TcpA has a methionine at its amino terminus instead of the more typical phenylalanine [26, 137]. A variety of pathogenic microbes express type IV pili, including Neisseria gonorrrhoeae, enteropathogenic E. coli, and Pseudomonas aeruginosa [133-135, 139, 140]. Type IV biogenesis appears to be conserved, and many of the gene products required for this process are shared among strains expressing these pili [141]. For TCP, these genes are expressed as part of an operon with tcpA from the genome of VPIc]) [17, 142]. Despite the general conservation of gene products among type IV pili, there are features associated with some type IV pili that have not been identified with TCP. For example, no adhesin molecule is associated with TcpA, which would confer host receptor binding specificity as is seen with the Pile protein associated with the pilus of N. gonorrhoeae [133, 134, 139]. Also, TCP has not been observed to undergo the unusual form of surface translocation, termed twitching motility, that is linked to type IV pilus expression in many other species and is postulated to arise as a consequence of pilus retraction [132]. In both enteropathogenic E. coli (EPEC) and N. gonorrhoeae, twitching motility is an important feature of the biology of the cell and may be important for pathogenesis [143, 144]. Type IV pili often serve as receptors for phages, which is also true for TCP; however, only in the case of TCP have phage structures been shown to be mediated by type IV pili.
2.
TCPA STRUCTURE AND HOST COLONIZATION
Mature TcpA is 199 residues in length and is synthesized as a prepilin with a signal sequence of 25 residues [141, 145]. A single disulfide bond between residues Cys-120 and Cys-186 results in a hairpin loop at the carboxy terminus of the protein. Formation of the disulfide bond is required for stable pilin production, because: (1) undetectable levels of TcpA are produced by strains expressing mutant alleles of tcpA in which the Cys codons were replaced with Ser codons [146], and (2) strains of V. cholerae carrying lesions in the gene encoding a periplasmic disulfide bond oxidoreductase {tcpG), which introduces disulfide bonds into proteins [147, 148], synthesize aberrant Tcp and are avirulent in the infant mouse model of cholerae [146]. The carboxy-terminal domain of TcpA in the vicinity of the hairpin loop formed by the disulfide bond between residues 120 and 186 has essential determinants of colonization. The crystal structure of the type IV pilin molecule from Neisseria gonorrhoeae predicts that the carboxy terminus of TcpA is exposed in the pilus structure [149]. Antibodies against this domain protect against infection [146,150, 151], implying that the exposed carboxy terminus may recognize a structure
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required for colonization or may mediate critical bacterial cell-cell contacts (see below). However, a Fab fragment from anti-Tcp IgG did not block association of wild-type V. choleme with human intestinal epithelium, and neither did pretreatment of the epithelium with purified TCP [152], suggesting that epithelial adherence is not Tcp-dependent. While this observation suggests that TCP does not act simply by adhering to an epithelial receptor, the utility of a cultured epithelium lacking a mucus layer calls into question the strength of that conclusion. An emerging theme in V. cholerae colonization is the association between TCP-mediated bacterial autoagglutination and host colonization, which is based on a number of mutant tcpA alleles carrying lesions that diminish both autoagglutination and colonization. These map both to the amino terminus (Leu-4 to Thr; Val-9 to Met; Val-20 to Thr) and carboxy terminus (Asp-129 to Ala; Asp-146 to Ala; Glu-158 to Ala) of the mature TcpA protein [146]. A role for TCP beyond solely mediating autoagglutination is supported by a mutation within the carboxyterminal loop of TcpA that diminished host colonization yet retained the ability to autoagglutinate [146]. Nevertheless, an accumulating body of evidence from work with type IV pili in other pathogens is that their ability to mediate cell-cell interactions among bacteria leading to microcolony formation is a major factor in host colonization [132, 144]. 3. TCP As THE
RECEPTOR FOR
CTXcj)
An important aspect of TCP function in the overall pathogenesis of V. cholerae is that it serves as the receptor for the filamentous phage encoding the cholera toxin genes [16]. Transduction from a strain carrying CTXcj) to one lacking it can occur in vivo provided the recipient cell expresses TCP [16]. This raises the issue of whether live vaccine strains lacking the ctxAB locus (see below) may be capable of reacquiring them during natural infection of a vaccinated individual with a wild-type {ctx^) strain. That TCP-expressing El Tor strains can be transduced with CTXcj) during infection proves that El Tor actually does express TCP in vivo, which has been a matter of controversy because El Tor strains express another type IV pilus, the mannose-sensitive hemagglutinin (MSHA), which was long thought to be of principal importance for colonization by El Tor organisms [153-157]. Reasons for this include the observation that TCP was not detected on El Tor organisms directly analyzed in stools of cholera patients or during experimental infection [155]. In addition, antibodies against the MSHA protected infant mice against infection by El Tor strains, but not classical strains [156, 157]. In addition to the transduction experiment proving that TCPs are indeed expressed by El Tor organisms in vivo, mutants lacking MSHA can colonize both experimentally infected animals and human volunteers, whereas mutants lacking TCP do not [153, 158, 159].
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It is now clear that TCP is an important colonization factor in both biotypes as well as playing a major role in virulence by acting as the receptor for the CTX(|). MSHA appears to be important for biofilm formation and may be involved in survival of Y cholerae outside the host [160]. Not surprisingly, given that type IV pili often act as phage receptors, MSHA acts as the receptor for a filamentous phage called 493 arising from 0139 strains [161].
D. Regulation of Toxin and Pilus Production 1.
THE
ToxR
REGULON
Expression of cholera toxin, the toxin-coregulated pilus, and several other gene products associated with virulence in V cholerae is under coordinate control of a transcription factor called ToxR and an effector of ToxR function called ToxS, which act through a complex regulatory cascade [162]. As the genes controlled by ToxR are in unlinked operons, they are collectively termed the ToxR regulon. In addition to the genes encoding toxin production or colonization, ToxR controls expression of two major porins, OmpU and OmpT (Table II) [163, 164]. It has been suggested that OmpU may be an adhesin, as antibodies directed against OmpU can block binding of V. cholerae to Hep2 cells [165, 166]. However, neither antibodies nor pretreatment with purified OmpU inhibit V. cholerae binding to rabbit small intestinal epithelium, as might be predicted for an adhesin [167]. A role for OmpT in virulence has not been determined. Although many of the genes of the ToxR regulon are on the genomes of the VPI or CTX phages [16, 17, 23, 24], toxR and other genes in the regulon, as well as other transcription regulators involved in toxin and pilus expression, are present on one of the megareplicons in V. cholerae [162, 168] and appear to be ancestral to the species.
2.
SIGNALS STIMULATING TOXIN AND PILUS PRODUCTION
In classical strains of V^ cholerae, growth at pH values between 6 and 7, at temperatures of 25-30°C, and under conditions of moderate osmolarity (-70 mM NaCl) and aeration favor maximal expression of the ToxR regulon [26, 28, 162, 163, 169, 170]. In El Tor strains, expression of the ToxR regulon requires more specialized conditions, designated AKI: growth in a rich medium that includes potassium and bicarbonate with poor aeration for several hours followed by several hours of extensive aeration [171, 172]. Although these conditions were developed for expression of toxin in El Tor isolates, they induce expression in classical strains as well. Are the signals that trigger expression of the ToxR regulon in vitro similar to those that cause activation in vivol While AKI conditions were developed to mimic the environment that V. cholerae may encounter during an infection, conditions promoting expression in classical strains
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Table II
Genes and Gene Products in the ToxR Virulence Regulon of V. cholerae
Genes acf
Functions Accessory colonization factor; motility regulators
aldA
Aldehyde dehydrogenase
ctxAB
Cholera toxin subunits
ompU/ompT
Porins; putative adhesin (OmpU)
tcpABQCRDSTEF
Toxin-coregulated pilus subunit (TcpA) and type II secretion system for pilus assembly
tcpl
Motility regulator
toxT-tcpJ
Transcription regulator (ToxT) and TcpA-prepilin peptidase (TcpJ)
are empirically derived. Given that the intestinal temperature is 37°C, it is not clear why the temperature optimum for classical strains is 25-30°C. In fact, at this elevated temperature the ToxR regulon is very poorly expressed. Part of the reason for decreased level of regulon expression at elevated temperatures in vitro comes from the fact that transcription of toxR is decreased at higher temperature. This appears to be due to divergent transcription of the heat shock gene htpG, whose promoter is activated by an RNA polymerase charged with the heat shock sigma factor a-32, which does not recognize the toxR promoter. Binding of this form of RNA polymerase may occlude RNA polymerase charged with appropriate sigma factor for toxR transcription [173]. Elevated temperature also affects the activity of ToxT, a regulatory protein required for toxin and pilus expression [174]. Expression of the ToxR regulon is also regulated by bile, an organic acid with detergent properties that plays a role in digesting fats and is found in the small intestine where V cholerae colonizes and causes disease. When present in culture media, bile strongly represses transcription of genes within the ToxR regulon [174, 175]. Thus, bile may act as a signal for V. cholerae resident in the lumen of the intestine, and not at the mucosal surface, where expression of toxin and pilus may have a more beneficial effect [174]. The concentration of bile may decrease as the organisms swim through the mucus layer to the epithelial surface and induction of toxin and pilus expression would ensue [174] (Fig. 1). Significant amounts of toxin can be produced from the replicative, extrachromosomal form (RF) of the CTXcj), and this expression is ToxR independent [176]. The significance of this observation with regards to pathogenesis is not clear yet, but it suggests that the ability to produce at least some cholera toxin is carried by the phage itself, and is independent of the host background. This could have
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MOLECULAR BASIS OF VIBRIO CHOLERAE PATHOGENESIS
483
implications for the emergence of new toxinogenic isolates of V. cholerae because, if toxin can be synthesized from the RF, then theoretically any host in which the phage could replicate would be toxinogenic. 3.
COORDINATE REGULATION BY THE TOXR/TOXT CASCADE
Regulation of virulence gene expression in V. cholerae involves a complex interaction of several activator proteins that ultimately control expression of a transcription activator called ToxT. The ToxT protein is the direct activator of both the cholera toxin operon and the large tcp operon that encodes the major pilus subunit Tcp A, as well as the secretion machinery required for its assembly [142, 177]. In addition to ToxR and ToxS, another pair of membrane-localized regulatory proteins, TcpP and TcpH, is required for activating toxT transcription [178]. Transcription of the tcpPH operon is itself controlled independently by two regulators called AphA and AphB [168, 179] (Fig. 6). The genes encoding the regulators ToxT, TcpP, and TcpH, as well as those encoding TcpA and the pilus assembly system, are all encoded within the genome of the VPI(|). Activation of toxT transcription appears to be the critical event determining whether or not toxin and pilus will be expressed, because mutations that abolish regulatory elements within the toxT promoter lead to loss of toxin and pilus production [180, 181]. Furthermore, expression of toxT from an unregulated promoter or from an externally regulatable promoter such as the IPTG-inducible tac promoter leads to toxin and pilus production independent of the signals that dictate El Tor-specific expression such as AKI growth conditions [168, 177, 182]. Yet, not all signals operate at the level of toxT transcription: repression of toxin and pilus production mediated by bile and, to a lesser extent, temperature, still occurs when toxT is expressed from a heterologous promoter [174], suggesting that these parameters may alter ToxT function. The toxT gene is embedded within the tcp gene cluster on the VPI(|) and is regulated by two promoters: one is located upstream of the toxT gene and is controlled by the concerted actions of ToxR/ToxS and TcpP/TcpH; the second promoter transcribes the large operon encoding the tcp genes [142, 181, 183] and is controlled by ToxT itself. The tcp operon has a weak transcription terminator, and therefore transcription may proceed into downstream genes, which include toxT and the gene encoding the TcpA prepilin peptidase TcpJ [137, 183]. Thus, expression of toxT involves an autoregulatory loop that requires activation by ToxR/S and TcpP/H to establish a threshold level of ToxT, which can then activate the tcp operon and provide for more of its own production by virtue of transcription reading through the terminator between the tcpA operon and the toxT-tcpJ optYon [142, 181] (Fig. 6). Sequence differences in the tcpA promoters between the two biotypes suggest that the readthrough mechanism of control from tcp transcription may be important in the biotype-specific regulation of virulence
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cAMP/CRP
Ule.
H(@)
-If
act
Fig. 6 Regulation of toxT, ct.xAB, and tcp expression by the ToxR/ToxT regulatory cascade. AphA and AphB activate expression of tcpPH, and the products of this operon cooperate with ToxR and ToxS for direct activation of toxT. ToxT controls its own transcription by virtue of its activation of the tcpA operon, transcription of which can read through a weak terminator structure, represented by the stem-loop, into the toxTgent downstream. The cAMP/cyclic AMP receptor protein (CRP) complex represses expression of the tcpA operon, and this repression is polar on /ojcr transcription. Some growth conditions, such as pH and the requirement for biotype-specific conditions (AKI) described in the text affect expression of toxin and pilus by altering toxT transcription, whereas bile salts and perhaps temperature affect the activity of ToxT.
genes [184, 185]. Alternatively, biotype-specific mechanisms of regulating tcpPH transcription may control differential expression of toxT [168, 179, 186]. Activation of the proximal ToxR/TcpP-dependent toxT promoter in El Tor strains requires the initial low oxygen phase of AKI growth, whereas subsequent transcription of the ctxAB operon requires the shaking, highly oxygenated phase [187]. Studies on expression of the ToxR regulon during experimental infection in vivo using a recombinase reporter system [188, 189, 189a] (see Chapter 4 in this book) show conclusively that regulated expression of toxT precedes cholera toxin gene expression.
10.
MOLECULAR BASIS OF VIBRIO CHOLERAE PATHOGENESIS
4.
485
CONTROL OF THE TOXR REGULON BY C A M P / C R P
Mutations in either the adenylate cyclase gene {cya) or the crp gene encoding the cyclic AMP receptor protein result in expression of toxin and pilus under repressing growth conditions [190]. CRP is a transcription factor that, when complexed with cAMP, binds DNA and activates transcription of many genes in response to low glucose levels. A consensus CRP-binding site in the tcpA promoter overlapping the RNA polymerase-binding site [162, 191] suggests that the likely mechanism by which this complex controls pilus and toxin transcription is through repression of tcpA transcription and consequent polarity on toxT expression under nonpermissive conditions for expression of the ToxR regulon [162, 190] (Fig. 6). This form of downregulation of toxT is also seen with polar insertions in tcpA and in tcpA promoter deletions: in each case, toxin levels are reduced even though activation of toxT hy ToxR/S and TcpP/H is unaffected [142, 181]. The ToxR-regulated ompT gene also appears to be controlled by cAMP/CRP, based on the presence of a consensus CRP-binding site in its promoter [191a], but, in contrast to its positioning in the tcpA promoter, in the ompT promoter this site is located where it likely results in activation by cAMP/CRP as opposed to repression. The physiological basis for cAMP/CRP repressing toxin and pilus expression is not clear. Assuming the system functions in V. cholerae as it does in E. coli, when glucose concentrations are low, production of toxin and pilus would be reduced and OmpT expression enhanced. Another source of catabolite control over the ToxR regulon comes from growth in the presence of maltose [192]. When grown in 0.4% maltose, 95% of the cholera toxin is associated with cells as opposed to being secreted into the supernatant, compared with growth in glucose, where over 99% of the toxin is found in the supernatant [192]. In contrast, the amount of surface-exposed TCP increases dramatically when the cells are grown in the presence of maltose as measured in a quantitative ELISA [192]. The increase in surface TCP production requires two genes of the maltose regulon, MalF and MalQ, components of the maltose transport and metabolism systems, respectively, but not the maltoporin OmpS [192, 193]. In addition to these in vitro effects, maltose regulation appears to be important for survival in vivo as both malF and malQ mutants are significantly less virulent in the infant mouse model of cholera [192]. This suggests a strong link between the metabolic behavior of V cholerae and regulation of toxin and pilus production. One interesting aspect of maltose control in terms of the evolution of V cholerae is that a number of the maltose genes are on the smaller of the two megareplicons, whereas the other toxin and pilus regulatory elements are on the larger one [40]. 5.
MEMBRANE LOCALIZATION OF TRANSCRIPTION FACTORS
In addition to increasing our knowledge of how V. cholerae controls virulence factor expression, the study of toxin and pilus regulation in this pathogen has
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VICTOR J. DIRITA
revealed a previously uncharacterized class of transcription regulatory system [194-197]. Proteins in this class of activator, represented by ToxR and TcpP, have a cytoplasmic domain that activates transcription and a periplasmic domain of undetermined function. The two domains are separated by a single stretch of hydrophobic amino acids that serves as a transmembrane domain. Activators in this family have been identified in E. coli. Salmonella typhimurium. Yersinia pseudotuberculosis, V. parahemolyticus, Vfischeri, and Photobacterium spp. [198-200]. These proteins frequently work in concert with another membrane protein that has a periplasmic domain but very minimal cytoplasmic domain; for ToxR and TcpP, these proteins are ToxS and TcpH, respectively [186, 194, 201] (Fig. 7). The cytoplasmic domain of ToxR and other membrane-localized transcription activators share similarity to the DNA-binding/activation domain of a class of regulatory proteins typified by the OmpR protein of E. coli [169, 202]. In the well-studied two-component gene family, one component, a sensor kinase, recognizes an environmental signal and in response phosphorylates itself on a conserved histidine residue. The phosphate group is then transferred to a
Effei^or (Toxs, TcpH, etc)
iU^tivator (ToxR, TepP, etoj
OinpR^like I ififliigtd helix
Fig. 7 Structure of membrane-localized transcription factors. Activator proteins like ToxR span the membrane and have significant domains in both the periplasmic space and the cytoplasm. The latter domain is similar to OmpR and has a winged helix DNA-binding/transcription activation domain. The periplasmic domains of different activators are not similar and have been proposed to be important for signal detection. The activators bind DNA directly (represented in the figure) and presumably stimulate RNA polymerase function through contact with one of the polymerase subunits. The effector proteins like ToxS are hypothesized to be important for stability and may play a role in multimerization of the activator.
10.
MOLECULAR BASIS OF VIBRIO CHOLERAE PATHOGENESIS
487
conserved aspartate residue on the second component, a response regulator, which is often a transcription factor (such as OmpR) that becomes activated when phosphorylated [203]. The similarity between ToxR and response regulators in the two-component family is within the domain that binds DNA and activates transcription (Fig. 7). This domain of ToxR is called a winged helix-tum-helix and is shared by other response regulators, including OmpR, whose winged helix-turn-helix structure has been solved by X-ray crystallographic analysis [204, 205]. Mutants of ompR with differing phenotypes have been generated over the years, and, combined with the available crystal structure, predictions regarding the nature of DNA binding and transcription activation by OmpR and OmpR-like proteins are possible. Mutations in toxR at residues that are shared between ToxR and OmpR have a severe effect on ToxR function [202], allowing a functional map of the similarities between ToxR and other winged-helix proteins to be generated for comparing which regions of the proteins are important for DNA binding and for interaction with RNA polymerase [204, 205]. It should be noted that as yet it has not been demonstrated that ToxR, or any other membrane-localized transcription activator, interacts directly with RNA polymerase; the requirement for the conserved residues between ToxR and OmpR remains the best evidence for such an activity [202, 204, 205]. Mutations abolishing expression of the membrane-localized effector proteins ToxS and TcpH cause downregulation of the ToxR regulon, as expected, but beyond that, their mechanism of action in transcription activation is unclear [181, 186, 201, 206]. Studies on the role of ToxS in ToxR function in heterologous backgrounds, such as E. coli or S. typhimurium, have led to the hypothesis that ToxS is required for the stability and/or multimerization of ToxR and that interaction of these proteins in the periplasmic space is required for this effect [201, 206, 207]. In these heterologous backgrounds, loss of ToxS function or the apparent ability for ToxR and ToxS to interact decreases the ability of ToxR to bind DNA and activate transcription [201, 206]. The ToxR protein can dimerize, and that dimerization is hypothesized to be a critical parameter in its ability to bind DNA [169, 201, 207-209]. The periplasmic domain of ToxR can be replaced by naturally dimerizing moieties, such as alkaline phosphatase, and the resulting fusion protein has ToxR activity that is independent of ToxS [169, 201]. Conversely, this domain can itself act as a dimerization domain [207]. These results notwithstanding, whether ToxR requires dimerization in order to be active is less clear because the periplasmic domain of ToxR can also be replaced with p-lactamase (Bla), a natural monomer, and the resulting fusion protein retains the ability to activate transcription in V^ cholerae [210]. Replacement of the periplasmic domain of TcpP does not abolish its ability to activate transcription either [197]. ToxR and ToxS very likely interact with one another in the periplasmic space [201, 206], and, given the similarities in topology, this is probably the case for
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VICTOR J. DIRITA
activators and effectors in other membrane-localized regulatory pairs as well. The most appealing hypotheses regarding the function of these proteins is that the periplasmic domain of one or both of them may be involved in sensing signals, such as those that are known to stimulate the ToxR regulon as discussed above, and that signal recognition is followed by conformational changes in the activator that allow it to bind DNA and activate transcription. There is limited experimental support for this hypothesis in that replacement of the periplasmic domains with proteins such as PhoA or Bla can result in constitutive (signal-independent) activity in some cases [169, 197], but the possibility that these fusions proteins may simply be more stable and therefore active under a broader range of conditions has not been ruled out. The CadC protein of E. coli is a membrane-localized transcription activator in which the periplasmic domain is important for signaling because point mutations in the periplasmic domain result in constitutive activation of the operon activated by CadC [211]. 6.
DNA BINDING AND ACTIVATION BY TOXR
The mechanism of transcription activation by membrane-localized ToxR and TcpP is of interest for its importance in the virulence of V cholerae and because of the general question of how membrane activators may engage the transcription complex to activate gene expression. ToxR and ToxS independendy control transcription of ompU and ompT, while activation of toxT transcription leading to toxin and pilus expression requires both the ToxR/ToxS and TcpP/TcpH pairs [162, 178-180, 186,212]. Control of omp gene expression by ToxR involves two different mechanisms, as befitting the opposite way that ompU and om/^r expression is regulated. ToxR activates ompU as a consequence of its binding to a series of sites extending positions -238 to -24 relative to the transcription initiation site at +1 [178], and the selectivity of ToxR for these sites may be dictated by the level of ToxR in the cell. Because ToxR appears to activate ompU transcription without the need for other factors from V. cholerae, it seems likely that binding to the multiple sites in the ompU promoter may position it for interaction with RNA polymerase, direcdy leading to transcription [178]. On the other hand, repression of ompT by ToxR is associated with its binding to a site extending from -95 to -30, and having limited similarity to the sites bound by ToxR in the ompU promoter. Binding of ToxR to the ompT promoter positions the protein downstream of the consensus cAMP/CRP site that may control om/^r acdvation [191a]. Taken together, these findings suggest that the relative amount of ompU or ompT expression is controlled by the level of ToxR and of cAMP/CRP within the cell: when ToxR levels are at their highest, the protein presumably occupies its sites on both the ompU and the ompr promoters, leading to activation of the former and repression of the latter. Within the regions required for ToxR binding in the ompU and om/^rpromoters, there are no elements such as direct or inverted repeats that might provide a strong clue as to how ToxR recognizes these sites [191a, 212]. Genetic and biochemical
10.
MOLECULAR BASIS OF V/B/?/c> c/yoL£/?/^£ PATHOGENESIS
489
analyses of two other promoters under ToxR control, those for the ctxAB and toxT genes (see below), provide little direction toward understanding specific requirements for ToxR binding. The promoter of the ctxAB operon contains a series of direct repeats of the sequence TTTTGAT, as well as sequences both upstream and downstream of these repeats, that are required for ToxR binding and transcription activation [169, 191a, 213]. The binding site for ToxR on the /6>xr promoter, on the other hand, includes an inverted repeat of primary sequence different than what is seen in the ctxAB, ompU, or ompT promoters [183, 214]. All of these binding sites are rich in A and T residues, indicating a general requirement for ToxR binding that may include recognition of bent DNA often associated with poly-A tracts [212, 213], but a consensus binding site for ToxR has not emerged from this work. The mechanism by which ToxR binding leads to activation of toxT is quite different compared to how ToxR activates ompU, because it requires another membrane-localized activator, TcpR ToxR binds to the toxT promoter between -93 and -58, which is rather far upstream from the RNA polymerase-binding site [183, 214]. But ToxR may play an indirect role because it cannot activate toxT'm the absence of TcpP, whereas TcpP can activate toxT in the absence of ToxR provided that TcpP is overexpressed [178, 183]. Thus, ToxR appears to act primarily by facilitating TcpP function in a novel cooperation between two different sets of the membrane regulatory proteins ToxR/S and TcpP/H. ToxR and ToxS activate the ctxAB promoter directly when expressed in E. coli that has a copy of a ctx-lacZ gene fusion [169, 196, 201]. However, in strains of V. cholerae expressing ToxR but lacking functional ToxT, cholera toxin expression is nearly undetectable, calling into question the significance of activation of CtxAB by ToxR [180]. Activation of ctxAB transcription by ToxR requires repeats of the sequence TTTTGAT present in the ctxAB promoter [169], which are not found in the other promoters regulated by ToxR. The lack of a consensus binding site for this protein may be the result of convergent evolution, in which genes present on three different genomes—the V cholerae ancestral chromosome and the genomes of VPIcj) and CTXcj)—have come under control by ToxR. The events that might have impelled this convergence toward ToxR control may have arisen during evolution of pathogenic V cholerae and its association with human hosts.
V. Natural and Induced Immunity against Vibrio cholerae Infection A. Immunity to Cholera Infection with V cholerae provides long-lasting immunity against subsequent infection: individuals infected once are much less likely to become infected again, although estimates of the protective capacity of natural infection vary widely
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[215-219]. Initial natural infection with classical strains may provide better protection against subsequent infection than does infection with El Tor strains, based on studies done in Bangladesh as part of a vaccine trial [218]. Immunity to V. cholerae is due to intestinal antibodies of the secretory IgA (sIgA) class generated against envelope components of the cell—particularly LPS and perhaps outer membrane proteins—and against toxin as well, notably the CT-B moiety. The question of whether antitoxin antibodies confer significant or relevant protection against disease is somewhat controversial because in some studies good protection against disease was conferred by preparations lacking the toxin, whereas in other studies toxin was shown to enhance the efficacy of vaccine preparations [220-224]. Whether or not antitoxin antibodies confer significant protection, current attempts at producing vaccines against cholera include CT-B as an antigen in the preparations. Serum antibodies of the IgG class capable of killing V. cholerae when mixed with complement (serum vibriocidal antibodies) have repeatedly been shown to have a significant correlation with protection against cholera, although it is unlikely that these antibodies contribute to long-term protection [225-227].
B. Killed or Subunit Vaccines A whole cell vaccine consisting of killed V. cholerae of different serotypes and biotyes has been licensed for use, but it is administered by injection (parenterally) and does not generate strong or long lived immunity [228]. To achieve these objectives, efforts have been made to produce an orally administered formulation that will stimulate the intestinal immune response required for protection against subsequent colonization. Each of the two major strategies that have been successful in combatting other infectious diseases—inoculation with either killed Vibrios or a subcellular fraction, or ingestion of live, attenuated organisms—has had proponents in development of anticholera vaccines. A killed whole cell vaccine (WC) that has been well studied is a preparation of formalin- or heat-killed cells of both Inaba and Ogawa serogroups and El Tor and classical biotype (each at 2.5 x 10^^; final cell amount therefore 1 x 10^'), which may also be mixed with 1 mg of purified CT-B (BS-WC). In volunteer studies, BS-WC given in two oral doses stimulated intestinal sIgA responses equivalent to what is observed in clinical cholera [222]. In extensive field trials in Madab, Bangladesh during the 1980s, the WC and BS-WC provided strong short-term protection against cholera, although the long-term protective efficacy was less compelling [220, 224, 225]. After three doses taken by more than 60,000 people, protective efficacy within the first year was as high as 85% but by the third year had dropped to 50% for WC-BS and to 52-58% for WC alone [220, 224]. Although these levels of efficacy are considered quite good, the fact that
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repeated doses of the vaccine must be given to achieve protection is viewed as a disadvantage of this type of vaccine [229]. Another subunit vaccine preparation was constructed specifically to generate serum vibriocidal antibodies of the IgG class, based on the hypothesis that these contribute to protection [230, 231 ] and that an injectable vaccine may be easier to administer to infants who routinely receive other parenteral vaccines. The preparation, called DeALPS-CT, is a protein-polysaccharide conjugate in which V cholerae LPS (treated to remove the toxic lipid A moiety) is chemically coupled to CT. Given in two injections spaced 6 weeks apart, DeALPS-CT elicited both vibriocidal (anti-LPS) and antitoxin antibodies within 1 month of the first injection, and these remained high (30-35% of initial titer) out to 230 days post-injection. Injection of a control cellular vaccine preparation (a mixture of killed Ogawa and Inaba V. cholerae) elicited an initially higher titer of vibriocidal antibodies that waned to a greater extent (to 22% of 28-day titer) over 230 days [231].
C. Live Vaccines The other major vaccine effort is aimed at developing recombinant live strains of V. cholerae that are attenuated for virulence but retain the ability to colonize the intestine and stimulate a protective immune response [91, 226, 232-234]. Thus, vaccine strains are engineered with deletions in the ctxA gene, but with an intact ctxB gene to allow for expression of the immunogenic B subunit. Concern for reacquisition of the ctxA gene has been taken into account in some strains by removing the att site for the CTXcj) or by introducing mutations into the recA gene, which is required for homologous recombination events that may introduce the ctxAB genes back into the chromosome of vaccine strains [37, 38, 235]. An additional concern regarding the homologous recombination system derives from the observation that, once acquired, the ctxAB locus is capable of amplification via homologous recombination mechanisms, and this amplification event is enriched for during in vivo growth [41 ]. A number of live candidate vaccines have been developed using both El Tor and classical isolates as the starting strain, as the choice of starting strain may be a critical parameter because of the reported biotype specificity of protection [218]. One of the better-analyzed strains is CVD103HgR, which has a deletion ofctxA but an intact ctxB gene expressed from the natural ToxR-regulated site [236, 237]. The remainder of the ToxR regulon in this strain was left intact, but the progenitor of this vaccine, the classical Inaba strain 569B, naturally carries a deletion of the toxS gene and therefore simultaneously expresses significant amounts of both OmpU and OmpT, a feature not usually seen in wild-type V cholerae [238]. CVD103-HgR has a gene for mercury resistance inserted in the gene for
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hemolysin hlyA. Mercury resistance was engineered into the strain in order to be able to monitor the presence of the vaccine in the environment [226]. When tested in volunteers in North America, a single dose of CVD103-HgR induced significant increases in serum vibriocidal antibodies after one dose. As predicted, it protected better against infection by classical V cholerae than against infection by El Tor V cholerae, with protective efficacies (at 1 month after immunization) of 87 and 62%, respectively [236]. In addition to its immunizing effect, colonization by CVD103-HgR was well tolerated by volunteers, meaning that no adverse effects such as headache, cramping, or nausea were observed. These side effects have been observed with other vaccine candidates, but their source is not clear. The CTX(t)-encoded products Zot and Ace affect ion flux in intestinal tissue in vitro, but deletion of the zot and ace genes from vaccine candidates does not alleviate the reactogenicity [37]. The recently identified Rtx toxin encoded in the El Tor genome near the ctxAB genes may also be a source of the reactogenicity, but this hypothesis is yet to be tested [21]. Reactogenicity may also arise as a consequence of colonization per se, because strains that colonize at high levels are associated with greater adverse reactions [37], and, as noted, there appears to be an association between motility of the vaccine strain and reactogenicity [33, 37-39]. CVD103-HgR was tested in over 67,000 persons of wide-ranging age in Indonesia for efficacy against natural cholera infection over the period from 1993 to 1997. There was no reported reactogenicity of the vaccine, but vaccination provided surprisingly little protection as compared with placebo controls. Of 93 cholera cases, 43 of the individuals had been vaccinated with CVD103-HgR, while 50 had received a placebo, for an efficacy rate of 14% (J. B. Kaper, personal communication, 1999). Nearly 70% of the vaccinees responded by producing serum vibriocidal antibodies, whereas individuals vaccinated with placebo exhibited a very low rate of vibricidal seroconversion. The reason for the unexpectedly poor performance of this vaccine in field trial is not clear. As the current cholera pandemic is caused by El Tor strains, use of a classical strain (such as 569B) as the progenitor of vaccine candidates may not be an optimal strategy, even if classical strains do tend to protect better against El Tor infection than the other way around. Accordingly, a bivalent vaccine consisting of CVD103-HgR and an attenuated derivative of an Ogawa El Tor strain N16961, called CVDl 11, has been tested. CVDl 11 has a deletion of the virulence cassette, which is the term used to collectively describe the genes encoding Ctx, Zot, and Ace [239]. By itself, this strain demonstrated a protective efficacy of more than 80% in U.S. volunteers but caused mild diarrhea in 12% of them, an unacceptable level of reactogenicity [240]. However, when the bivalent CVD103HgR/CVDl 11 vaccine was tested in a range of doses given to volunteers in the United States and Peru, it induced rises in anti-LPS against both strains and caused no adverse reactions [241].
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Theoretically, any V. cholerae strain may serve as the starting point for vaccine development, as the genetic tools necessary to introduce the relevant mutations to construct a vaccine strain are applicable to most isolates. This has led to the development of a number of vaccine strains based on El Tor isolates from different geographical areas where cholera is epidemic. These strains, with names such as Peru-3, Bah-3 (for Bahrain), and Bang-3 (for Bangladesh), were engineered by removing the CTX(|) genes as well as the natural site for phage integration (attRSl) to keep the vaccine strains from reacquiring toxin genes. The ctxB gene was reintroduced by insertion into the recA gene or the lacZ gene under control of a heat shock promoter or the natural ctx promoter, respectively [37, 38]. These candidate vaccines induced a rise in vibriocidal antibodies and, in many volunteers, stimulated an antitoxin response, albeit at low titers [37]. There were adverse reactions in some volunteers with a correlation between symptoms and how well the particular strain colonized in the infant mouse model. Those strains with the lowest level of reactogenicity were Peru-3 or 5 and Peru-14, which is a nonmotile variant of Peru-3, and another nonmotile strain isolated independently called Peru-15. These strains showed protective efficacy against challenge with an El Tor strain ranging from 80 to 87% [37, 38]. Thus, they represent strong vaccine candidates against infection by El Tor V cholerae and validate the use of epidemic isolates with defined genetic alterations for vaccine protection. The approach of engineering vaccine strains from epidemic isolates also allowed for rapid development of vaccine candidates against the emergent 0139 strains of pathogenic V cholerae [39]. One of these strains, Bengal-15, a nonmotile strain deleted for the CTXcj), attRSl, and the recA genes, showed promise as an 0139 vaccine. It caused mild symptoms (headache, malaise, cramps) but no diarrhea in volunteers who ingested 10^ cfu and produced a high, although short-lived, seroconversion to vibriocidal antibody production. When challenged with 5 x 10^ wild-type organisms, those vaccinated with Bengal-15 were protected from cholera symptoms with an efficacy of 83% [39].
W. Future Studies: Ttie Past Is Prologue After more than a century of studying Vibrio cholerae as a pathogen of man and with all that has been learned from doing so, a good deal remains to be done to fully understand the pathophysiology of infection. Even given the vast amount of knowledge that has been obtained from the study of molecular mechanisms in V cholerae infection, basic questions remain, and answering these will contribute both to an understanding of cholera and to solution of broader problems in molecular pathogenesis. For example, how does the Eps system identify the cholera toxin and other secreted proteins as targets for extracellular secretion, and
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how does the Eps system actually work to deliver molecules across the outer membrane? Also, an understanding of cellular intoxication by CT remains incomplete for lack of definitive knowledge regarding the trafficking pathway and how toxin ultimately crosses the membrane, whatever its source, to reach its target. As for the behavior of V cholerae within the host, new methods for studying the microbe during its transient period of interaction within the host hold great promise for filling out our knowledge of the niches it colonizes during infection [188, 189]. Another area for future study includes the role of TCP and bacterial autoaggregation in pathogenesis of cholera. Examining this will likely serve as a model for understanding other pathogens in which a similar phenotype has been observed. How the organism perceives the intestinal environment and integrates signals such as temperature, bile concentration, and the levels and kinds of metabolites through the ToxR/ToxT system, leading to expression of cholera toxin and toxin-coregulated pilus, remains unclear. Finally, the roles of recently identified regulators of virulence gene expression have yet to be completely integrated into the already complex regulatory scheme [242, 243]. Clarifying the mechanism of action of membrane-localized transcription factors, first identified in V. cholerae with the discovery of ToxR [169, 196], will expand our general understanding of an unusual regulatory mechanism even as it contributes to what we know about cholera. Recent work has generated a much greater appreciation for the fact that V. cholerae is essentially an environmental organism that has become lysogenized with virulence-converting phages such as CTXcj) and VPIcj), and perhaps others as well [16, 17, 244]. The phage work has already yielded novel mechanisms (such as the capacity for lysogeny by filamentous phages), and unraveling those mechanisms will expand both our understanding of cholera and of phage biology in general [245, 246]. Of particular interest and importance in the study of CTXcj) biology is the role that phage immunity may play in evolution and, eventually, control of V cholerae, because it is quite clear now that heteroimmunity among phages is important in the horizontal transfer of cholera toxin genes among V cholerae isolates. Phage immunity also has important ramifications for development and use of live vaccine strains, because classical strains of V cholerae are not immune to infection by CTXcj) from El Tor strains due to differences in the phage repressor that controls lysogeny [246]. With the completion of the nucleotide sequence of its genome [246a], we are at the beginning of the postgenomic era of V. cholerae. Important discoveries have already come from the sequencing project, including vcpD, the prepilin peptidase critical for toxin secretion and MSHA assembly, and Rtx, the cytotoxin that may be associated with reactogenicity in vaccine strains [21, 108]. The application of genomic approaches will result in discovery of other virulence determinants [243, 247].
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Acknowledgments I thank the many colleagues who discussed various aspects of this chapter with me during its preparation, including Peter Christie, Maria Sandkvist, David Karaolis, Jim Kaper, Andy Camilli, and Ron Taylor. I also appreciate the indulgence of both my editor and my lab while I was writing it. Work in my laboratory is supported by PHS NIH grants AI 31645 and 45125.
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182. DiRita, V. J., Neely, M., Taylor, R. K., and Bruss, R M. (1996). Differential expression of the ToxR regulon in classical and El Tor biotypes of Vibrio cholerae is due to biotype-specific control over /ojcJ expression. Proc. Natl. Acad. Sci. U.S.A. 93, 7991-7995. 183. Higgins, D. E., and DiRita, V. J. (1994). Transcriptional control of toxT, a regulatory gene in the ToxR regulon of Vibrio cholerae. Mol. Microbiol. 14, 17-29. 184. Ogierman, M. A., Zabihi, S., Mourtzios, L., and Manning, R A. (1993). Genetic organization and sequence of the promoter-distal region of the tcp gene cluster of Vibrio cholerae. Gene 126, 51-60. 185. Iredell, J. R., and Manning, R A. (1994). Biotype-specific tcpA genes in Vibrio cholerae. FEMS Microbiol. Lett. 121, 47-54. 186. Carroll, R A., Tashima, K. T., Rogers, M. B., DiRita, V. J., and Calderwood, S. B. (1997). Phase variation in tcpH modulates expression of the ToxR regulon in Vibrio cholerae. Mol. Microbiol. 25, 1099-1111. 187. Medrano, A. I., DiRita, V. J., Castillo, G., and Sanchez, J. (1999). Transient transcriptional activation of the Vibrio cholerae El Tor virulence regulator toxT in response to culture conditions. Infect. Immun. 67, 2178-2183. 188. Camilli, A., Beattie, D. T, and Mekalanos, J. J. (1994). Use of genetic recombination as a reporter of gene expression. Proc. Natl. Acad. Sci. U.S.A. 91, 2634-2638. 189. Camilli, A., and Mekalanos, J. J. (1996). Use of recombinase gene fusions to identify Vibrio cholerae genes induced during infection. Mol. Microbiol. 18(4), 671-683. 189a. Lee, S. H., Hava, D. L., Waldor, M. K., and Camilli, A. (1999). Regulation and temporal expression patterns of Vibrio cholerae virulence genes during infection. Cell 99, 625-634. 190. Skorupski, K., and Taylor, R. K. (1997). Cyclic AMP and its receptor protein negatively regulate the coordinate expression of cholera toxin and toxin-coregulated pilus in Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 94, 265-270. 191. Ogierman, M. A., Voss, E., Meaney, C , Faast, R., Attridge, S. R., and Manning, P. A. (1996). Comparison of the promoter proximal regions of the toxin-co-regulated tcp gene cluster in classical and El Tor strains of Vibrio cholerae Ol. Gene 170, 9-16. 191a. Li, C. C , Crawford, J. A., DiRita, V. J., and Kaper, J. B. (2000). Molecular cloning and transcriptional regulation of ompT, a ToxR-repressed gene in Vibrio cholerae. Mol. Microbiol. 35, 189-203. 192. Lang, H., Jonson, G., Holmgren, J., and Palva, E. T. (1994). The maltose regulon of Vibrio cholerae affects production and secretion of virulence factors. Infect. Immun. 62, 4781-4788. 193. Lang, H., and Palva, E. T. (1993). The ompS gene of Vibrio cholerae encodes a growth-phasedependent maltoporin. Mol. Microbiol. 10, 891-901. 194. Miller, V. L., DiRita, V. J., and Mekalanos, J. J. (1989). Identification of toxS, a regulatory gene whose product enhances ToxR-mediated activation of the cholera toxin promoter. J. Bacteriol. 171, 1288-1293. 195. Miller, V. L., and Mekalanos, J. J. (1985). Genetic analysis of the cholera toxin-positive regulatory gene toxR. J. Bacteriol. 163, 580-585. 196. Miller, V. L., and Mekalanos, J. J. (1984). Synthesis of cholera toxin is positively regulated at the transcriptional level by ToxR. Proc. Natl. Acad. Sci. U.S.A. 81, 3471-3475. 197. Hase, C. C , and Mekalanos, J. J. (1998). TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae. Proc. Natl. Acad. Sci. U.S.A. 95, 730-734. 198. Reich, K. A., and Schoolnik, G. K. (1994). The light organ symbiont Vibrio fischeri possesses a homolog of the Vibrio cholerae transmembrane transcriptional activator ToxR. J. Bacteriol. 176, 3085-3088.
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218. Clemens, J. D., Van Loon, K, Sack, D. A., Rao, M. R., Ahmed, F., Chakraborty, J., Kay, B. A., Khan, M. R., Yunus, M. D., and Harris, J. R. (1991). Biotype as determinant of natural immunising effect of cholera. Lancet 337, 883-884. 219. Woodward, W. (1971). Cholera reinfection in man. J. Infect. Dis. 123, 61-66. 220. Clemens, J. D., Harris, J. R., Khan, M. R., Kay, B. A., Yunus, M., Svennerholm, A. M., Sack, D. A., Chakraborty, J., Stanton, B. R, Khan, M. U., Atkinson, W., and Holmgren, J. (1986). Field trial of oral cholera vaccines in Bangladesh. Lancet ii, 124-127. 221. Holmgren, J., and Svennerholm, A. M. (1983). Cholera and the immune response. Prog. Allergy 33, 106-112. 222. Svennerholm, A. M., Jertbom, M., Gothefors, L., Karim, A. M. M. M., Sack, D. A., and Holmgren, J. (1984). Mucosal antitoxic and antibacterial immunity after cholera disease and after immunization with a combined B subunit-whole cell vaccine. J. Infect. Dis. 149, 884-893. 223. Levine, M. M., Nalin, D., Craig, J. R, Hoover, D., Bergquist, E. J., Waterman, D., Holley, H. P., Homick, R. B., Pierce, N. F., and Libonati, J. P. (1979). Immunity to cholera in man: Relative role of antibacterial versus antitoxic immunity. Trans. R. Soc. Trop. Med. Hyg. 73, 3-9. 224. Clemens, J. D., Sack, D. A., Harris, J. R., Van Loon, F., Chakraborty, J., Ahmed, F., Rao, M. R., Khan, M. R., Yunus, M., Huda, N., Stanton, B. F., Kay, B. A., Walter, S., Eeckels, R., Svennerholm, A. M., and Holmgren, J. (1990). Field trial of oral cholera vaccines in Bangladesh: Results from three-year follow-up. Lancet 335, 270-273. 225. Clemens, J. D., Van Loon, F., Sack, D. A., Chakraborty, J., Rao, M. R., Ahmed, F., Harris, J. R., Khan, M. R., Yunus, M., and Huda, S. (1991). Field trial of oral cholera vaccines in Bangladesh: Serum vibriocidal and antitoxic antibodies as markers of the risk of cholera. J. Infect. Dis. 163, 1235-1242. 226. Levine, M. M., and Jacket, C. O. (1994). Recombinant live cholera vaccines. In ''Vibrio cholerae and Cholera" (I. K. Wachsmuth, P A. Blake, and 0. Olsvik, eds.), pp. 395-413. ASM, Washington, DC. 227. Glass, R. I., Svennerholm, A. M., Khan, M. R., Huda, S., Huq, M. I., and Holmgren, J. (1985). Seroepidemiological studies of El-Tor cholera in Bangladesh: Association of serum antibody levels with protection. / Infect. Dis. 151, 236-242. 228. Swerdlow, D. L., and Ries, A. A. (1992). Cholera in the Americas. Guidelines for the clinician. JAMA 267, 1495-1499. 229. Sanchez, J. L., and Taylor, D. N. (1997). Chlolera. Lancet 349, 1825-1830. 230. Szu, S. C , Gupta, R., and Robbins, J. B. (1994). Induction of serum vibriocidal antibodies by 0-specific polysaccharide-protein conjugate vaccines for prevention of cholera. In ''Vibrio cholerae and Cholera" (I. K. Wachsmuth, P A. Blake, and 0. Olsvik, eds.), pp. 381-394. ASM, Washington, DC. 231. Gupta, R. K., Taylor, D. N., Bryla, D. A., Robbins, J. B., and Szu, S. C. (1998). Phase 1 evaluation of Vibrio cholerae 0 1 , serotype Inaba polysaccharide-cholera toxin conjugates in adult volunteers. Infect. Immun. 66, 3095-3099. 232. Kaper, J. B., Lockman, H., Baldini, M. M., and Levine, M. M. (1984). Recombinant nontoxinogenic Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature 308, 655-658. 233. Taylor, R., Shaw, C , Peterson, K., Spears, P, and Mekalanos, J. (1988). Safe, hve Vibrio cholerae vaccines? Vaccine 6, 151-154. 234. Mekalanos, J. J. (1992). Bacterial mucosal vaccines. Adv. Exp. Med. Biol. 321, 43-50. 235. Ketley, J. M., Kaper, J. B., Herrington, D. A., Losonsky, G., and Levine, M. M. (1990). Diminished immunogenicity of a recombination-deficient derivative of Vibrio cholerae vaccine strain CVD103. Infect. Immun. 58, 1481-1484.
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CHAPTER 1 1
H. pylori Pathogenesis TIMOTHY L. COVER DOUGLAS E. BERG MARTIN J. BLASER HARRY L. T. MOBLEY
I. II. III. IV.
V. VI. VII.
VIII.
IX.
X.
XI.
XII.
Introduction Epidemiology Gastric Histology and Physiology Clinical Diseases Associated with//. pv/or/Infection A. Acute Infection B. Peptic Ulcer Disease C. Gastric Adenocarcinoma D. Gastric Non-Hodgkins B-Cell Lymphoma Microbiology Genetic Diversity and Population Structure of H. pylori Initial Gastric Colonization A. Motility B. Urease C. Multiple Mechanisms of Acid Resistance D. Effects of H. pylori on Gastric Acid Production E. Adherence Gastric Inflammation A. Mechanisms B. Role of the cag Pathogenicity Island Interactions of H. pylori with the Gastric Epithelium A. Cytoskeletal Changes and Tyrosine Phosphorylation B. Apoptosis Vacuolating Cytotoxin A. Structure B. Allelic Variation in vacA C. Mechanism of Action D. Role of VacA in Vivo Persistence of H. pylori Infection A. Resistance to Clearance by Host Immune Defenses B. Characteristics of H. pylori Lipopolysaccharide C. Bacteria-Host Equilibrium Factors Influencing Development of Clinically Evident Disease A. Bacterial Factors B. Host Factors C. Environmental Factors D. Perspectives o n / / . pv/t^n-Related Diseases References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
SOQ
510 510 512 516 516 516 517 518 519 521 524 524 526 527 527 528 529 529 530 531 531 532 532 532 533 534 535 536 536 537 538 539 539 540 541 541 542
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L Introduction Helicobacter pylori are extraordinary among bacteria in their ability to colonize the human gastric mucosa, an inherently inhospitable acidic environment, and to persist in this niche for many decades, despite the development of host immune and inflammatory responses. To our knowledge, no other bacteria, with the possible exception of the closely related H. heilmanii, can establish long-term residence in the human gastric mucosa. More than half of the world's human population is persistently colonized with H. pylori, usually without disease. However, in about 10% of cases, colonization, probably in combination with other critical risk factors, leads to serious illnesses, ranging from peptic ulcers to two different forms of gastric cancer. Because persistent (chronic) colonization with H. pylori has been recognized as a significant risk factor for serious gastroduodenal diseases (Table I), the interaction between //. pylori and humans has been considered an "infectious disease." Consistent with this interpretation, persistent H. pylori colonization nearly always elicits a specific host immune and inflammatory response. However, based on the recognition that the majority of//. /?}^/6>r/-colonized persons never develop any clinical symptoms, persistent colonization can also be viewed as a commensal process, perhaps analogous to colonization by Bacteroides spp., alpha streptococci, and lactobacilli. In addition, there has been speculation that //. pylori colonization could be beneficial for humans in some circumstances. These different perspectives illustrate the complexity of interactions between H. pylori and humans, and the lack of current consensus about how these bacteria should be viewed. Regardless of whether the term "infection" or "colonization" is used, the salient feature is that //. pylori are extremely well adapted to life in the human stomach. //. pylori occupy an interesting position in the history of medicine and biomedical research. Bacteria were detected by microscopy in human gastric tissue more than a century ago, but these observations were essentially forgotten. Only since 1982 has there been interest in the idea that these bacteria might cause disease [1, 2]. Reports that gastric inflammation and peptic ulcers might be the consequences of a bacterial infection [3] were initially met with considerable skepticism in the medical community. However, human volunteer studies have shown that //. pylori ingestion indeed results in gastric inflammation [4-6], and most gastroenterologists now agree that //. pylori play a major role in the pathogenesis of peptic ulcers [7] and gastric cancer [8]. In this chapter, we review our current understanding of how //. pylori persistently colonize the human gastric mucosa and discuss factors that determine whether infections progress to clinically evident disease or remain asymptomatic.
//. Epidemiology H. pylori are present in nearly all human populations throughout the world. In the United States, about 30-50% of adults are persistendy colonized, whereas in
11.
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H. PYLORI PATHOGENESIS
Table I
Histological Features and Clinical Diseases Associated with H. pylori
Condition
Definition
Estimated lifetime incidence in H. pylohpositive persons in the U.S.
Predisposing factor
Gastritis
Inflammation of the gastric mucosa
Duodenal ulcer
Defect in the duodenal mucosa
Gastric ulcer
Defect in the gastric mucosa
Atrophic gastritis
Loss of gastric glands
5-10%
Prolonged H. pylori carriage
Intestinal metaplasia
Intestinal epithelium in gastric mucosa
5-10%
Prolonged H. pylori carriage
Gastric epithelial dysplasia
Abnormal epithelial morphology and organization
<5%
Prolonged H. pylori carriage
Gastric adenocarcinoma
Gastric tumor derived from epithelial cells
<1%
Atrophic gastritis, intestinal metaplasia, dysplasia, dietary factors. particular cag^ strains
Gastric lymphoma
Gastric tumor derived from lymphocytes
<1%
None known
100%
Always associated with H. pylori
5-10%
H. pylori colonization of the duodenum, ulcerogenic strains
5%
Ulcerogenic strains
many developing countries the prevalence is 80% or higher [9, 10]. In all geographic areas studied, the prevalence of H. pylori increases with age [9-11]. The rate of acquisition of H. pylori among adults in developed countries is typically quite low [12]; therefore, the high prevalence of the organism among adults over 50 years of age in the United States [13] probably represents a cohort effect, in which currently infected adults acquired the organisms during childhood [14]. Initial colonization probably occurs preferentially during childhood [11, 15, 16], either because toddlers characteristically put everything in their mouths, or because of increased susceptibility to infection due to age-related characteristics of the stomach or immune system. The incidence of H. pylori infection has declined with the societal changes that accompanied industrial and socioeconomic development, including sewage disposal, water chlorination, hygienic food
512
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preparation, diminished crowding, and education [10]. Thus, children are colonized significantly more frequently and at an earlier age in developing countries than in developed countries [16, 17]. Despite an apparently decreasing incidence in developed countries, H. pylori continues to be present among children in the United States [17], and is especially common in children of poorer socioeconomic groups. Once established, H. pylori usually persists for decades, unless eradicated with antimicrobial therapy. Humans constitute the only major reservoir for H. pylori. Early studies demonstrated intrafamilial clustering of infections [18]. More recently, DNA fingerprint analyses of isolates have confirmed that most transmission occurs locally within families or small population groups [19, 20]. This is consistent with an apparent inability of H. pylori to proliferate or survive for long periods in the environment. Person-to-person transmission may involve direct ingestion of H. pylori found in saliva, vomitus, or feces, or ingestion of recently contaminated foods or beverages. The relative contributions of fecal-oral and oral-oral transmission are not known, but they may vary among different populations. Isolation of H. pylori from saliva, dental plaque, or feces has been reported [21-22], albeit rarely. H. pylori is commonly found in the stomachs of nonhuman primates [23], but these are unlikely to be important sources from which humans acquire the organism. Various strains of many bacterial pathogens can be distinguished from one another based on antigenic properties (serotype), enzymatic properties (biotype), or phage sensitivity (phage type). Such classifications often help to identify particular clonal lineages associated with increased virulence (e.g., E. coli 0157:H7 or Vibrio cholerae 01 and 0139). None of these typing methods have been widely used for classifying strains of H. pylori. When H. pylori isolates from different patients are analyzed by appropriate DNA fingerprinting methods, each isolate appears to be distinctly different genetically from all independent isolates [24, 25]. DNA-based typing methods thus provide a useful approach for differentiating H. pylori strains and tracing patterns of transmission among epidemiologically linked individuals. Certain genetic markers are also potentially useful in predicting clinical outcome, as will be discussed below.
///. Gastric Histology and Ptiysioiogy The human stomach is generally free of viable bacteria other than H. pylori and a related organism, H. heilmanii [26, 27], despite the huge number of bacteria that are swallowed along with food each day. To put the capacity of H. pylori to
11.
H. PYLORI PATHOGENESIS
513
colonize the human stomach into context, we will first review fundamental concepts of gastric anatomy and physiology. The stomach wall consists of four major layers (serosa, muscle layer, submucosa, and mucosa) [28]. The mucosa, or innermost layer bordering the lumen, is comprised of a surface epithelium, an underlying connective tissue layer (lamina propria), and a thin smooth muscle layer (muscularis mucosae). Secretory gastric glands lined with epithelial cells are found throughout the mucosa of the stomach. Based on histologic features of the mucosa, the stomach can be divided into three parts (Fig. 1). The major part (known as the body or corpus) is characterized by unbranched glands, which contain parietal cells that secrete hydrochloric acid and chief cells that secrete pepsinogen (a precursor for the proteolytic enzyme pepsin). The mucosa of the lower part of the stomach (known as the antrum) does not secrete substantial amounts of pepsin or acid, but has branched glands that contain specialized endocrine G cells and D cells, which secrete gastrin and somatostatin, respectively. Gastrin stimulates acid secretion by parietal cells, and somatostatin inhibits acid secretion. The third part of the stomach is a small non-acid-secreting region, known as the cardia, which borders the esophagus. At the antrum-corpus junction, there is a narrow transitional zone that separates the two types of mucosa. The mucosa of the duodenum (located just distal to the stomach) and esophagus (proximal to the stomach) are typically lined with different types of epithelium than are found in the stomach, and transitional zones occur at both the gastroduodenal and esophagogastric junctions. A remarkable feature of the stomach is its capacity to resist damage from hydrochloric acid and pepsin. This is attributable in part to a protective layer of Esophagus'
Body (corpus) Antrum
Duodenum Fig. 1 Anatomy of the human stomach. The stomach can be divided into three parts (cardia, body, and antrum). The gastric corpus is characterized by unbranched glands containing parietal cells that secrete HCl and intrinsic factor, and chief cells that secrete pepsinogen. The antrum is characterized by branched glands containing G cells that secrete gastrin and D cells that secrete somatostatin.
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TIMOTHY L. COVER ETAL
mucus, about 0.2 to 0.6 mm thick, that overlies gastric-type epithelial cells throughout the stomach. Secretion of bicarbonate by epithelial cells results in a strong pH gradient across the mucus layer, ranging from pH <2 at the lumenal surface to nearly neutral pH at the epithelial cell surface [29]. The secretion of HCl through the mucus layer is thought to occur via a process known as "viscous fingering," in which HCl traverses the mucus via a series of discrete channels [30]. Within the stomach, most H. pylori appear to be free-living within the mucus layer [31], but some organisms adhere to gastric epithelial cells [32]. Very rarely, organisms appear to be within epithelial cells, but it is not clear that this is significant biologically. H. pylori are found almost exclusively overlying gastrictype epithelium (Fig. 2), but not areas of the stomach where the normal gastric surface has been replaced by metaplastic intestinal-type epithelium. Conversely, H. pylori can colonize portions of the gastrointestinal tract other than the stomach if metaplastic gastric-type epithelium is present, which perhaps occurs as a consequence of epithelial damage. The most clinically important site for gastric metaplasia is the duodenum, where H. pylori overlies gastric epithelium but not the normal intestinal-type epithelium [33-35]. H. pylori is not found associated with squamous epithelium of the esophagus or with intestinal epithelium of the small intestine. This tropism of H. pylori for gastric epithelium might reflect specific gastric epithelial cell-surface properties, or the production of relevant factors, such as specific gastric mucins. In the absence of H. pylori, the gastric mucosa contain very few immune or inflammatory cells, whereas all H. pylori-xnitcitd persons exhibit an inflamma-
Fig. 2 Scanning electron micrograph of H. pylori in the human gastric mucosa. H. pylori is found in the mucus layer overlying gastric epithelial cells, but does not invade tissue.
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Fig. 3 Histology of gastric mucosa from an H. pylori-'infecied human. Compared to gastric mucosa from noncolonized persons, there is infiltration of immune and inflammatory cells. Photograph provided by K. Tham.
tory response in the gastric mucosa (termed gastritis) [36, 37] (Fig. 3). The intensity of the host response and the relative predominance of different types of inflammatory and immune cells vary considerably among infected persons. The inflammation tends to be predominantly lymphocytic in children [38], but often includes abundant neutrophils in adults. Chronic atrophic gastritis is a histologic abnormality characterized by the loss of glandular components within the gastric mucosa. Atrophic gastritis is frequently accompanied by the replacement of normal gastric epithelium with intestinal-type epithelium, a condition termed intestinal metaplasia. These histologic abnormalities are more common in the stomachs of H. pylori-infcciQd persons than in noninfected persons, and are thought to occur primarily in the setting of long-standing infection over a period of decades [39]. With the occurrence of gastric atrophy and loss of gastric acid secretory capacity, there is proliferation of other bacterial species in the gastric lumen and the progressive loss of H. pylori. Atrophic gastritis and intestinal metaplasia are risk factors for the development of gastric adenocarcinoma, one of the most frequently lethal
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malignancies worldwide [40]. Thus, analysis of the process leading to these pathologic abnormalities is of great importance.
IV, Clinical Diseases Associated with H. pylori Infection A. Acute Infection At present, we know relatively Httle about the symptoms and tissue reaction that accompany initial colonization of the stomach by H. pylori. Two volunteers who ingested H. pylori each described an acute dyspeptic illness, characterized by nausea and abdominal pain, which lasted for 1 to 2 weeks and then resolved [4, 5]. Similar symptoms occurred in several persons who were inadvertently exposed to H. pylori during the course of medical procedures [41, 42]. Inflammation of the gastric mucosa was detected within about 1 week following ingestion of the organism, and persisted for years after symptoms resolved. The concept that initial H. pylori acquisition is accompanied by a dyspeptic illness is based solely on these case reports involving adults. Whether or not initial acquisition is typically accompanied by symptoms in children is not known. B. Peptic Ulcer Disease A peptic ulcer is a visible defect in the integrity of the mucosal surface of the stomach or duodenum, which often (but not always) leads to abdominal pain, bleeding, or perforation [43]. Nearly all duodenal ulcers occur in a portion of the duodenum known as the duodenal bulb. The majority of gastric ulcers occur in a region close to the junction (transition zone) between antral and corpus mucosae, which is just distal to acid-producing mucosa. Differences in the pathogenesis of gastric and duodenal ulcers have been recognized for a long time. Most notably, duodenal ulcers often are associated with excess gastric acid secretion, whereas most patients with gastric ulcers have either normal or decreased gastric acidity. Over the past 200 years, there have been marked changes in the epidemiology of peptic ulcer disease in Western countries [43-45]. Prior to 1800, peptic ulcers were quite rare, but during the nineteenth century the incidence of gastric ulcers increased markedly, particularly among young women. Later in that century the incidence of duodenal ulcer disease began to increase, and the proportion of gastric ulcers decreased. During the twentieth century, there was a progressive increase in the age for development of peptic ulcers, such that the peak now occurs among patients over 50 years of age. Currendy, the lifetime prevalence of duodenal ulcer in the United States is estimated to be about 10% for men and 4% for women [43]. The epidemiology of peptic ulcer disease is not uniform throughout the world [43]. For example, the incidence of ulcer disease is currendy increasing in some
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parts of the world (e.g., Hong Kong, Singapore, and South Africa) but seems to be decreasing in others (e.g., the United States). Peptic ulcers occur rarely in some populations (e.g., Eskimos in northern Greenland, southwestern Native Americans, and Australian aborigines) even though there is a high prevalence of H. pylori infection. Gastric ulcers (not associated with nonsteroidal antiinflammatory drugs such as aspirin) currendy account for only a small proportion of peptic ulcers in the United States but have been more common than duodenal ulcers in Japan. Throughout the world, peptic ulcers occur more commonly among men than women, but this male-to-female sex ratio varies from about 18:1 in India to about 1:1 in the United States and Australia. These striking geographic and temporal variations in the epidemiology of ulcer disease provide potentially important clues regarding possible environmental factors that may influence ulcer pathogenesis. Prior to studies of H. pylori in the 1980s, the recognized causes of peptic ulceration included ingestion of nonsteroidal antiinflammatory drugs (including aspirin), excessive alcohol consumption, and a rare tumor (gastrinoma) associated with increased gastric acid production. Most ulcers not attributable to these causes were simply termed "idiopathic" ulcers (i.e., of unknown cause). It is this large group of "idiopathic" peptic ulcers that are now known to be associated with H. pylori. Three lines of evidence indicate an important role for //. pylori in the pathogenesis of peptic ulcer disease. First, more than 95% of patients with "idiopathic" duodenal ulcers and more than 80% of patients with gastric ulcers are infected with H. pylori; these prevalences are significantly higher than among patients without ulcer disease [46, 47]. Second, cohort studies have shown that duodenal ulcers develop more frequently in H. pylori-positivt persons than in noninfected persons [48]. Finally, eradication of H. pylori with antimicrobial therapy promotes ulcer healing and results in a markedly decreased rate of ulcer recurrence [7,49-51]. Taken together, these data demonstrate that H. pylori plays an etiologic role in peptic ulcer disease, and is not simply an opportunistic colonizer of damaged tissue. Nevertheless, H. pylori infection per se is not sufficient (or even necessary) for peptic ulceration. Particular interactions between microbe and host, which are largely undefined at present, appear to be critical for the development of ulcer disease.
C.
Gastric Adenocarcinoma
Throughout much of the world, gastric cancer (gastric adenocarcinoma) is currently one of the leading causes of cancer-related deaths [52]. As with peptic ulcer disease, there have been striking changes in the epidemiology of gastric cancer over the past century in many Western countries [53, 54], which may reflect in part the changing epidemiology of H. pylori. During the early part of the twentieth century, gastric cancer was the leading cause of cancer-related
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deaths in the United States, but there has been a steady decline in the incidence of this tumor over the past 60 years. A similar trend has been observed in many European countries. The incidence of gastric cancer currendy varies considerably throughout the world. For example, in Japan, the annual incidence of gastric cancer among men is greater than 70 cases per 100,000 population per year, whereas in the United States the incidence is about sevenfold lower [52]. Longitudinal studies conducted several decades ago established that gastric inflammation is a risk factor for the development of gastric cancer [55-57], and it now is clear that //. pylori is the major cause of such inflammation. Throughout the world, the risk of gastric adenocarcinoma (particularly of the distal stomach) is higher among H. /7y/(9n-positive persons than among H. pylori-ntgdXwt persons [58, 59]. In Europe and in China, there also is an association between the prevalence of H. pylori in a geographic region and the incidence of gastric cancer in that region [60, 61]. However, a high prevalence of H. pylori in a geographic region does not dictate that the rate of gastric cancer will be high. For example, in India and many parts of Africa, there is a high prevalence of//, pylori infection but a relatively low rate of gastric cancer. The progressive decline in the incidence of distal gastric cancer in the United States and many European countries during the twentieth century is often attributed to a decreasing prevalence of //. pylori as well as to a later age of //. pylori acquisition, but other cofactors also are probably involved. In contrast to the overall declining incidence of gastric cancer in many developed countries, in several Western countries there has been an increase over the past few decades in the incidence of cancers located in the cardia (proximal stomach), distal esophagus, and gastroesophageal junction [63]. The reason for an increased occurrence of these cancers is not known, but these tumors are not associated with //. pylori. Some data indicate that the prevalence of //. pylori and incidence of these tumors may be inversely related, suggesting that infection with certain types of //. pylori strains might have a protective effect against proximal gastric cancer [64-66].
D.
Gastric Non-Hodgkins B-Cell Lymphoma
Gastric lymphoma is a relatively rare malignancy in which gastric tumors arise from proliferation of monoclonal B lymphocytes [67]. There is a considerable spectrum of aggressiveness with gastric lymphomas, ranging from indolent localized proliferations of B lymphocytes that infiltrate into the gastric epithelium, to large tumors that invade the gastric wall and metastasize outside the stomach. Low-grade localized gastric lymphomas often are termed MALT (mucosa-associated lymphoid tissue) and are histologically similar to collections of lymphoid tissue known as Peyer's patches, which are found normally in the intestines. //. pylori is present in patients with gastric lymphomas significantly more frequendy
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than in matched controls [67-70]. In one prospective study, the presence of//. pylori increased the risk of developing gastric lymphomas sixfold [69]. Eradication of //. pylori results in regression of gastric MALT lymphomas [71-73] and sometimes also leads to regression of low-grade MALT lymphomas at distant sites [74]. In vitro studies have shown that proliferation of cells derived from these lymphomas is dependent on the presence of H. pylori [75]. Gastric MALT lymphoma is the only known malignancy for which the course can be directly modified by elimination of a microbe. Nevertheless, //. pylori infection clearly leads to lymphoid proliferation in all hosts, and monoclonality per se is not malignancy. Thus, the boundary between benign and malignant proliferation of lymphoid cells needs better definition.
V. Microbiology Helicobacter spp. are small curved Gram-negative bacteria that are classified as epsilon-Proteobacteria. Isolates are characterized biochemically by the presence of urease, catalase, and oxidase activities. Based on analysis of ribosomal RNA sequences, the most closely related organisms are Wolinella succinogenes, Campylobacter spp., and Arcobacter spp. Following the successful culture of //. pylori from humans, many other Helicobacter species have been recovered from various other mammals [76, 77] (Table II). In each mammalian species studied thus far, gastric colonization by Helicobacter spp. is associated with gastric inflammation. //. heilmanii (formerly Gastrospirillum hominis) is a tightly spiraled organism that can colonize the human stomach [26, 27] but is less prevalent than //. pylori. Mixed infections with //. pylori and //. heilmanii may occur, and seem to be especially common in rhesus monkeys. Several Helicobacter species can colonize the intestines or biliary tracts of humans and other mammals, where they cause an inflammatory response [78, 79]. //. hepaticus colonization of the biliary tract of mice is associated with hepatic tumor formation [80]. These various Helicobacter spp. are of considerable interest as models for understanding chronic inflammatory and neoplastic processes, and some of these related organisms potentially may play roles in the pathogenesis of other human diseases. //. pylori colonies become visible after growth on complex media for 2-7 days, depending on the strain and how well adapted it is for growth in vitro. Growth in vitro typically requires atmospheres with increased CO2 and reduced oxygen tension. Defined media for the culture of //. pylori have been described [81] but have not yet been widely used. In gastric biopsy specimens, //. pylori appear as spiral forms, whereas fresh cultures of H. pylori grown in vitro appear predominantly as curved rods. Older cultures contain an increased proportion of coccoid forms, which cannot be subcultured [82]. The mechanisms by which rod-shaped
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Currently Recognized Helicobacter Species
Species H. pylori H. nemestrinae H. bizzozeronii H. acinonyx H. heilmannii H.felis H. mustelae H. salomonis H. siincus H. rappini H. muridarum H. trogontum H. hepaticus H. bills H. cholecystus H. rodentium H. canis H. fennelliae H. cinaedi H. pullorum H. pametensis
Host Primates Macaque Dog, human Cheetah Many mammals Cat, dog Ferret Dog House shrew Many mammals Rodents Rat Mouse Mouse Hamster Mouse Dog Human Rodents, human Chicken Birds
Urease activity + + + + + + + + + + + + + +
-
Major sites Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Intestine, stomach Intestine Intestine Intestine, liver Intestine, liver Gall bladder Intestine Intestine Intestine Intestine Intestine Intestine
H. pylori undergo transition into coccoid forms are not yet well understood. Although there has been continuing speculation that coccoid forms represent dormant bacteria involved in transmission to new human hosts, coccoid forms may simply reflect the morphology of dying or dead organisms [83]. The complete genome sequences of two H. pylori strains (strain 26695 and J99) have been determined [84, 84a], and provide considerable insight into H. pylori biology. These H. pylori genomes consist of circular chromosomes about 1.7 Mb in size, similar to the size of the H. influenzae Rd chromosome, and substantially smaller than that oiE. coli K12. The relatively small size of the H. pylori genome probably reflects a limited metabolic repertoire and biosynthetic capacity, and is consistent with the specialization of //. pylori for growth in a restricted niche (the gastric mucosa). Of the 1590 predicted coding sequences in the H. pylori 26695 genome, 594 lack homology to E. coli or H. influenzae genes [85], and 499 of these lack obvious homology to any sequences in databases at the time of the annotation in 1997. Some of these species-specific genes undoubtedly play an important role in adaptation of H. pylori to the human stomach.
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Analysis of the H. pylori 26695 genome sequence reveals considerably fewer global regulatory proteins than expected, based on an E. coli model. For example, no homologs of the OxyR, SoxR, or SoxS oxidative stress regulators and no homolog of Lex A (SOS DNA damage response regulator) have been found [86]. This may reflect exposure of H. pylori to a smaller number of environments than are encountered by organisms such as E. coli. One interesting feature of the H. pylori genome is that numerous predicted proteins, including urease, are most closely related to corresponding proteins from Gram-positive organisms, Archaea, or eukaryotes, rather than from other Gramnegative organisms [84]. This suggests horizontal gene transfer from disparate phylogenetic groups into the H. pylori lineage during the evolution of this species. Although H. pylori strain 26695 does not contain detectable plasmids, about half of H. pylori strains contain one or more plasmid DNAs, with sizes ranging from 1.5 to >20 kb [87, 88]. No virulence or drug-resistance factors are currently known to be plasmid-bome, but only a few small H. pylori plasmids have been thoroughly studied thus far. A bacteriophage has been detected in one strain of H. pylori [89], but this observation has not yet been generalized to other strains. Although genetic manipulation of H. pylori in vitro is more difficult than manipulation of organisms such as E. coli, techniques have been developed that enable a considerable range of experiments to be performed. Insertional mutagenesis of genes, use of shuttle vectors, use of transcriptional reporters, and site-directed mutagenesis of chromosomal genes via a positive selection method have all been described. However, there is currently no well-developed system for efficient random mutagenesis of chromosomal H. pylori genes.
W. Genetic Diversity and Populotion Structure of H. pylori Early analyses of //. pylori isolates from different patients indicated that nearly every strain is unique at the DNA level [24], and subsequent sequence analyses have confirmed this result. For example, analysis of flaB from 54 different H. pylori isolates yielded 54 different sequences [19]. Alignments of representative orthologous sequences [including ureC (glmM), cysS, floA, and flaB] from different H. pylori isolates indicate levels of nucleotide identity in the range from 95 to 98%, with most nucleotide substitutions occurring at synonymous sites [19, 90, 91]. This level of polymorphism is higher than that observed in similar studies of clinical isolates of E. coli, and similar to that detected in Neisseria meningitidis [19]. Portions of several H. pylori genes, including vacA (encoding a vacuolating cytotoxin) [92-94], hspA (encoding a GroES homolog) [95], vapD [96], cagA [97, 98], and iceA [99], reveal a substantially higher level of genetic diversity among
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strains (60-90% nucleotide identity). Much of the diversity in these genes represents nonsynonymous nucleotide substitutions, which may reflect selection for different structures, functions, or antigenic properties. In addition to the many point mutations that distinguish individual H. pylori strains, large gene clusters are present in some strains but not in others. Early studies showed that about 60% of H. pylori isolates from patients in the United States and Western Europe produce an immunodominant 120-140-kDa protein of unknown function (CagA), whereas the other 40% of strains do not [100, 101]. Subsequent experiments showed that the gene for this antigen is present in strains that express CagA, but absent from strains that do not express CagA [102, 103]. As will be discussed further, cagA is at one end of a ~40-kb DNA segment (termed the cag pathogenicity island) [104, 105], many of whose genes help H. pylori activate proinflammatory signal transduction pathways in gastric epithelial cells [106-108], and thus contribute to an enhanced host inflammatory response. Infection with strains that contain the cag island is more likely to result in overt cHnical disease than is colonization by cag~ strains [100, 101, 103]. Thus, presence or absence of the cag pathogenicity island is an important genetic distinction among H. pylori strains. The cag pathogenicity island has a lower G+C content than that of the entire H. pylori chromosome (35 and 39%, respectively), and contains terminal 31-bp direct repeats [84]. These features suggest that this segment was acquired from an unrelated bacterial species. However, in contrast to several of the best-studied bacterial pathogenicity islands, which tend to be inserted within or adjacent to tRNA genes or other small RNA-related genes, the cag island is located between two protein-encoding genes. Among cag"" strains, the cag pathogenicity island gene content and arrangement are rather well conserved [84, 104, 105]. However, in at least one strain, the cag island has been split into two parts and rearranged due to insertion of a transposable element (IS605) [104, 105]. Many other cag"" strains contain a specific DNA inversion that separates cagA from the adjacent p/cA gene [105]. Additional chromosomal sites of high-level diversity have been identified by comparing a monkey-colonizing H. pylori strain (J 166) with a fully sequenced reference strain (26695), using a PCR-based subtractive hybridization method. This resulted in the identification of 18 different strain-specific DNA fragments, 7 of which seemed to encode DNA restriction-modification enzyme systems [109]. This suggests that restriction-modification systems may vary considerably among different H. pylori strains. Currently, the roles of these enzymes in H. pylori are not well understood, and it is not known why a single strain (e.g., 26695) contains more than 20 different genes encoding putative restriction-modification enzymes [86]. Numerous additional strain-specific genes have been identified by comparing the complete genome sequences of two different H. pylori strains [84a]. There is growing evidence that recombination occurs between different H. pylori strains [91, 92, 110]. Simultaneous colonization of human stomachs with
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more than one strain of H. pylori is detectable in about 5-10% of patients in the United States [25], and may occur more commonly in other populations [111]. Such mixed infections, even if transient, provide an opportunity for genetic exchange between strains. Analysis of single cell clones from a patient who was naturally infected with two different H. pylori strains, one of which was cag^ and the other cag~, revealed evidence for at least six different genetic exchanges [20]. One exchange resulted in replacement of the entire cag island with DNA containing the "empty site allele" from the cag~ strain. Several others involved a region encoding putative outer membrane proteins that could be involved in interactions with the host. These findings demonstrate that recombination indeed occurs in the setting of mixed infections. Genetic exchange may play an important role in the biology of H. pylori by generating new genotypes much more rapidly than is possible by mutation alone, and thereby allowing organisms to rapidly adapt to new sites in the gastric environment or to new hosts. For several genes, including flaA, flaB, and portions of vacA, phylogenetic analyses of orthologous sequences from different strains have yielded a "bush"rather than a "tree"-like pattern [19], indicating considerable interstrain recombination. Further analysis of sequence data indicates that polymorphic sites within each gene locus analyzed are all essentially at linkage equilibrium [19]. Recombination is thought to occur more commonly in H. pylori than in any other bacterial species analyzed thus far [19]. Thus, the high level of allelic variation observed in H. pylori can be attributed to at least two factors. First, large populations of H. pylori have probably evolved within millions of individual human stomachs over hundreds of thousands of years or longer, resulting in considerable mutational diversity. Second, additional diversity has accumulated as a result of extensive intragenic recombination in the setting of mixed infections. As with several other bacterial species with recombinational population structures, including Neisseria spp. and Streptococcus pneumoniae, H. pylori are naturally competent for genetic transformation in vitro [112]. This property indicates that the bacteria can take up exogenous DNA, which can subsequendy replicate (e.g., plasmids) or incorporate into the chromosome by homologous recombination. Two loci that have been implicated in the transformation capacity of H. pylori are comB [113, 114] and dprA [115]. In addition to genetic exchange via transformation, H. pylori strains may exchange DNA via a contact-dependent mechanism [116]. This latter mode of DNA transfer is unidirectional and not eliminated by DNAse treatment, and thus resembles bacterial conjugation. Recombination occurs sufficiently frequently among H. pylori strains to result in linkage equilibrium for most polymorphisms [19], but linkage disequilibrium is evident at several loci. Specifically, strains that possess the cag pathogenicity island nearly always contain type si vacA (vacuolating toxin) alleles [92], and most cag'^ strains adhere to Lewis B antigens on host cells [117]. The relevant genes (cag island, vacA, and babAl) are far apart in the H. pylori chromosome [84], and their products are not thought to interact directly. Therefore, it seems
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likely that these are co-adapted genes, for which either concomitant expression or concomitant lack of expression confers a selective advantage. Although identification of phylogenetic lineages of individual H. pylori strains in a given human population is complicated by recombination, geographic differences in //. pylori gene pools have been detected [118]. Strains that are cag~ and that contain type s2 vacA alleles are found in about one-third of patients from Europe and North America [92] but are quite rare in many Asian populations [119, 120]. When vac A or cagA sequences from Asian and Western strains are compared, the highest levels of relatedness are found among isolates from the same geographic region [93, 94, 119, 120]. This suggests that there may be different selective forces in East Asian and Western populations or, alternatively, that geographic partitioning of H. pylori gene pools reflects characteristics of the H. pylori strains that colonized human ancestors of the current populations. Several genome-mapping studies have concluded that individual H. pylori strains differ markedly in chromosomal gene order [121, 122]. However, a comparison of two completely sequenced H. pylori genomes indicated that the overall genomic organization and gene order were quite similar, with only 10 major inversions or transpositions [84a]. The mechanisms by which rearrangements of H. pylori chromosomes occur are not yet known, but two different species of IS elements and numerous duplicate and divergent genes could be involved. Thus, it is parsimonious to imagine that rearrangements have arisen by transposition or by homologous recombination.
W/. Initial Gastric Colonization The major innate host defenses against microbial colonization of the stomach are gastric acid, peristalsis, and continual shedding of the cells and mucus that line the gastric lumenal surface. Gastric Helicobacter spp. must have developed mechanisms for resisting these defenses (Table III). The lumenal pH of the fasting human stomach is <2, but within the gastric mucus there is a pH gradient that ranges from pH 2 at the luminal surface to nearly neutral pH at the epithelial cell surface [29]. Entry of H, pylori into the gastric mucus layer is presumably an important feature that permits H. pylori to escape extremely low pH.
A.
Motility
Flagella confer motility on H. pylori and enable the organisms to penetrate and colonize the gastric mucus layer. H. pylori typically produce 4-6 unipolar flagella, which are encased in a membranous sheath and capped by terminal bulbs
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Table III Well-Characterized H. pylori Constituents Potentially Important for Initial Colonization or Persistence
Constituent Urease Flagella HspB HspA BabA2 HpaA Porins Hp-NAP Superoxide dismutase Catalase cag pathogenicity island VacA Lewis LPS antigens
Function
Substantial intraspecies variation
Acid resistance Motility Chaperone Chaperone, nickel binding Adhesin Adhesin Transport, proinflammatory Neutrophil adhesin, bacterioferritin, DNA binding Decreased oxidative damage Decreased oxidative damage Cytokine induction in epithelial cells
Alterations in epithelial cell function, monolayer permeability, and antigen processing Molecular mimicry
[123, 124]. The bulbs are believed to be an extension of the sheath, which may protect the flagellar filament from depolymerization in acidic pH [124]. The flagellum is comprised of three main components (filament, hook, and basal body). The flagellar filament is comprised primarily of repeating subunits of two polypeptides: FlaA (the major component) and FlaB (a minor component) [125-128]. Mutation of flaA results in production of short stubby flagellar structures comprised of FlaB, whereas mutation of flaB allows synthesis of FlaA-composed flagellae with normal appearance and function in vitro. The flagellar filament is connected directly to the flagellar hook, which is comprised primarily of one protein, FlgE [129]. The flagellar hook connects the flagellar filament to the basal body, which is a multiprotein structure that serves as the proton motive force-driven motor that propels rotation. Transcription of flaA seems to be driven by a 023 (classic flagellar) promoter 3.nd flaB expression by a 054 (typical nitrogen-regulated) promoter [126-128]; the significance of this difference is not known. Transcription of flagellar genes appears to be regulated by a protein known has FlhA (also known as Fib A) [132], which is predicted to contain both inner membrane and cytoplasmic domains. It
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is thought that FlbA may sense environmental cues and regulate flagellar transcription by interacting with another uncharacterized protein. Flagellar-mediated movement of H. pylori through gastric mucus may be facilitated by a corkscrew bacterial shape [31]. //. pylori are motile in vitro under conditions of elevated viscosity, whereas viscous media retard the movement of many other motile rod-shaped bacteria, including E. coli [31]. /AZ vitro studies have demonstrated H. pylori chemotaxis toward urea and bicarbonate [133]. In early studies, motility was implicated in colonization when a nonmotile variant of an H. pylori strain was used to challenge gnotobiotic piglets. Only 2 of 8 pigs were colonized by the nonmotile isolate, whereas 9 of 10 piglets were colonized by the wild-type isolate [130]. In a subsequent study, strains with null insertion mutations in either/ZflA or flaB colonized piglets only in very low numbers, and infection with these mutant strains was short-lived compared to the wild-type strain [131]. T>o\xh\t flaA-flaB mutants were completely unable to colonize this animal model.
B.
Urease
Urease is an important colonization factor that is produced in abundance by all fresh clinical H. pylori isolates but not required for H. pylori growth in vitro. This enzyme catalyzes the hydrolysis of urea (the primary nitrogenous waste product of humans) to ammonium and CO2 [134]. Urease is a ~550-kDa nickel metalloenzyme comprised of two distinct subunits—UreA (26.5 kDa) and UreB (60.3-61.6 kDa)—which are present at six copies each per enzyme [135-138]. H. pylori urease, like ureases from other bacterial species, is a cytoplasmic enzyme [135]. However, bacterial autolysis may result in the release of urease, which can bind to the surface of viable bacteria or be shed into the gastric mucosa [139, 140]. With a K^ value for urea of 0.17-0.48 mM, H. pylori urease is well suited to physiological gastric urea concentrations (1.7-3.4 mM), and is probably always saturated and working at maximum efficiency [135, 136, 141]. One of the principal actions of urease may be to protect H. pylori from the deleterious effects of gastric acid. One possible mechanism is that ammonia, produced by hydrolysis of urea, is released and directly neutralizes low gastric pH. Urease activity might also contribute to acid resistance by altering bacterial intracytoplasmic pH. Another alternative role of urease activity may be to provide a source of ammonia for nitrogen metabolism in H. pylori [84]. For example, glutamine synthetase catalyzes the conversion of ammonia and glutamate into glutamine, which can be directly incorporated into protein. The role of urease in colonization has been assessed by comparing wild-type strains and defined urease-negative mutants in animal models [142]. In all animal models tested thus far, urease-negative mutants are unable to colonize the gastric mucosa. Colonization with urease-negative strains does not occur, even when the gastric pH of piglets is neutralized by treatment with omeprazole (a proton pump
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inhibitor) [143]. These experiments demonstrate that urease is required for colonization, and suggest that this protein may have another role in addition to neutralizing gastric acidity.
C. Multiple Mechanisms of Acid Resistance Like E. coli and related organisms, H. pylori are thought to possess an intracellular pH near neutrality [144,145]. In vitro experiments indicate that urease activity is essential for survival of//, pylori at extracellular pH <3, whereas mechanisms independent of urease are operative at moderately acidic extracellular pH [146-148]. These may include the generation of a proton motive force across the cytoplasmic membrane, and the generation and transport of bases or acids [84, 144]. The survival of Salmonella species at pH 3 is markedly enhanced if the bacteria have an opportunity to preadapt to moderately acidic pH [149]. This phenomenon, known as the acid-tolerance response, involves increased expression of more than 40 different proteins on exposure to acidic pH. Preliminary studies indicate that H. pylori also is capable of an acid-tolerance response [150]. One //. pylori gene upregulated in response to acid encodes a protein required for LPS 0-antigen biosynthesis [151]. This suggests that acidic pH may stimulate changes in //. pylori LPS assembly or structure, which potentially could increase the acid tolerance of the organism.
D. Effects of H. pylori on Gastric Acid Production In addition to possessing numerous intrinsic adaptations that provide resistance to gastric acidity, //. pylori is able to alter the pH of its gastric environment. Human volunteers experimentally challenged with H. pylori, as well as persons who have inadvertendy ingested //. pylori, experience a reduction in gastric acidity (hypochlorhydria) during the earliest phase of infection [4, 5, 41, 42]. A similar phenomenon is observed in ferrets that are experimentally infected with their cognate gastric Helicobacter, H. mustelae [152]. This hypochlorhydria associated with initial acquisition of //. pylori is typically transient, and normal gastric acidity probably returns in most persons within several months [4, 5, 41, 42, 153]. Reduction in gastric acidity during initial //. pylori acquisition may contribute to establishment of colonization. The mechanisms by which //. pylori transiently decreases gastric acid secretion are not yet known. One hypothesis is that urease may be expressed at high levels during the early phase of infection, and that levels of expression subsequently diminish. H. pylori also may produce specific factors that directly inhibit HCl secretion by gastric parietal cells [154]. Lipopolysaccharide from Pseudomonas aeruginosa and E. coli inhibit gastric acid secretion when administered intrave-
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nously to dogs or rats [155], and, thus, LPS from H. pylori could be one factor that inhibits parietal cell function. A^-a-histamine methyltransferase activity of H. pylori may affect gastric acidity by catalyzing conversion of histamine (an H2 histamine receptor agonist that stimulates gastric acid secretion) to A^-a-methyl histamine (an H3 histamine receptor agonist) [156, 157]. Finally, the inhibitory effects of H. pylori on acid secretion may occur indirectly, via the activities of various inflammatory mediators. Interleukin 1-beta in particular is known to be a potent inhibitor of gastric acid secretion when administered intravenously or intraperitoneally to rats [158]. Several alterations in gastric physiology persist after resolution of the initial hypochlorhydria. In the setting of chronic H. pylori infection, antral concentrations of somatostatin (a hormone that inhibits acid secretion) are decreased, and serum levels of gastrin, a hormone that stimulates acid secretion, are increased compared to noninfected persons [159-162]. Eradication of//, pylori is associated with normalization of levels of both gastrin and somatostatin. Autoantibodies to gastric parietal cells have been detected in mice experimentally infected with //. pylori, and their presence is associated with parietal cell loss [163]. Whether autoimmune phenomena are biologically important in chronically //. pylori-infected humans is not yet clear, but antiparietal cell antibodies [164] could be important in altering gastric acid production. Thus, //. pylori infection seems to affect gastric acid secretion and its regulatory pathways in several different ways.
E.
Adherence
Adherence to cells is a requirement of many pathogens that colonize mucosal surfaces. One probable function is to decrease loss of organisms due to mucus turnover and peristalsis. In vivo, most //. pylori are localized within the gastric mucus layer, but a small fraction of organisms adhere direcdy to gastric epithelial cells [32]. In an in vitro adherence assay involving binding of bacteria to formalin-fixed or frozen tissue, //. pylori bind to surface mucus cells in the upper pit and luminal surface of human stomach tissue, and bind weakly to colonic epithelium, but do not bind to epithelium from esophagus, kidney, cervix, or endometrium [165]. Binding of H. pylori to gastric epithelium in this assay is mediated by fucosylated host cell-surface glycoproteins, corresponding to Lewis B (Le^) and H-1 antigens [166, 167]. These antigens, which were first found on erythrocytes (where they define the Lewis blood group system), are also expressed on epithelial cell surfaces (including the gastric epithelium), as "histo-blood group" antigens. The best-characterized //. pylori adhesin thus far is a 75-kDa protein that binds to the Lewis b histo-blood group antigen on gastric epithelial cells [117]. This adhesin is expressed in low quantities on the surface of//, pylori, and was isolated via a novel procedure termed receptor activity-directed affinity tagging [117].
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Screening a genomic library with a probe corresponding to the N terminus of the 75-kDa adhesin identified several different ORFs {babAl, babA2, and babB), and mutational inactivation experiments identified the functional adhesin as the babAl product. These bab genes belong to a family of about 30 related genes, whose products show extensive amino-acid homology in the amino- and carboxy-terminal domains [84]. It is notable that only a subset of H. pylori strains possess Le^ binding activity. Of 95 clinical strains studied, nearly all of those that bound Le^ were cag+ [117]. However, deletion of the entire cag pathogenicity island did not affect Le^ adhesin activity. This indicates that the presence of the cag pathogenicity island and the capacity to bind Le^ are co-adapted traits, but not mechanistically related. In addition to BabA-Le^ interactions, there may be multiple other //. pylori adhesins and cognate receptors. One of the first of these other putative adhesins to be described was HpaA, a hemagglutinin that binds to sialic acid-containing cell-surface components [168]. Subsequent studies have shown that HpaA is a lipoprotein and that isogenic HpaA-negative H. pylori mutants do not differ from wild-type strains in hemagglutination or adherence to gastric epithelial cells in vitro [169, 170]. This suggests that other adhesins might have overlapping roles. A 53-kDa lipoprotein (AlpA) that mediates H. pylori adhesin to KatoIII cells also has been described [171]. In addition, H. pylori binds to class II major histocompatibility complex molecules on gastric epithelial cells [172]. The possibility that H. pylori adherence to gastric epithelial cells may not be essential for colonization is suggested by comparative analysis of transgenic mice expressing Le^ and nonexpressing wild-type littermates [163]. Both types of mice were experimentally infected with an H. pylori strain that expressed the Le^ adhesin. In the transgenic mice, many bacteria adhered to epithelial cells, whereas in the nontransgenic mice no adherence was detected and bacteria were found only in the mucus layer. Importantly, H. pylori colonized and persisted at comparable microbial densities in both types of mice. Thus, adherence of H. pylori to the Le^ receptor on epithelial cells is not needed for colonization of the mouse gastric mucus layer, at least by this one //. pylori strain [163].
W//. Gastric Inflammotion A.
Mechanisms
In all persons with persistent H. pylori infection, the gastric mucosa contains a proliferation of inflammatory and immune cells, consisting predominantly of lymphocytes, macrophages, and neutrophils [36, 37]. Gastric inflammation also is detected when gnotobiotic piglets, mice, or gerbils are experimentally infected
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with H. pylori [173-175]. The inflammatory response is probably greatest immediately after H. pylori acquisition, and then diminishes [4-6]. The mechanisms by which H. pylori elicit an inflammatory mucosal reaction are not yet well understood [176], but several models merit consideration: (1) direct recruitment of immune and inflammatory cells into the gastric mucosa in response to secreted or released bacterial components, (2) autoimmune-mediated damage via antibodies that are crossreactive between H. pylori components and host tissue, and (3) recruitment of inflammatory cells in response to cytokine expression that is stimulated by bacterial attachment to gastric epithelial cells. H. pylori undergo autolysis during growth in vitro [139], and a similar phenomenon in vivo [140] would provide abundant bacterial components for entry into the gastric mucosa. Urease in particular has been detected within the lamina propria of//, py/on-infected persons, and is proinflammatory [177, 178]. In vitro, urease stimulates chemotaxis of monocytes and neutrophils, and activates mononuclear cells [177-180]. H. pylori porins also possess chemotactic properties [181]. Various H. pylori constituents, including a protein designated Hp-NAP, promote neutrophil-endothelial cell interactions [182-184]. LPS also may be released from //. pylori, perhaps in the form of membrane vesicles, and its immunomodulatory activities are discussed below. Since //. pylori are found predominandy within the gastric mucus layer rather than in tissue, there are probably pathways for uptake of bacterial components by the mucosa, where they incite an inflammatory response. This might entail uptake of //. pylori components across an intact gastric epithelial layer via transcytosis, entry via interruptions in the epithelial layer, or transport of antigens into the mucosa through tight junctions between cells. Various //. /?j/or/-specific antibodies react with gastric mucosal tissue, which suggests that inflammation could result from autoimmune processes [164, 185, 186]. For example, growth in a mouse of a hybridoma secreting antibodies to a component of//, pylori LPS (anti-Lewis^) resulted in histopathologic evidence of gastritis [185]. In contrast, H.felis induced similar levels of lymphocytic gastric inflammation in immunodeficient (SCID) and immunocompetent mice [187], which suggests that humoral autoimmunity is not required for mononuclear cell infiltration of the gastric mucosa. However, the validity of extrapolating from //. /^//i'-infected mice to //. /7_y/6>n-infected humans is not known.
B. Role of the cag Pathogenicity Island A number of cytokines—including IL-lp, IL-2, IL-6, IL-8, IL-10, and TNF-a— are present in higher concentrations in the gastric mucosa of //. /7j/or/-positive persons than of//. /?y/on-negative persons [188, 189]. Several of these cytokines are proinflammatory, and IL-8 in particular has potent chemotactic and stimulatory properties for neutrophils. Levels of IL-8 are significandy higher in the
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gastric mucosa of patients infected with cag"" H. pylori strains than in patients infected with cag' strains [190], a difference that accounts, at least in part, for a more intense neutrophilic mucosal inflammatory response in patients with cag'^ strains compared to those with cag~ strains [101, 190]. The capacity of H. pylori to induce cytokine production has been explored by studying interactions of H. pylori with epithelial cells in vitro [104-108]. Direct contact of viable H. pylori with gastric epithelial cells in vitro results in the synthesis and release of several proinflammatory cytokines, including IL-8, and H. pylori strains containing the cag pathogenicity island are much more efficient in stimulating epithelial IL-8 expression in vitro than are cag~ strains. Adherence of cag'^ H. pylori strains to epithelial cells stimulates a cascade of signalling events that include activation of N F - K B , which leads to induction of IL-8 gene transcription [191-195]. Insertion mutations in many (but not all) genes of the cag island markedly diminish this induction of cytokine production [104-108]. Five proteins encoded by the cag pathogenicity island have substantial homology to components of type IV bacterial secretion systems, including Ptl proteins of Bordetella pertussis (which mediate secretion of pertussis toxin), and Vir proteins of Agrobacterium tumefaciens (which mediate T-DNA transport into plant cells). It is currently thought that the cag pathogenicity island encodes products that secrete an IL-8-inducing factor, but this putative factor is yet to be identified. Genes in the cag pathogenicity island do not have the same conserved contiguous arrangement found in the vir and/?^/ operons, and homologs of many Vir/Ptl genes are not seen in the H. pylori genome. Whether H. pylori homologs of Vir and Ptl have functional roles similar to those of the corresponding proteins in Bordetella and Agrobacterium, and what other H. pylori proteins are involved, is not known.
/X. Interactions of H. pylori witti thie Gastric Epittielium A.
Cytoskeletal Changes and Tyrosine Phosphorylation
Adherence of H. pylori to gastric epithelial cells in vitro can result in several changes in cell architecture, including the formation of adherence pedestals and effacement of microviUi at the site of bacterial attachment [107, 108, 196]. Actin, alpha-actinin, and talin cytoskeletal elements are rearranged direcdy beneath adherent bacteria [107, 108]. These changes seem similar to those caused by adherence of enteropathogenic E. coli (EPEC) to intestinal epithelial cells. However, H. pylori does not possess genes with significant homology to the EPEC genes that mediate these effects.
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Adherence of cag'^ H. pylori to cultured cells is also associated with tyrosine phosphorylation of a 145-kDa protein [107, 108]. This 145-kDa protein has been identified as H. pylori CagA, which is delivered into epithelial cells by the cag type IV secretion system [196a]. The kinase inhibitor staurosporine inhibits IL-8 induction but not tyrosine phosphorylation, which suggests that these represent two distinct pathways [107, 108]. B.
Apoptosis
Integrity of the mucosa in the gastrointestinal tract depends on a balance between production of new cells (proliferation) and cell loss. Apoptosis (programmed cell death) is an important mechanism for limiting unrestricted proliferation of epithelial cells, and many bacterial and viral pathogens can affect this process. The rate of apoptosis is increased in the gastric epithelium of H. pylori-infccied persons, compared with noninfected persons [197]. Apoptosis also is detected when gastric epithelial cells bind H. pylori in vitro [172, 198, 199]. In KatoIII cells (a gastric epithelial line), apoptosis results from binding of H. pylori to class II MHC components on the cell surface [172]. The expression of class II MHC components, in turn, is upregulated by adherence of H. pylori [200]. That there may be multiple pathways by which H. pylori induces apoptosis is indicated by the finding that H. pylori induces apoptosis in AGS cells (a gastric epithelial cell line that does not express class II MHC components) [201]. The rate of gastric epithelial cell proliferation is found to be higher in H. pylori-inftcied persons than in noninfected persons in some [202] but not all studies [203]. These seemingly conflicting reports might reflect important differences, such as presence or absence of the cag pathogenicity island, among H. pylori strains. In one study, cagA'^ strains were associated with higher rates of epithelial proliferation and lower rates of apoptosis than cagA' strains [204]. Both the rates of cell division and apoptosis may be modified by the complex cytokine network. For example, increased inflammation might induce enhanced cell proliferation. These effects of H. pylori on epithelial proliferation and apoptosis may be relevant to the development of gastric neoplasms that arise during the course of long-standing H. pylori infection.
X. Vacuolating Cytotoxin A.
Structure
Among H. pylori isolates from patients in the United States and Western Europe, about half produce detectable vacuolating toxic activity in a HeLa cell assay [205] (Fig. 4). This effect is mediated by a ~90-kDa secreted protein (VacA) [206-208]. Sequence analyses of the vacA gene revealed an ORF that encodes a predicted
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Fig. 4 Vacuolation of HeLa cells induced by the H. pylori vacuolating toxin (VacA). HeLa cells were incubated with H. pylori culture supernatant containing VacA, and then stained with crystal violet. Prominent intracellular vacuoles form in response to VacA.
product 140 kDa in size [209-211]. Proteolytic cleavage of the VacA protoxin at its N and C termini yields a 33-aa signal sequence, a mature 90-kDa secreted protein, and a C-terminal fragment that remains localized in the bacterial cell [209-211]. VacA lacks extensive homology with other known bacterial toxins, but its processing and secretion resemble those of the IgA protease family of secreted proteins [209,211]. Purified VacA migrates as a ~90-kDa protein under denaturing conditions, but in its nondenatured state it exists as a large six- or seven-sided complex, comprised of 12 or 14 identical 90-kDa subunits [212, 213]. During prolonged storage, the 90-kDa monomers undergo specific cleavage into 34- and 58-kDa fragments, which remain physically associated in an oligomeric complex [210213]. These two fragments may represent distinct domains or subunits of VacA.
B.
Allelic Variation in vacA
Most H. pylori strains that lack vacuolating activity for HeLa cells in vitro nevertheless produce and secrete an immunoreactive vacA product, albeit in lesser
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amounts than tox+ strains [92, 214]. These apparently tox~ strains also commonly elicit anti-VacA antibody responses in vivo [215]. Decreased production and secretion of VacA by tox~ strains seems to be attributable in part to either decreased vacA transcription or decreased stability of vacA transcripts in tox~ strains [92, 214]. In addition, there are potentially important differences between the vacA sequences of tox^ strains and tox~ strains [92]. Two major families of alleles (designated si and s2) are distinguished by differences in vacA signal sequences [92]. Essentially all isolates with type s2 vacA signal sequences fail to produce detectable vacuolation of HeLa cells. In addition, two families of alleles (designated ml and m2) are distinguished by striking sequence differences in the mid-portion of vacA [92]. Strains with type ml alleles typically produce more prominent vacuolation of HeLa cells than strains with type m2 alleles. Some strains with type m2 alleles produce vacuolation of RK-13 cells but not HeLa cells, which suggests that variation in VacA sequences may be related to cell-type specificity [216].
C. Mechanism of Action Purified VacA induces cell vacuolation when microinjected into HeLa cells, and, similarly, HeLa cells transfected with plasmids containing the vacA gene develop intracellular vacuoles [217]. These results provide strong evidence that vacuole formation is the consequence of an interaction between VacA and an intracellular target. In general, bacterial toxins that interact with intracellular targets are thought to act via a series of events: binding to the plasma membrane, internalization and translocation into the cytoplasm, and enzymatic modification of the intracellular target [218]. This framework provides a useful approach for understanding VacA action. Binding of VacA to cells probably involves interaction with a specific cell-surface receptor, as well as with an assortment of negatively charged lipids [219-221]. Probably both types of binding interactions are mediated primarily by amino-acid sequences within the domain of VacA corresponding to its 58-kDa proteolytic fragment [222]. How VacA gains access to the cytoplasm of cells is not yet known. VacA can insert into membrane vesicles, which suggests that toxin translocation might occur direcdy across the plasma membrane [221]. However, VacA is internalized slowly into vesicular compartments at 37°C [222], which suggests entry by receptor-mediated endocytosis. Nearly all bacterial toxins that act intracellularly do so by enzymatically modifying intracellular target molecules. No enzymatic activity has yet been identified for VacA. Interestingly, transfection of HeLa cells with plasmids encoding either of the two putative VacA subunits (34- and 58-kDa fragments) fails to induce cell vacuolation [217, 223], which suggests that a large portion of the 90 kDa VacA molecule is required for any putative enzymatic function.
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The activity of VacA is increased markedly by exposure of the protein to acidic pH before addition to cells [224]. Acid activation is associated with conformational changes, including disassembly of the dodecameric VacA structure into monomeric subunits [213] and increased exposure of hydrophobic domains [225]. Acidification of VacA enhances its insertion into lipid membranes [225], and is associated with the formation of ion-conductive channels in lipid bilayers [220, 221, 226]. These phenomena may help to explain why low pH increases the toxin's vacuolating activity. The effects of low pH on VacA activity may be particularly relevant in the acidic gastric environment, where secreted VacA could be activated before interacting with epithelial cells. The cell vacuoles induced by VacA become visible within 2 hours after addition of high concentrations of the toxin to cells in vitro. The vacuole membranes contain both the small GTP-binding protein Rab7 (a late endosomal marker) and the membrane glycoprotein LgpllO (a lysosomal marker), which suggests that the vacuoles represent postendosomal hybrid compartments [227, 228]. HeLa cells overexpressing dominant negative Rab7 mutants do not develop vacuoles on exposure to VacA, indicating that functional Rab7 is required for VacA-induced vacuolation [229]. Accordingly, VacA may disrupt normal membrane trafficking at or near the level of late endosomes [230], perhaps by modifying a cellular constituent that normally regulates membrane trafficking within the endocytic pathway [231].
D. Role of VacA in Vivo As a general rule, protein toxins produced by bacterial pathogens may facilitate colonization of the host, enhance transmission to new hosts, or cause damage to host tissue. In one study, the rate of colonization of gerbils by H. pylori vacA null mutants was slighdy lower than by wild-type strains [232], but both types of strains persisted for several weeks in this model. In contrast, no differences between mutant and wild-type strains were detectable in short-term colonization of gnotobiotic piglets [233]. These data suggest that VacA plays a relatively minor role in early colonization events. Nevertheless, immunization of mice with VacA provides effective protective immunity against subsequent experimental challenges with H. pylori [174]. It seems more likely that VacA may play a role in promoting persistence of H. pylori in the gastric mucosa. VacA interferes with the processing involved in antigen presentation in vitro [234], which suggests that one action of this toxin may be to enable H. pylori to resist clearance by the host immune system. VacA also selectively increases the permeability of polarized epithelial cell monolayers for small molecules with molecular mass <400 daltons [235], which is proposed to increase the release of certain nutrients into the mucus layer.
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XL Persistence o/H. pylori Infection Aremarkable characteristic of//, pylori is its capacity to persist in the human stomach for decades. Several features of the gastric environment, including gastric acidity and peristalsis, should operate to restrict bacterial growth and persistent bacterial colonization, were H. pylori not so well adapted. Thus, bacterial motility within the mucus layer and urease activity are probably required for persistence, as well as for initial colonization. Mathematical modeling suggests that persistent colonization necessitates the maintenance of both nonadherent and adherent populations of organisms [236], and, therefore, the ability of bacteria to attach to cells (and perhaps also the capacity of progeny bacteria to detach) may be important. Restricted nutrient availability is an effective force for limiting bacterial proliferation, and the gastric mucus layer potentially contains only a limited supply of nutrients that can be used by //. pylori. Nutrient availability may be increased by specific bacterial interactions with the host [237], For example, adherence to epithelial cells indirectly promotes inflammation, and inflammation may in turn lead to host cell damage and increased release of nutrients into the mucus layer. Thus, the gastric inflammation that accompanies //. pylori infection may simultaneously represent a normal (but futile) response of the host to eliminate bacteria, and a process that renders the gastric mucus layer more hospitable for H. pylori by increasing available nutrients. A. Resistance to Clearance by Host Immune Defenses One of the most intriguing aspects of//, pylori persistence is the capacity of these bacteria to resist clearance by the host immune system. //. pylori colonization of humans and experimentally challenged primates may sometimes be transient [4, 175], which perhaps reflects effective host responses. However, it is clear that //. pylori, once established, frequently persist for decades in the stomach despite local and systemic humoral immune responses and recruitment of inflammatory cells into the gastric mucosa. Several explanations have been proposed to account for the resistance of H. pylori to host humoral immune defenses: (1) secretory IgA antibodies may not enter gastric mucus very efficiendy, making this environment an immunologically protected niche; (2) antibodies may function inefficiently in the low pH of gastric mucus; (3) //. pylori autolysis may release soluble bacterial proteins, which either protectively coat viable bacteria or serve as decoys to divert immune responses away from important target antigens on the bacterial surface; and (4) //. pylori may express surface antigens that are closely related to those present on host tissue (molecular mimicry). In //. /7_y/6>n-infected persons, polymorphonuclear neutrophils and mononuclear cells are found predominantly in the lamina propria rather than in the gastric mucus layer. Limited contact between these cells and //. pylori may be another factor that helps to explain why //. pylori are not eradicated. //. pylori also produce factors that impair phagocytic activity [238] and possess enzymes such
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as catalase and superoxide dismutase [239, 240], which may confer resistance to killing by phagocytes. The differentiation of CD4"^ T cells into Thl- or Th2-type cells is an important determinant of whether cell-mediated or humoral immunity will predominate. Optimal clearance of H. pylori should require an efficient secretory IgA response, which is dependent on recruitment of helper T cells belonging to the Th2 subset. In contrast, H. pylori infection is associated with mucosal recruitment of predominantly Thl-type T cells [241, 242], which are best suited for boosting cell-mediated immunity rather than secretory immune responses to extracellular pathogens. How H. pylori infection preferentially elicits a Thl-type response is not yet understood, but this phenomenon may help to explain persistence of the organism. The expression of a broad repertoire of surface antigens by H. pylori may represent another important mechanism for evading host defenses. Analysis of the H. pylori 26695 genome reveals genes encoding at least 32 putative outer membrane proteins, many of which are closely related [84]. Recombination between these genes could potentially generate proteins with new amino-acid sequences and novel antigenic properties, perhaps analogous to the antigenic diversity seen in Neisseria gonorrhoeae, Borrelia spp., Campylobacter fetus, and Mycoplasma genitalium. In addition to antigenic diversity arising via recombination, on/off (phase variation) mechanisms may be operative. Several genes encoding putative surface structures contain homopolymeric tracts or dinucleotide repeats, which tend to change in number as a result of mutations arising via slipped-strand mispairing [84, 243, 244]. This translational regulation of outer membrane protein expression would contribute to phase variation in bacterial surface components, and thus permit evasion of immune responses directed specifically against any of these antigens.
B.
Characteristics of H, pylori Lipopolysaccharide
The important role of LPS from organisms such as E. coli and Salmonella in inducing inflammation (and ultimately bacterial clearance from host tissue) has led to interest in analyzing the LPS structure of H. pylori. Gram-negative LPS typically consists of an innermost portion (lipid A) embedded in the outer membrane, an adjacent core oligosaccharide, and an external surface-exposed polysaccharide composed of repeating oligosaccharide units (0-antigen). H. pylori LPS has a general structural organization similar to that of LPS from Enterobacteriaceae, but has several unique characteristics [245, 246]. In comparison with E. coli lipid A, H. pylori lipid A is mono- rather than diphosphorylated, has six rather than four fatty-acid sidechains, and has longer fatty-acid sidechains (16 to 18 carbons instead of 14) [247-250]. In E. coli. Salmonella, and related genera of the family Enterobacteriaceae, lipid A has potent endotoxic and immunomodulatory properties, including the capacity to induce fever (pyrogenicity) and activate macrophages. H. pylori LPS has from
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500- to 30,000-fold less endotoxic activity than either E. coli or Salmonella LPS [251-254], depending on the strain and the method of assay. The differences in lipid A structure may be responsible for the low toxicity of //. pylori LPS, and may help to explain the relatively mild mucosal inflammatory responses that accompany H. pylori infection (compared to infections with organisms such as Salmonella), which in turn could contribute to persistence. Consistent with this view, the LPS of Bacteroides species, which also persistently colonize the human gastrointestinal tract, has very low endotoxic activity. The number of 0-antigen structures is much more limited in //. pylori than in most genera of Enterobacteriaceae. Most //. pylori strains express either the fucosylated trisaccharide Lewis^, or its related fucosylated tetrasaccharide Lewis^ [245-256]. Three genes encoding fucosyl transferases, which catalyze production of these antigens, have been identified [84]. Polymeric cytosine tracts are present near the 5' ends of these genes, which suggests the potential for DNA slippage and on/off phase variation [244]. Other 0-antigen components include the mono-fucosylated H antigen, and the nonfucosylated backbone (i). The full repertoire of 0-antigens has not yet been established, but within a single clonal population of H. pylori cells there are often subclones that express more than one of these structures. In addition to being present as components of//, pylori LPS, Lewis x and Lewis y antigens are present on host tissue, including gastric epithelial cells [257]. The presence of these antigens on the surface of //. pylori may be a form of camouflage or molecular mimicry that serves to diminish host immune responses and thereby facilitates bacterial persistence, because of host tolerance to these "self antigens. There is evidence in some (but not all) studies that the predominant Lewis expression of the //. pylori population and of the particular colonized host are related [258, 259].
C. Bacteria-Host Equilibrium In acute bacterial infections, the intensity of inflammation typically increases progressively until pathogenic bacteria are eradicated, or until bacterial proliferation leads to destruction of tissue and/or death of the host. In the case of //. pylori, persistent infection must involve an equilibrium in which //. pylori persist in the stomach without any overall change in bacterial population size [236], and gastric inflammation neither progresses nor resolves. It seems possible that over hundreds of thousands of years there may have been selection for human hosts that can control //. pylori proliferation without extensive accompanying gastric inflammation and damage, as well as for //. pylori strains that elicit relatively mild inflammation [260]. Thus, comparisons can be drawn between persistent colonization of the human stomach by //. pylori and colonization of the intestine by commensal organisms.
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XIL Factors Influencing Development of Clinically Evident Disease In most H. pylori-mfQciod persons, colonization of the gastric mucosa and associated inflammation are tolerated for decades without causing any symptoms. Why serious gastroduodenal illnesses occur in a subset of infected persons, in an apparendy unpredictable or sporadic fashion, is not yet understood. However, it is currendy thought that characteristics of individual H. pylori strains, characteristics of individual human hosts, and environmental factors are each important determinants of clinical outcome [261].
A. Bacterial Factors
Due to extensive allelic polymorphism and frequent genetic recombination, each H. pylori strain has unique characterisdcs. Therefore, a spectrum of clinical outcomes can be reasonably attributed to the diversity that exists among H. pylori strains. To identify markers for strains associated with adverse clinical outcomes, investigators have compared H. pylori strains isolated from padents with ulcer disease with strains isolated from asymptomatic padents. Four bacterial markers that predict clinical outcomes have been idendfied thus far: the cag pathogenicity island [100, 101, 104, 262], type si vacA signal sequences [92, 262], the Le^ binding phenotype [117], and type iceAl alleles [99, 262]. The former three markers are frequently found together, probably as coadapted traits, in H. pylori strains associated with an increased risk for pepdc ulcer disease. Strains containing the cag island also have been associated with an increased risk for distal gastric adenocarcinoma [263-265]. iceAl alleles seem to constitute an independent marker for ulcerogenic strains [99, 262]. These markers have proven somewhat useful for predicting clinical outcome in the United States and Western Europe, but have limited udlity in many parts of Asia, where nearly all H. pylori isolates are cag'^ and possess type si vacA alleles [266]. Moreover, it is clear that only a minority of persons infected with putatively "ulcerogenic strains" (i.e., cag'^, vacA type si, type iceAl, with Le^-binding properties) ever develop ulcer disease or gastric cancer. Thus, these genedc markers provide important insights into the pathogenesis of H. pylori-sissociated ulcer disease and gastric cancer, but do not endrely account for differences in clinical outcome.
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Host Factors
Considerable diversity exists in the human population, and host differences are likely to influence clinical outcome. Consistent with this hypothesis is the epidemiologic observation that peptic ulcer disease and gastric cancer occur significandy more frequently in males than in females [43]. The increased incidence of these diseases in males is not attributable to a higher prevalence of H. pylori infection and is unlikely to result from gender-related differences in H. pylori strains. Clustering of pepdc ulcer disease in families occurs [43, 267], and could be due to either host-related genetic predispositions or to transmission of ulcerogenic strains within family groups. Higher rates of concordance for pepdc ulcer disease between monozygodc twins than between dizygodc twins suggest that host factors are indeed relevant [43]. Geographic variations in the incidence of ulcer disease and gastric cancer also may reflect host differences [43], but it is difficult to control for differences in H. pylori strains or environmental exposures in epidemiologic surveys. Of particular relevance to the clinical outcomes of H. pylori infection are host variables that affect bacterial growth or localization in the gastric mucosa. Levels of gastric acid production clearly vary among humans. High levels of gastric acidity are associated with growth of metaplastic gastric tissue in the duodenum, which provides a site for H. pylori colonization [33-35]. The risk of duodenal ulceradon is increased in padents whose duodenal mucosa is colonized by H. pylori compared to H. /?_y/on-infected patients without duodenal colonizadon [33-35]. Thus, by promoting growth of//, pylori in the duodenum, high levels of gastric acid producdon may predispose to duodenal ulcer disease. Low levels of gastric acid producdon are associated with increased //. pylori growth in the gastric corpus, which could be an important initiating step in the pathogenesis of gastric ulcers. The risk of gastric cancer is significandy increased in patients who have a history of gastric ulcers compared to patients without any history of pepdc ulcer disease, whereas the risk of gastric cancer is significantly decreased in padents with a history of duodenal ulcers [268]. This suggests that duodenal ulcer disease and gastric ulcer disease/gastric atrophy/gastric cancer represent two disdnct pathways in //. pylori-'mftcitd persons. Potentially these two pathways are determined by underlying host characteristics, such as high and low gastric acid producdon, respecdvely. An alternate hypothesis is that the time of life when //. pylori is acquired (early in childhood or later) is an important determinant of clinical outcome [269]. The latter model holds that early childhood infection predisposes to development of atrophic gastritis, gastric ulcers, and distal gastric cancer in adult years, and that acquisition of //. pylori later in life is associated with development of duodenal ulcers. Humans differ in expression of histo-blood group andgens, one of which (Le^) is a host cell receptor for H. pylori adherence. Presence or absence of Le^ may influence the extent to which //. pylori adhere to gastric epithelium. Studies in
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mice indicate that bacterial adherence to Le^ receptors is associated with the development of parietal cell autoantibodies and parietal cell loss [163], which would lead to reduced gastric acid production. Thus, an increased proportion of adherent H. pylori may also result in enhanced autoimmune-related phenomena or enhanced inflammatory responses in humans. Immune responses to bacteria can vary considerably among individuals. Immune responses to //. pylori seem to be generally ineffective in eradicating the organisms, but the intensity or nature of the immune response may influence growth or localization of H. pylori. For example, vigorous immune responses might be very effective in controlling bacterial growth, but may have undesired consequences related to tissue injury. Conversely, ineffective immune responses might lead to gastric mucosal damage due to excessive bacterial growth. C. Environmental Factors Environmental exposures probably play an important role in modulating clinical outcomes of H. pylori infection. In particular, the striking temporal changes in rates of ulcer disease and gastric cancer in many Western countries over the past two centuries are most easily explained by changing environmental conditions, rather than changes in bacterial or host characteristics. Determining which environmental factors are most relevant is a difficult task, but several factors are currently known to influence clinical outcome. Cigarette smoking and nonsteroidal antiinflammatory drugs are two well-documented risk factors for development of peptic ulcer disease. The invention of automated methods for manufacturing cigarettes in the late 1800s and the associated increases in smoking throughout the United States and Europe may have contributed to the high rates of ulcer disease earlier in this century. Dietary factors also may be relevant. In particular, gastric cancer has been associated with high salt intake and with low consumption of fruits and vegetables [54]. D. Perspectives on H, pj/ori-Related Diseases At least 20 different Helicobacter species are currently known, and various species colonize the gastrointestinal tracts of many different mammals, including several different primate species. Thus, it seems likely that individual Helicobacter species have evolved within the alimentary tracts of different mammals over a very long time period, and, in particular, //. pylori have probably colonized human stomachs for hundreds of thousands of years or longer [260, 261]. Currently, about 60% of the world's human population is infected with H. pylori, and this proportion is probably considerably lower than in previous centuries. Thus, even though the presence of H. pylori is a risk factor for potentially fatal diseases such as peptic ulcers and distal gastric cancer, the human species has proliferated quite successfully. One relevant factor may be that mortality due to peptic ulcer disease and gastric cancer occurs primarily in old age, and, therefore.
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these illnesses do not substantially alter survival of children or reproductive-age adults [261]. In addition, it is quite possible that mortality rates from H. pylori-rt\2iitd diseases have been higher during the twentieth century than during most previous periods. During the last decades of the twentieth century, there has been a gradual decrease in the prevalence of H. pylori in many developed countries, and this trend will probably continue worldwide as sanitation and hygiene continue to improve and as family sizes shrink. Thus, we are in the midst of a long-term natural experiment that will help to clarify the role of H. pylori in human health and disease. The continuing loss of//, pylori from much of the human population should lead to commensurate declines in peptic ulcer disease and distal gastric cancer. The possibility of other less fortunate consequences, such as an increased rate of proximal gastric cancer and esophageal adenocarcinoma [63-66], must also be followed closely. Therefore, at present it seems prudent to recognize that //. pylori and humans have shared a long evolutionary history, and that our current understanding of "disease" in the context of this ancient relationship is quite limited.
Acknowledgments Research on H. pylori in our laboratories is supported by grants from the National Institutes of Health (ROl AI39657, DK 53623, DK 53727, AI 38166, DK 53707, and AI 25567) and the Medical Research Service of the Department of Veterans Affairs.
References 1. Warren, J. R. (1983). Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i, 1273. 2. Marshall, B. J. (1983). Unidentified curved bacillus on gastric epithelium in active chronic gastritis. L««c^r i, 1273-1275. 3. Marshall, B. J., and Warren, J. R. (1984). Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet i, 1311-1313. 4. Marshall, B. J., Armstrong, J. A., McGechie, D. B., and Glancy, R. J. (1985). Attempt to fulfil Koch's postulates for pyloric Campylobacter. Med. J. Aust. 142, 436-439. 5. Morris, A., and Nicholson, G. (1987). Ingestion of Campylobacter pyloridis causes gastritis and raised fasting gastric pH. Am. J. Gastroenterol. 82, 192-199. 6. Morris, A. J., Ali, M. R., Nicholson, G. I., Perez-Perez, G. I., and Blaser, M. J. (1991). Long-term follow-up of voluntary ingestion of Helicobacter pylori. Ann. Intern. Med. 114, 662-663. 7. NIH Consensus Development Panel on Helicobacter pylori in Peptic Ulcer Disease (1994). Helicobacter pylori in peptic ulcer disease. JAMA 272, 65-69.
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210. Telford, J. L., Ghiara, P., Dell'Orco, M., Comanducci, M., Burroni, D., Bugnoli, M., Tecce, M. R, Censini, S., Covacci, A., Xiang, Z., Papini, E., Montecucco, C , Parente, L., and Rappuoli, R. (1994). Gene structure of the Helicobacter pylori cytotoxin and evidence of its key role in gastric disease. J. Exp. Med. 179, 1653-1658. 211. Schmitt W., and Haas R. (1994). Genetic analysis of the Helicobacter pylori vacuolating cytotoxin: Structural similarities with the IgA protease type of exported protein. Mol. Microbiol. 12,307-319. 212. Lupetti, P., Heuser, J. E., Manetti, R., Massari, P., Lanzavecchia, S., Bellon, P. L., Dallai, R., Rappuoli, R., and Telford, J. L. (1996). Oligomeric and subunit structure of the Helicobacter pylori vacuolating cytotoxin. / Cell Biol. 133, 801-807. 213. Cover, T. L., Hanson, P. I., and Heuser, J. E. (1997). Acid-induced dissociation of VacA, the Helicobacter pylori vacuolating cytotoxin, reveals its pattern of assembly. J. Cell Biol. 138, 759-769. 214. Forsyth, M. H., Atherton, J. C., Blaser, M. J., and Cover, T. L. (1998). Heterogeneity in levels of vacuolating cytotoxin gene (vacA) transcription among Helicobacter pylori strains. Infect. Immun. 66, 3088-3094. 215. Perez-Perez, G. I., Peek Jr., R. M., Atherton, J. C , Blaser, M. J., and Cover, T L. (1999). Detection of anti-VacA antibody responses in serum and gastric juice samples using type si/ml and s2/m2 Helicobacter pylori VacA antigens. Clin. Diag. Lab. Immunol. 64, 489-493. 216. Pagliaccia, C , de Bernard, M., Lupetti, P., Ji, X., Burroni, D., Cover, T. L., Papini, E., Rappuoli, R., Telford, J. L., and Reyrat, J-M. (1998). The m2 form of the Helicobacter pylori cytotoxin has cell type-specific vacuolating activity. Proc. Natl. Acad. Sci. U.S.A. 95, 10212-10217. 217. de Bernard, M., Arico, B., Papini, E., Rizzuto, R., Grandi, G., Rappuoli, R., and Montecucco, C. (1997). Helicobacter pylori toxin VacA induces vacuole formation by acting in the cell cytosol, Mol. Microbiol. 26, 665-674. 218. Montecucco, C , Papini, E., and Schiavo, G. (1994). Bacterial protein toxins penetrate cells via a four-step mechanism. FEBS Lett. 346, 92-98. 219. Massari, P, Manetti, R., Burroni, D., Nuti, S., Norais, N., Rappuoli, R., and Telford, J. L. (1998). Binding of the Helicobacter pylori vacuolating cytotoxin to target cells. Infect. Immun. 66, 3981-3984. 220. Czajkowsky, D. M., Iwamoto, H., Cover, T. L., and Shao, Z. (1999). The vacuolating toxin from Helicobacter pylori forms hexameric pores in lipid bilayers at low pH. Proc. Natl. Acad. Sci. U.S.A. 96, 2001-2006. 221. Moll, G., Papini, E., Colonna, R., Burroni, D., Telford, J., Rappuoli, R., and Montecucco, C. (1995). Lipid interaction of the 37-kDa and 58-kDa fragments of the Helicobacter pylori cytotoxin. Eun J. Biochem. 234, 947-952. 222. Gamer, J. A., and Cover, T. L. (1996). Binding and internalization of Helicobacter pylori cytotoxin by epithelial cells. Infect. Immun. 64, 4197-4203. 223. de Bernard, M., Burroni, D., Papini, E., Rappuoli, R., Telford, J., and Montecucco, C. (1998). Identification of the Helicobacter pylori VacA toxin domain active in the cell cytosol. Infect. Immun. 66, 6014-6016. 224. de Bernard, M., Papini, E., de Filippis, V, Gottardi, E., Telford, J., Manetti, R., Fontana, A., Rappuoli, R., and Montecucco, C, (1995). Low pH activates the vacuolating toxin of Helicobacter pylori, which becomes acid and pepsin resistant. J. Biol. Chem. 270, 23937-23940. 225. Molinari, M., Galli, C , de Bernard, M., Norais, N., Ruysschaert, J.-M., Rappuoli, R., and Montecucco, C. (1998). The acid activation oi Helicobacter pylori toxin VacA: Structural and membrane binding studies. Biochem. Biophys. Res. Commun. 248, 334-340.
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226. Tombola, F, Carlesso, C , Szabo, I., de Bernard, M., Reyrat, J. M., Telford, J. L., Rappuoli, R., Montecucco, C , Papini, E., and Zoratti, M. (1999). Helicobacter pylori vacuolating toxin forms anion-selective channels in planar lipid bilayers: Possible implications for the mechanism of cellular vacuolation. Biophys. J. 76, 1401-1409. 227. Papini, E., deBernard, M., Milia, E., Bugnoli, M., Zerial, M., Rappuoli, R., and Montecucco, C. (1994). Cellular vacuoles induced by Helicobacter pylori originate from late endosomal compartments. Proc. Natl. Acad. Sci. U.S.A. 91, 9720-9724. 228. Molinari, M., Galli, C , Norais, N., Telford, J. L., Rappuoli, R., Luzio, J. P., and Montecucco, C. (1997). Vacuoles induced by Helicobacter pylori toxin contain both late endosomal and lysosomal markers. J. Biol. Chem. 212, 25339-25344. 229. Papini, E., Satin, B., Bucci, C , de Bernard, M., Telford, J. L.. Manetti, R., Rappuoli, R., Zerial, M., and Montecucco, C. (1997). The small GTP binding protein rab7 is essential for cellular vacuolation induced by Helicobacter pylori cytotoxin. EMBO J. 16, 15-24. 230. Satin, B., Norais, N., Telford, J., Rappuoli, R., Murgia, M., Montecucco, C , and Papini, E. (1997). Effect oi Helicobacter pylori vacuolating toxin on maturation and extracellular release of procathepsin D and on epidermal growth factor degradation. J. Biol. Chem. 272, 2502225028. 231. Montecucco, C , Papini, E., and Schiavo, G. (1996). Bacterial protein toxins and cell vesicle trafficking. Experientia 52, 1026-1032. 232. Wirth, H.-P, Beins, M. H., Yang, M., Tham, K. T, and Blaser, M. J. (1998). Experimental infection of Mongolian gerbils with wild-type and mutant Helicobacter pylori strains. Infect. Immun. 66, 4856-4866. 233. Eaton, K. A., Cover, T L., Tummuru, M. K. R., Blaser, M. J., and Krakowka, S. (1997). Role of vacuolating cytotoxin in gastritis due to Helicobacter pylori in gnotobiotic piglets. Infect. Immun. 65, 3462-3464. 234. Molinari, M., Salio, M., Galli, C, Norais, N., Rappuoli, R., Lanzavecchia, A., and Montecucco, C. (1998). Selective inhibition of li-dependent antigen presentation by Helicobacter pylori toxin Vac A. J. Exp. Med. 187, 135-140. 235. Papini, E., Satin, B., Norais, N., de Bernard, M., Telford, J. L., Rappuoli, R., and Montecucco, C. (1998). Selective increase of the permeability of polarized epithelial cell monolayers by Helicobacter pylori vacuolating toxin. J. Clin. Invest. 102, 813-820. 236. Kirschner, D. E., and Blaser, M. J. (1995). The dynamics of Helicobacter pylori infection of the human stomach. / Theor Biol. 176, 281-290. 237. Blaser, M. J. (1992). Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation. Gastroenterology 102, 720-727. 238. Knipp, U., Birkholz, S., Kaup, W., and Opferkuch, W. (1993). Immune suppressive effects of Helicobacter pylori on human peripheral blood mononuclear cells. Med. Microbiol. Immunol. 182, 63-76. 239. Hazell, S. L., Evans, D. J., and Graham, D. Y. (1991). Helicobacter pylori catalase. J. Gen. Microbiol. 137,57-61. 240. Spiegelhalder, C , Gerstenecker, B., Kersten, A., Schiltz, E., and Kist, M. (1993). Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene. Infect. Immun. 61,5315-5325. 241. Bamford, K. B., Fan, X., Crowe, S. E., Leary, J. F, Gourley, W. K., Luthra, G. K., Brooks, E. G., Graham, D. Y, Reyes, V. E., and Ernst, P. B. (1998). Lymphocytes in the human gastric mucosa during Helicobacter pylori have a T helper cell 1 phenotype. Gastroenterology 114, 482-492.
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242. Sommer, F., Faller, G., Konturek, P., Kirchner, T, Hahn, E. G., Zeus, J., Rollinghoff, M., and Lohoff, M. (1998). Antrum- and corpus mucosa-infiltrating CD4^ lymphocytes in Helicobacter pylori gastritis display a Thl phenotype. Infect. Immiin. 66, 5543-5546. 243. Saunders, N. J., Peden, J. F, Hood, D. W., and Moxon, E. R. (1998). Simple sequence repeats in the Helicobacter pylori genome. Mol. Microbiol. 27, 1091-1098. 244. Appelmik, B. J., Shiberu, B., Trinks, C , Tapsi, N., Zheng, P Y., Verboom, T., Maaskant, J., Hokke, Ch., Schiphorst, W. E., Blanchard, D., Simoons-Smit, I. M., van den Eijnden, D. H., and Vandenbrouche-Grauls, C. M. (1998). Phase variation in Helicobacter pylori lipopolysaccharide. Infect. Immun. 66, 70-16. 245. Moran, A. P., and Aspinall, G. O. (1998). Unique structural and biological features of Helicobacter pylori lipopolysaccharides. Prog. Clin. Biol. Res. 397, 37-49. 246. Moran, A. P. The role of lipopolysaccharide in Helicobacter pylori pathogenesis. Aliment. Pharmacol. Then 10 (Suppl. 1), 39-50. 247. Moran, A. P, Lindner, B., and Walsh, E. J. (1997). Structural characterization of the lipid A component of Helicobacter pylori rough- and smooth-form lipopolysaccharides. J. Bacteriol. 179, 6453-6463. 248. Suda, Y., Ogawa, T., Kashihara, W., Oikawa, M., Shimoyama, T., Hayashi, T., Tamura, T., and Kusumoto, S. (1997). Chemical structure of lipid A from Helicobacter pylori strain 206-1 lipopolysaccharide./ Biochem. 121, 1129-1133. 249. Aspinall, G. O., and Monteiro, M. A. (1996). Lipopolysaccharides of Helicobacter pylori strains P466 and MO 10: Structures of the O antigen and core oligosaccharide regions. Biochemistry 35, 2498-2504. 250. Aspinall, G. O., Monteiro, M. A., Pang, H., Walsh, E. J., and Moran, A. P (1996). Lipopolysaccharide of the Helicobacter pylori type strain NCTC 11637 (ATCC 3504): Structure of the O antigen chain and core oligosaccharide regions. Biochemistry 35, 2489-2497. 251. Moran, A. P., Helander, I. M., and Kosunen, T. U. (1992). Compositional anlysis oiHelicobacter pylori rough-form lipopolysaccharides. J. Bacteriol. 174, 1370-1377. 252. Mattsby-Baltzer, L, Mielniczuk, Z., Larsson, L., Lindgren, K., and Goodwin, S. (1992). Lipid A in Helicobacter pylori. Infect. Immun. 60, 4383-4387. 253. Birkholz, S., Knipp, U., Netzki, C , Adamek, R. J., and Opferkuch, W. (1993). Immunological activity of lipopolysaccharide of Helicobacter pylori on human peripheral mononuclear blood cells in comparison to lipopolysaccharides of other intestinal bacteria. FEMS Immunol. Med. Microbiol. 6, 317-324. 254. Perez-Perez, G. I., Shepherd, V. L., Morrow, J. D., and Blaser, M. J. (1995). Activation of human THP-1 and rat bone marrow-derived macrophages by Helicobacter pylori lipopolysaccharide. Infect. Immun. 63, 1183-1187. 255. Sherburne, R., and Taylor, D. E. (1995). Helicobacter pylori expresses a complex surface carbohydrate, Lewis X. Infect. Immun. 63, 4564-4568. 256. Wirth, H.-R, Yang, M., Karita, M., and Blaser, M. J. (1996). Expression of the human cell surface glycoconjugates Lewis X and Lewis Y by Helicobacter pylori isolates is related to cagA status. Infect. Immun. 64, 4598^605. 257. Monteiro, M. A., Chan, K. H., Rasko, D. A., Taylor, D. E., Zheng, P Y, Appelmik, B. J., Wirth, H.-P, Yang, M., Blaser, M. J., Hynes, S. O., Moran, A. P, and Perry, M. B. (1998). Simultaneous expression of type 1 and 2 Lewis blood-group antigens by Helicobacter pylori lipopolysaccharides: Molecular mimicry between H. pylori lipopolysaccharides and human gastric epithelial cell surface glycoforms. / Biol. Chem. IIX 11533-11543. 258. Wirth, H. P, Yang, M., Peek Jr., R. M., Tham, K. T, and Blaser, M. J. (1997). Helicobacter pylori Lewis expression is related to the host Lewis phenotype. Gastroenterology 113, 1091-1098.
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259. Taylor, D. E., Rasko, D. A., Sherburne, R., Ho, C , and Jewell, L. D. (1998). Lack of correlation between Lewis antigen expression by Helicobacter pylori and gastric epithelial cells in infected patients. Gastroenterology 115, 1113-1122. 260. Blaser, M. J. (1997). Ecology oi Helicobacter pylori in the human stomach. J. Clin. Invest. 100, 759-762. 261. Blaser, M. J. (1998). Helicobacters are indigenous to the human stomach: Duodenal ulceration is due to changes in gastric microecology in the modern era. Gut 43, 721-727. 262. van Doom, L. J., Figueiredo, C , Sanna, R., Plaisier, A., Schneeberger, R, de Boer, W., and Quint, W. (1998). Clinical relevance of the cagA, vacA, and iceA status oi Helicobacter pylori. Gastroenterology 115, 58-66. 263. Blaser, M. J., Perez-Perez, G. L, Kleanthous, H., Cover, T. L., Peek, R. M., Chyou, P H., Stemmermann, G. N., and Nomura, A. (1995). Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 55, 2111-2115. 264. Kuipers, E. J., Perez-Perez, G. I., Meuwissen, S. G., and Blaser, M. J. (1995). Helicobacter pylori and atrophic gastritis: Importance of the cagA status. J. Natl. Cancer Inst. 87, 1777-1780. 265. Parsonnet, J., Friedman, G. D., Orentreich, N., and Vogelman, H. (1997). Risk for gastric cancer in people with CagA positive or CagA negative Helicobacter pylori infection. Gut 40, 297-301. 266. Pan, Z.-J., van der Hulst, R. W. M., Feller, M., Xiao, S.-D., Tytgat, G. N. J., Dankert, J., and van der Ende, A. (1997). Equally high prevalences of infection with c«^A-positive Helicobacter pylori in Chinese patients with peptic ulcer disease and those with chronic gastritis-associated dyspepsia. J. Clin. Microbiol. 35, 1344-1347. 267. Brenner, H., Rothenbacher, D., Bode, G., and Adler, G. (1998). The individual and joint contributions of Helicobacter pylori infection and family history to the risk for peptic ulcer disease. J. Infect. Dis. Ill, 1124-1127. 268. Hansson, L.-E., Nyren, O., Hsing, A. W., Bergstrom, R., Josefsson, S., Chow, W.-H., Fraumeni, J. F, and Adami, H.-O. (1996). The risk of stomach cancer in patients with gastric or duodenal ulcer disease. New Engl. J. Med. 335, 242-249. 269. Blaser, M. J., Chyou, P. H., and Nomura, A. (1995). Age at establishment oi Helicobacter pylori infection and gastric carcinoma, gastric ulcer, and duodenal ulcer risk. Cancer Res. 55, 562-565.
CHAPTER 12
Neisseria SCOTT D . GRAY-OWEN CHRISTOPH DEHIO THOMAS RUDEL MICHAEL NAUMANN THOMAS F. MEYER 1. Introduction A. Neisserial Morphology and Physiology B. Neisserial Infections II. Natural Competence for Transformation III. Surface Structures A. Lipooligosaccharide B. The Meningococcal Capsule IV. Tissue Colonization A. Neisserial Type 4 Pilus B. Opa-Mediated Interactions C. Opc-Mediated Interactions D. Interactions Mediated by a Novel Multiple Adhesin Family V. PorB A. Influence of PorB on Cellular Interactions B. PorB Induction of Apoptosis VI. IgAl Protease VII. Iron Acquisition in Vivo VIII. Immune Response A. Cellular Response to Neisserial Infections B. Humoral Response to Neisserial Infection IX. Summary References
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/. Introduction The genus Neisseria contains two human pathogenic species, as well as a number of other species that are either pathogenic to animals or are normal flora in either humans or animals. These fascinating organisms are exquisitely adapted to life within their host, a fact that is manifested by their limited biosynthetic capabilities as compared to enteric or free-living bacteria. Because of this, each species is Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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usually able to colonize only a single host species. The pathogenic Neisseria spp. are also capable of evading the massive immune response that is typical of neisserial disease. In part, this is due to their ability to undergo both the frequent change of immunodominant epitopes (i.e., antigenic variation) and the high-frequency, reversible switching "on" and "off of the expression of various surface-exposed virulence factors (i.e., phase variation). In addition to presenting the immune system with an alternative antigenic make-up, each of these mechanisms can also result in a change in a bacterium's functional characteristics. For example, the phase-variable expression of adhesin molecules may result in generation of bacteria capable of invading tissues that the parental phenotype could not. This could potentially allow a subpopulation of the bacteria to become sequestered in a site that is relatively more protected from the immune system. The highly efficient exchange of chromosomal sequences between neisserial bacteria strains and species during mixed infections also provides a mechanism for the strain to adapt over a longer time-frame. Together, these features provide the Neisseria with a powerful arsenal of weapons to persist within their target population. A. Neisserial Morphology and Physiology The members of genus Neisseria, which belongs to the family Neisseriaceae, are Gram-negative diplococci with adjacent sides flattened. Since the bacteria divide in two planes, tetrads can also be observed. Single bacteria range from 0.6 to 1.5 |Lim in size, are nonmotile, and do not produce endospores. They are typically considered to be aerobic, with optimal growth occurring between 35 and 37°C in the presence of high humidity and 5% CO2. A^. gonorrhoeae can, however, also survive in an anaerobic environment. Some species may express a carbohydrate capsule, while others cannot. All species are oxidase positive, and most are catalase positive. Some strains are also highly sensitive to fatty acids, often necessitating incorporation of soluble starch into the growth medium. Consistent with their limited host specificity and the fact that they are obligate parasites, Neisseria have limited metabolic capacities. The species can therefore be differentiated based on their varying abilities to produce polysaccharide from sucrose, their catalase and DNase activities, and their ability to oxidatively (i.e., not fermentatively) produce acid from various carbohydrates, reduce nitrate and nitrite, oxidize fatty acids, and produce certain enzymes [1]. B. Neisserial Infections Despite their close evolutionary relationship [2], N. gonorrhoeae primarily infects the urogenital or anorectal mucosa following intimate sexual contact, while A^. meningitidis instead colonizes the nasopharynx after the inhalation of infected respiratory droplets. These associations are at least partially the result of their respective modes of transmission rather than to a tropism for these loci, since
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gonococcal pharyngitis and meningococcal anogenital infections have also been described [3, 4]. The meningococcal polysaccharide capsule (Fig. 1 A) is a major virulence factor in this respect, since it does contribute to this organism's abihty to be spread via the aerosol route, while the absence of such a capsule makes the gonococci highly susceptible to drying when outside of the host. Although they are infrequently described as being the etiologic agents of various opportunistic infections, other neisserial species are generally considered as normal flora of the oro- and nasopharynges. The virulence mechanisms and primary interactions that occur between the pathogenic Neisseria and various mucosal surfaces are generally quite similar, and are depicted schematically in Figure IB. In this chapter, we will thus discuss neisserial virulence mechanisms in general terms, with specific differences between N. gonorrhoeae and N. meningitidis being highlighted as appropriate. 1.
NEISSERIA GONORRHOEAE
N gonorrhoeae is the second leading cause of sexually transmitted disease in the United States, one of the few countries in which gonorrhea is reportable. Over 325,000 cases were reported to the American Centers for Disease Control in 1996; however, this is considered a conservative estimate due to significant underreporting. Gonorrhea is also a major concern in non-Western countries, as evidenced by the fact that it is the most common sexually transmitted disease in China [5]. The incidence of gonococcal infection generally correlates with low socioeconomic status, being highest in poor regions of both developed and developing nations. In Africa, gonococci remain endemic in poorer regions, with a recent report showing 3.4% of men in a study group of transport workers in Kenya to have a urethral gonococcal infection [6]. For epidemiological studies, gonococcal strains are typically characterized by auxotyping and/or serotyping. Auxotyping is based on the different growth requirements of various gonococcal strains for specific nutrients or cofactors. Using different chemically defined growth media, over 30 different auxotypes have been identified [7]. A more common typing scheme is based on the antigenic characterization of protein I (PorB; Fig. lA), since variant alleles of this constitutively expressed protein are generally stably maintained in different strains. Currendy, the major serogroups of protein I, termed lA and IB, have been further subdivided into 26 and 31 serovars, respectively (e.g., serovar IA-21) [8, 9]. In men, gonorrhea typically presents as an acute urethritis after an incubation period of 2-5 days, with symptoms including purulent discharge and dysuria. Acute epididymitis is the most common complication of untreated gonococcal infection; however, disseminated gonococcal disease can also occur. In women, the majority of disease results from uncomplicated infections of the lower genital tract, with the primary site of infection being the endocervix. Estimates suggest that up to 80% of women may be either asymptomatic or possess minor symptoms that do not induce them to seek medical attention [10-121. Symptomatic disease is thought to present within 10 days of infection, and is characterized by cervicitis
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Fig. 1 Neisserial colonization of host tissues. (A) Virulence factors involved in neisserial interactions with host cell surfaces. The highly variable PilE subunit forms the fiber of neisserial type 4 pili. The pilus-associated protein PilC functions in pilus biogenesis and mediates pilus attachment to various human cell types. The phase-variable, colony opacity-associated Opa proteins are integral outer membrane proteins that mediate intimate binding to and invasion into various human cell types (e.g., epithelial, endothelial, and phagocytic cells). Some Opa protein variants can also mediate transcellular traversal of bacteria across epithelial monolayers in vitro, and could therefore potentially allow penetration into submucosal layers in vivo. Ope is an integral outer membrane protein that is structurally distinct from the Opa proteins, but it carries cellular binding and invasion functions that are indistinguishable from some Opa protein variants in vitro. The glycolipid adhesins of the multiple adhesin family (Maf) may also contribute to neisserial interactions with various cell types; however, their role is not well characterized. Although their role in cellular interactions are less obvious, PorB, LOS, and the meningococcal capsule influence the efficacy and outcome of the adhesins binding to their cellular receptors. The porin PorB can translocate into host cell membranes to form an ATP-regulated ion channel that apparently plays a role in both cellular invasion and intracellular accommodation of Neisseria. The lipopolysaccharide of Neisseria spp. lacks any repetitive O sidechains, and is thus termed lipooligosaccharide (LOS). In addition to its endotoxin activity, some LOS variants can be sialylated to render the bacterium resistant to the bactericidal activity of serum. The polysaccharide capsule, which can be expressed by N. meningitidis but not by A^. gonorrhoeae, allows the bacterium to resist serum bactericidal activity, phagocytosis and desiccation, and masks many surface epitopes which may be targets of antibody. The expression of either sialylated LOS or capsule does, however, also block most interactions between the bacterium and target cells, thus impeding their colonization and/or invasion. Fig. 1 (B) (opposite) Mucosal colonization by the pathogenic Neisseria. Primary adherence to mucosal epithelial cells is thought to occur via pili. A secondary, more intimate contact can then be established between other adhesins (e.g., Opa) and their cellular receptors. This interaction may lead to bacterial invasion and transcytotic passage to subepithelial tissues. Neisserial interactions with endothelial cells may then allow the bacteria to enter the bloodstream and disseminate to sites distal from the primary locus of infection. The sialylation of LOS and/or meningococcal expression of capsule can protect the bacterium from the bactericidal activity of serum complement. It may also protect the bacterium from being decorated by opsonic antibodies, thus protecting the bacteria from phagocytosis by professional phagocytes (e.g., polymorphonuclear neutrophils and/or monocytes) present either at the site of infection or encountered during disseminated neisserial disease. Interestingly, the bacterial Opa proteins do mediate an opsonin-independent engulfment of bacteria by professional phagocytes. Although these cells are likely able to effectively kill the phagocytosed bacteria, a subpopulation of bacteria may be able to remain viable due to their ability to influence phagolysosomal maturation and/or escape the membranebound vesicle and enter into the cell cytoplasm.
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and/or urethritis with increased vaginal discharge, dysuria, and cervical edema, which bleeds easily on gentle swabbing. Approximately 40% of women with uncomplicated gonococcal disease are also anorectally colonized, likely by contamination from infected cervical secretions [13]. Spread of the organism into the upper genital tract can result in salpingitis or pelvic inflammatory disease (PID), a major cause of morbidity in women of reproductive age and one of the major reasons why control of gonorrhea is desired [14]. PID is often found to be a polymicrobial infection, also containing aerobic or anaerobic flora that appear to synergistically affect gonococcal growth at this site [15]. The onset of PID can lead to high fever, chills, nausea and/or vomiting with pelvic adnexal tenderness, and often follows the beginning of menses by only a few days [16]. Left untreated, PID may result in fallopian tube scarring and blockage, which leads to tubal infertility, ectopic pregnancy, and/or chronic pelvic pain; however, early treatment is generally successful at preventing any permanent damage to the reproductive system [17]. Disseminated gonococcal infection (DGI) is estimated to develop in 0.5-3% of Americans infected with N. gonorrhoeae. It is most common in young women, but may develop in sexually active people of any age. Classic symptoms include dermatitis, tenosynovitis, and migratory polyarthritis. Strains causing DGI are frequendy serum resistant, owing to their ability to sialylate their lipooligosaccharide (LOS) (Fig. lA). This modification reduces bacterial susceptibility to bactericidal antibodies, downregulates the activation of complement, decreases phagocytosis, and reduces bacterial adherence to and induction of an oxidative burst by neutrophils [18] (see §111.A). Such strains are typically serotype lA, have a requirement for arginine, hypoxanthine, and uracil in the growth medium, and are more sensitive to penicillin [16, 19, 20]. A characteristic dermatitis may also be present, with discrete papules and pustules seen primarily in the extremities. Other disseminated complications of gonococcal infection may include perihepatitis [21] and endocarditis [22]; however, both are relatively infrequent. Gonococcal conjunctivitis in adults is a localized infection that can lead to corneal scarring or perforation causing visual loss if not rapidly treated with aggressive antibiotic therapy [23, 24]. Ophthalmia neonatorum, an ocular infection caused by exposure of neonates to gonococci during passage through the birth canal of an infected mother, remains a common cause of blindness in some developing countries [25]. In developed nations, this problem has been largely eliminated by the routine application of prophylactic silver nitrate drops or erythromycin ointment to the eyes of a newborn. Although the incidence of gonococcal disease in the United States has declined in recent years, there has been an increased level of antibiotic resistance that has influenced suggested treatment regimens. In 1994, 30.5% of gonococcal isolates had either chromosomal or plasmid-mediated resistance to penicillin and/or tetracycline, and the prevalence of penicillin resistance itself increased from 8.4% in 1988 to 15.6% in 1994. Perhaps more troubling is the recently reported isolation of strains resistant to the fluoroquinolone antibiotic ciprofloxacin, one of the currently recommended treatments for gonorrhea [26].
12. NEISSERIA 2.
565
NEISSERIA MENINGITIDIS
The frequency of meningococcal disease differs according to geographical area, ranging from 0.001% of the population per year up to 1% during epidemic outbreaks. The Sahel in Africa and China are the hardest hit regions in this respect, typically experiencing an epidemic wave in each decade [27]. An inverse correlation between atmospheric humidity in Zaire and rate of meningococcal carriage and disease [28] suggests that environmental factors may at least partially contribute to the particular susceptibility of these regions. The meningococci can be separated into at least 13 serogroups based on serum agglutination. Most epidemics are caused by meningococci of serogroup A; however, epidemics caused by capsular types B and C can also occur. The diversity of virulent serogroup A meningococci appears to be quite limited, and only a few strains have been responsible for the waves of epidemic that have struck the Sahel and China in recent decades [29, 30]. In nonepidemic conditions, serogroup B organisms predominate as the cause of sporadic meningococcal meningitis [31, 32]. A^. meningitidis has been isolated from the oro- or nasopharynx of up to 30% of healthy individuals in nonepidemic regions [33, 34], yet a high incidence of carriers does not necessarily correlate with an elevated risk of meningococcal meningitis in a population [35, 36]. Because of this, meningococci are typically considered as normal flora when isolated from the throat, but are a major concern once disseminating from this site. The exception to this rule is that patients who experience close contact with invasive meningococcal disease may be 500-800 times more likely to develop serious meningococcal disease than normal asymptomatic carriers [37]. Such individuals are, therefore, generally prophylactically treated with antibiotics and vaccination whenever possible. The clinical manifestations of meningococcal disease can be quite varied. Mild systemic disease can occur in young children [38], and there has been a report of children who spontaneously recovered from meningococcemia without antibiotic treatment [39]. Fulminant meningococcemia can, however, progress very rapidly, ultimately resulting in the death of a previously healthy individual within a few hours of the onset of symptoms. Symptomatic meningococcemia may present with fever, lethargy, shock, coma, intravascular coagulopathy, petechial or purpuric skin rash, and/or gross adrenal hemorrhage (Waterhouse-Friderichsen syndrome) [40, 41]. There appears to be a high incidence of early myocardial depression during disseminated meningococcal infection as compared to that seen during bacteremia caused by other Gram-negative bacteria [42], and myocarditis is present in over half of the patients who die of meningococcal disease [43,44]. Symptoms of meningococcal meningitis can include high fever, headache, and stiff neck, which may develop during a period of between several hours to 2 days. Early treatment can significantly influence outcome; however, case-fatality rates of as high as 21% are still being reported in industrialized nations that have modem medical techniques available [45-47]. Although children below the age of 5 years are most prone to meningococcal disease, their mortality rate is typically lower than that of 16-24 years olds. In this latter age group, the infection most commonly presents as men-
566
SCOTT D. GRAY-OWEN ETAL
ingitis or meningococcemia [48], and N. meningitidis has been reported to cause as much as 93% of all bacterial meningitis in 16-20 year olds [47]. Pericarditis and septic arthritis can both arise as a complication of meningococcemia or meningococcal meningitis; however, they have also been reported to occur as primary events [49, 50]. Endemic disease in older adults often involves immunocompromised individuals, and meningococcal pneumonia, sinusitis, or tracheobronchitis may be seen.
//. Natural Competence for Transformation The constant antigenic and functional variation of neisserial surface components is likely vital for the success of Neisseria as a parasite. In addition to the high-frequency intrabacterial genetic variation that occurs in some genetic loci (see below), variation can also occur via the horizontal exchange of genetic information between heterologous neisserial strains. The Neisseria are naturally competent for DNA transformation [51, 52], and the efficiency of this process is such that up to 1% transformation efficiencies are easily attainable. The exchange of chromosomal DNA has been shown to occur during simple cocultivation of different neisserial strains in vitro, resulting in the transformation-dependent exchange of DNA with a frequency on the order of 10"^ per cell and genetic locus after only 1 hr of incubation. Importantly, this study also demonstrated that exchange between different neisserial species can occur [53], and epidemiological data suggest that such exchanges have happened in vivo [27, 54]. This phenomenon is of central importance to the biology of the genus, affecting its population structure and genetic flexibility. For example, there appears to be a continuous horizontal flow of genetic information between different neisserial strains and species, resulting in the existence of some loci containing genes that are a mosaic of multiple previously described alleles [55-60]. Both antigenic and functional differences can result from such protein chimeras, as has been most clearly demonstrated for the pilin-encoding gtntpilE [61, 62] (see §IV.A). It seems likely that the commensal Neisseria spp. may function as intermediates in the exchange of chromosomal segments between pathogenic strains, and so coinfection by two virulent strains is not a prerequisite for such processes to occur. Natural transformation in vitro is prevented by the addition of DNase into the culture medium [53], confirming that DNA must be released by one bacterium in order to be taken up by another. This appears to occur via spontaneous autolysis of the bacteria, which is a gonococcal response to nutrient depletion or other adverse conditions [63-67]. Interestingly, a chromosomally encoded peptidoglycan hydrolase similar to bacteriophage endolysins has been shown to be involved in autolysis. Mutagenesis of the atlA gene that encodes this protein results in significantly increased levels of gonococcal survival following entry into stationary growth phase [68]. A 10-bp recognition sequence (5'-GCCGTCTGAA), which is widely dispersed throughout the neisserial chromosome and is contained as a palindrome within transcriptional terminators, must be present within the DNA fragments for efficient transformation to occur [69, 70]. An 11-kDa outer
12. NEISSERIA
567
membrane protein that binds DNA fragments containing the neisserial uptake sequence has been identified, and mutagenesis of the gene that encodes it (dudl) blocks DNA uptake [71,72]. Following DNA binding, the expression of neisserial pilus and pilus-biosynthetic machinery (see §IV.A.l) are required for efficient penetration of DNA across the outer membrane [73]. The function of these components in this process, however, is still unknown. Other factors distinct from pilus are then essential for the transfer of DNA across the periplasmic space (i.e., ComL and Tpc [74, 75]) and through the cytoplasmic membrane (i.e., ComA) [76]. Once inside the cell cytoplasm, RecA uses homologous chromosomal sequences to recircularize the linear molecule of transforming DNA into a plasmid or to recombine it into homologous sequences in the chromosome itself [77, 78]. Together with the restriction barrier posed by the existence of several restriction/modification systems in Neisseria [79], the requirement for uptake sequences and sequence homology provides an effective mechanism for the bacteria to discriminate between neisserial and unrelated foreign DNA fragments. The combination of autolysis and competence have been considered to be part of the bacterium's "catastrophe kit" [80], since the lysed bacteria may theoretically provide nutrients to extend the viability of the surviving population while also contributing DNA to reshuffle the gene pool in an attempt to generate a new clone capable of surviving the adversity.
///. Surface Structures A.
Lipooligosaccharide
Neisseria produce a short type of lipopolysaccharide, known as lipooligosaccharide (LOS), which lacks any repetitive O sidechains. Nonetheless, its structural heterogeneity is evident by the multiple size classes and antibody reactivity patterns seen in bacteria cultured in vitro [81]. LOS variation relies on the phase-variable expression of the glycosyl transferases that are involved in the biosynthesis of the variable a-chain carbohydrates of LOS (IgtA, IgtC, and IgtD). This occurs due to slipped-strand mispairing of a homopolymeric tract of guanines within the coding sequence, which thereby influences the reading frame of these genes [82-85]. In vitro, the phase variation of LOS occurs spontaneously at a frequency between 0.02 and 0.2% [81]. Several lines of evidence suggest that this variation also occurs in vivo. In meningococcal carriers, more than 70% of bacteria isolated from the nasopharynx preferentially express a short LOS species [86], whereas 97% of clinical isolates from the blood and CSF instead display the long LOS form [87]. Likewise, experimental gonococcal infection of human volunteers demonstrates that bacteria isolated early in the infection have short LOS, whereas after the development of an inflammatory response the long LOS species predominates [88].
568 1.
SCOTT D . GRAY-OWEN ETAL. LOS AS A BACTERIAL "SAFEGUARD"
A major difference between the variant LOS molecules is the presence of additional carbohydrates in the longer LOS forms, including a terminal galactose residue. This galactose can be externally modified by the membrane-associated bacterial sialyltransferase using host-derived or endogenous cytidine 5'-monophospho-A^-acetylneuraminic acid (CMP-NANA) as a sialyl donor [89, 90]. LOS sialylation does occur during natural infection, and greatly affects the biological properties of bacteria seen in vitro, including both their ability to enter epithelial and endothelial cells and to resist the host immune defenses. For example, Opa5o/HSPG-dependent entry into host cells (see §IV.B.2) is less efficient if LOS is sialylated, whereas invasion levels can be enhanced if sialylation is prevented due to either the absence of a terminal galactose residue or the unavailability of CMP-NANA substrate [90, 91]. This effect may be due to the steric masking of outer membrane proteins such as Opa or Ope [92]. Unsialylated bacteria are, however, more susceptible to killing by antibodies and complement [93a], likely due to the sialylated LOS form having antigenic similarities with host cell structures. This phenotype is also stably expressed in gonococcal sialyl-transferase-deficient mutants [93b, 93c]. Indeed, the Gal(pl-4)Glc-NAc(pl-3)Gal(pl4)Glc (lacto-A^-neotetraose) carbohydrate structure present at the nonreducing terminus of LOS variants is identical to the nonreducing terminus of oligosaccharides found on many human glycolipids and glycoproteins, and its sialylation on the terminal galactose mimics human I and i antigens [94, 95]. Together, these findings suggest that LOS variation may serve as a mechanism that enables bacterial switching between invasive and immunoresistant phenotypes [90]. This hypothesis is supported by clinical observations showing poor virulence of sialylated gonococci in experimentally infected human volunteers [96], despite the fact that such phenotypes do predominate during inflammatory disease [97]. Purified LOS has been shown to activate and/or be cytotoxic to a variety of host cell types. In primary endothelial cells, meningococcal LOS-mediated toxicity was found to be modulated by pilus-dependent but Opc-independent adherence, suggesting that pili have a synergistic effect that contributes to the overall damage caused by LOS [98]. Interestingly, a viable meningococcal mutant producing no LOS or endotoxin was recently reported [99]. This mutant should allow differentiation between LOS-mediated cytotoxicity and cytotoxicity caused by other mechanisms, such as porin-induced apoptosis (see below). It should also allow the study of the interactions occurring between neisserial surface adhesins (e.g., pili. Pile, Opa, Ope, and Maf) and host-cell receptors in the absence of any interference by toxicity or steric hindrance by the LOS structure. 2.
NEISSERIAL LOS AS AN ''ADHESIN"
In addition to its influence on cellular interactions mediated by Opa, Ope, and pilus, the unsialylated form of lacto-A^-neotetraose-containing LOS may also play a more direct role in host-cell binding in the absence of these proteins. Near the
12. NEISSERIA
569
end of their functional lives, human glycoproteins and glycolipids are converted from the sialo-[NANA(a2-3)Gal(pl-4)Glc-NAc(Pl-3)Gal(pl-4)Glc] to the asialo-[Gal(pl-4)Glc-NAc(pl-3)Gal(pl-4)Glc] form, by the loss of their terminal NANA moieties. When this occurs, these compounds are bound by and removed from the circulation via asialoglycoprotein receptors. Similarly, the asialo-lactoA^-neotetraose-containing LOS has been demonstrated to interact with both the asialoglycoprotein receptor and an additional 70-kDa receptor protein on the surface of hepatic HepG2 cells. Moreover, this variant LOS type does mediate gonococcal adherence and invasion into HepG2 cells by an Opa-independent mechanism [100, 101]. Whether a similar LOS-dependent uptake mechanism can also operate at the level of the mucosa has not yet been investigated. B. The Meningococcal Capsule In contrast to the gonococci, A^. meningitidis and many commensal Neisseria spp. may express a polysaccharide capsule. The capsular polysaccharides of the A^. meningitidis serogroups B and C, which predominate in the Northern Hemisphere, are homopolymers of sialic acids with a-2,8 and a-2,9 linkages, respectively [102]. A^. meningitidis serogroup A, which is responsible for most meningococcal epidemics, expresses a capsule that lacks sialic acid [103]. Polysialic capsules mediate resistance to both phagocytosis and complement-mediated killing via the alternative pathway of complement activation [104, 105]. HSPG-specific Opaand Opc-mediated invasion into host cells is also blocked by the expression of a capsule. Consistent with this, selection of meningococcal variants that efficiently invade into primary mucosal cells results in the recovery of variants that: (1) are nonpiliated, (2) are unencapsulated, (3) express a short LOS form that is not sialylated, and (4) express an Opa protein that can bind to both HSPG and CEACAM (formerly CD66 [105a]) host-cell receptors [91, 106]. Although CEACAM-specific Opa proteins have been shown to bind their receptors even in the presence of capsule, this interaction was enhanced in unencapsulated variants [107]. Together, these findings suggest that capsule expression may be downregulated following primary contact with the mucosa [108, 109], and then re-expressed following transmigration across the epithelial barrier in order to provide protection against the host's immune defenses. Certain capsule types appear to be selected for by various environmental stimuli [110]; however, spontaneous phase variation of capsule expression is also observed under standard growth conditions [111]. A novel mechanism of genetic variation that allows reversible phase (on and off) switching of both polysialic capsule synthesis and endogenous LOS sialylation has been identified in meningococci [111]. The fluctuation between a sialylated and a nonsialylated bacterial phenotype operates by reversible insertion/excision of a naturally occurring insertion sequence element into the essential sialic acid biosynthesis gene siaA. The in vivo relevance of this particular adaptive mechanism for the regulation of
570
SCOTT D. GRAY-OWEN ETAL.
meningococcal penetration into the mucosal barrier does, however, remains to be demonstrated.
IV. Tissue Colonization A. Neisserial Type 4 Pilus Data obtained from the experimental urethral inoculation of human male volunteers with N. gonorrhoeae suggests that pili play an important role in neisserial colonization of the host mucosa [112, 113]. Neisserial piH are long (<6 |Lim) filamentous structures that extend from the bacterial surface. This structure allows the organelle to mediate primary binding to the host tissues, since it can overcome the electrostatic barrier that exists because the bacteria and eukaryotic cells are both negatively charged [114]. The pilus fiber is primarily composed of a single pilin protein subunit (PilE), which is assembled into a helical arrangement with a diameter of 6 nm [115]. Other minor pilus components, including the PilC protein that has been implicated in both pilus biogenesis and adhesion, are also copurified with neisserial pili. Several other apparently unrelated phenotypes also depend on pilus expression. A form of movement known as twitching motility correlates with neisserial piliation, and appears to be a consequence of the retraction of pilus fibers [116]. As described in section II, pilus also appears to function in the uptake of DNA across the outer membrane, since piliated bacteria are 1000-fold more competent for transformation by DNA than are nonpiliated variants [73]. The strict correlation between these functions and neisserial piliation does, however, make it difficult to determine the specific contribution of each of these phenotypes to neisserial virulence. 1.
PILUS BIOGENESIS
Due to its morphology, mechanism of assembly, and polarized arrangement on the bacteria [117], neisserial pili are related to the type 4 family of pili, members of which have also been demonstrated in many other important mucosal pathogens [118-121]. Type 4 pili from various species possess a highly conserved amino-terminal domain within the major structural subunit protein (PilE) [122]. At least 22 gene products are typically involved in the assembly and function of the pilus fiber (Fig. 2) [123], and this biosynthetic machinery can assemble pilin subunits from the type 4 pili of heterologous species [124-126]. For example, the gonococcal pili can be correctly assembled by Pseudomonas aeruginosa [126]. Homologs of one or more components of the pilus assembly apparatus have also been implicated to function in competence for DNA transformation of Bacillus subtilis [127], and in the general secretion pathway responsible for secretion of a wide variety of hydrolases and toxins by Gram-negative bacteria [128].
12.
571
NEISSERIA
A high conservation between these systems has allowed the function of some neisserial pilus biosynthetic and assembly proteins to be determined by complementation of defined mutants in the well-characterized R aeruginosa machinery [129]. Two major complexes are involved in pilus biosynthesis, being localized to the inner and outer membranes. The inner membrane complex consists of PilD, PilF, and PilG [130]. The PilD peptidase functions in amino-terminal processing of prepilin to its mature form. Roles for the cytoplasmic membrane protein PilG and the cytosolic protein PilF have not yet been defined, but a function in prepilin transport through the inner membrane is likely. Interestingly, the PilQ protein is a member of the large GspD protein family, the members of which are involved in filamentous phage assembly and transport [131], pullulanase secretion by
piiD -PiiF-PiiG-pirr Complex Fig. 2 Hypothetical model of pilus biogenesis. The pre-PilE molecules contain a 7-aa long positively charged signal sequence, a hydrophobic helix that spans the cytoplasmic membrane (CM), and a globular domain that is exposed in the periplasmic space. The prepilin peptidase PilD removes the signal sequence, allowing the hydrophobic helices to polymerize so that the hydrophilic globular domains form the outer surface of the pilus fiber. Since the PilE hydrophobic domain is maintained within a hydrophobic environment, the reversible polymerization and depolymerization of pilus fibers can occur without significant energy being expended. This process, known as twitching motility, is thought to be under the control of a cytoplasmic membrane-localized multi-subunit complex that includes PilD, PilF, PilG, and PilT. The assembled pili penetrate the outer membrane (OM) through a gated pore formed by the PilQ and PilP proteins. Alternative cleavage of pre-PilE at position Ala40 instead leads to the release of soluble S-pilin into the periplasm and, presumably, through the outer membrane complex. Although several proposals do exist for the role of S-pilin in vivo (see text), its actual function is still uncertain. Signal peptidase 1 (SPl) is thought to be responsible for this cleavage event.
572
SCOTT D. GRAY-OWEN ETAL
Klebsiella oxytoca [132], the type III contact-dependent secretion systems of various Enterobacteriaceae [133, 134], transformation oi Haemophilus influenzae [135], and S-layer production in Aeromonas hydrophila [136]. Based on these homologies, it appears that PilQ probably forms a gated channel consisting of 10 to 12 subunits that is stabilized by the PilP protein [137, 138]. Consistent with such a function, PilQ and PilC appear to interact during the terminal stages of pilus biogenesis, since PilQ mutants release the pilus-associated PilC protein into the culture supernatant [137]. 2.
PILUS STRUCTURE, ANTIGENIC AND PHASE VARIATION
Pilus expression is phase variable due to the frequent RecA-independent frame shifts that occur within a poly-C stretch in pilE (see Fig. 3B). This leads to the reversible on and off switching of pilin expression at a rate of about 10"^ [78, 139]. Pilus antigenic variation also occurs. The observation that pilin phase and antigenic variants exhibit different colony morphologies has both stimulated and gready facilitated the study of pilin variation. It has resulted in the identification of a large number of isogenic gonococcal variants that express distinct pili, thus allowing the pilin subunit (PilE) primary structure, pilus structure, and pilus-associated phenotypes of these variants to be compared. PilE has a highly conserved
silent gene 1 expression gend'^^"'®"^ expression gen§ progeny
Fig. 3 Variable expression of neisseria! adhesins. (A) Gene conversion. The nonreciprocal transfer of DNA sequences from one of the multiple silent ipilS) loci into an expressed ipilE) locus leads to a "conversion" of pilE to generate a novel allele. Depending on the sequence of these newly generated chimeras, this change may result in generation of pilus fibers with an altered functional and/or antigenic profile. Altered sequences may also result in differences in the generation of strains that no longer express PilE, or that express prepilin that is aberrantly processed to S-pilin or L-pilin (P^ or
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53-aa amino terminal that is involved in its multimerization into the pilus fiber [115]. The extreme heterogeneity of pili from different strains is due to the presence of variable regions (minicassettes), one of which is flanked by the two cysteine residues that join to generate a highly divergent loop that becomes exposed on the surface of the pilus fiber [115]. This structure contains the epitopes that are recognized by most type-specific antibodies. Pilus antigenic variation usually rests on the nonreciprocal transfer of DNA sequences from one of several silent pilin genes (pilS) into an expressed/?//£ locus (Fig. 3A) [140]. This "gene conversion" involves the RecA protein [78]. Also, DNA transformation can lead to apparent gene conversion [61, 62, 141-143], suggesting that the autolytic release of chromosomal DNA fragments may contribute to this process. Several studies, however, suggest that DNA transformation cannot account for all observed pilus variations [76, 139, 144], indicating that nonreciprocal exchange may truly occur between loci of a single bacterium. Whether a specialized system catalyzes this process is still uncertain. In addition to their influence on antigenic variation, changes within pilE can also influence the processing of pilin, and thereby affect biosynthesis of the pilus fiber. A naturally occurring variant of PilE is differentially processed, resulting in an additional 40 amino acids being removed from the amino terminus (i.e., in addition to the seven amino acids normally removed). This results in the generation of a short, soluble variant (S-pilin) that is secreted directly into the growth medium in vitro [145]. Originally, the nonpiliated appearance of S-pilin variants led to the assumption that they do not produce pili; however, most, if not all, S-variants can form pili. They do form much less pili than strains not secreting pilin, and it appears that the amount of S-pilin produced inversely correlates with the number of pilus fibers formed. Although the biological function of S-pilin production has not been defined, it may influence the infection process. For example, S-pilins might be produced as a molecular shield that protects the bacterium because it binds anti-pilin antibodies. This would likely be most effective during the early stages of infection, when antibody titers are still low. Alternatively, S-pilin may interact with the target cell, initiating or amplifying a signal transduction pathway that somehow benefits the bacteria [146]. Interestingly, a long (L-pilin) variant also exists. The allele encoding this protein typically results from a duplication in the hypervariable region of pilin, resulting in a protein that is not properly processed. The resulting protein does not form a pilus polymer, but instead remains associated with the bacterial membrane [56]. The functional relevance of this splice variant is still uncertain. Neisserial pilin is posttranslationally modified by the covalent attachment of carbohydrates [115, 147], a function frequently described in eukaryotic cells but which rarely occurs in bacteria. Several different attached glycans have been characterized, including phosphodiester-linked glycerol, a very unusual modification that has not been detected on any other bacterial or eukaryotic protein to date [148]. Interestingly, the gene that encodes the glycosyltransferase responsible for pilin glycosylation (pglA) contains a tract of 11 guanosine residues [149],
12. NEISSERIA
575
suggesting that its expression may be phase variable by a mechanism similar to that seen for the glycosyltransferases involved in LOS variation (see §III.A). The function of these pilin carbohydrate structures are still unknown, however. Mutants that lack the carbohydrate moieties synthesize normal pili and exhibit an adherence phenotype on epithelial and endothelial cells that are indistinguishable from the wild-type forms [149, 150]. It is therefore possible that pilin glycosylation instead plays a role in either immunoevasion or some as-yet-undetermined aspect of the natural infection process. 3.
CELLULAR INTERACTIONS MEDIATED BY PILUS
Neisserial pili interact with many different cell types, including epithelial cells, endothelial cells, granulocytes, macrophages and erythrocytes [151-155]. There are at least two different binding epitopes present in the pilus, since isogenic strains expressing distinct PilE variants differ substantially in their adherence to epithelial and endothelial cells [152, 153, 156], while binding to erythrocytes is variation independent [152, 157]. A region of low sequence variation within pilin has been shown to be involved in erythrocytic and granulocytic receptor recognition [158], and subsequent elucidation of the three-dimensional structure of gonococcal pili confirmed that this epitope is exposed along the pilus fiber [115, 159]. The second binding function is mediated by the PilC protein, which is present in low quantities in the pilus fiber [160, 161]. Piliated PilC knockout mutants still bind erythrocytes but fail to interact with epithelial and endothelial cells [161, 157]. Purified PilC binds to target cells with the same species specificity as do piliated gonococci, and prevents adherence of piliated strains in a competitive manner. Together these results clearly demonstrate that PilC mediates cellular attachment. Very little is known about the receptors that are recognized by the two binding functions of pilus, or about the cellular response to pilus-mediated adherence. Biochemical studies aimed at characterizing the receptors were unclear, with different studies concluding that the receptor was either carbohydrate or proteinaceous in composition [162, 163]. The membrane cofactor protein (MCP or CD46) has recently been shown to function as a cellular receptor for the pili of both pathogenic Neisseriae [164]. Whether MCP binds to the pilus-associated PilC adhesin or to the pilus fiber (i.e., PilE) is still unknown. MCP is a transmembrane C3b/C4b-binding glycoprotein that functions to control the activation state and deposition of complement, and thereby protects host cells from potential damage caused by the human complement system [165]. A soluble form of MCP is also found in serum; however, its function is unknown [166]. Consistent with the phase-variable binding function of piliated strains, MCP is expressed on almost every human cell type with the exception of erythrocytes. Two splice variants of MCP, designated BCl and BC2, are recognized by pili, while two others, CI and C2, are not [164]. It is interesting that BCl and BC2 express different carboxy-terminal cytoplasmic tails [165], since this suggests that different intracellular signaling and cellular responses may follow binding to each of these receptor types.
576
B.
SCOTT D. GRAY-OWEN ETAL
Opa-Mediated Interactions 1.
DIVERSITY AND PHASE-VARIABLE EXPRESSION OF NEISSERIAL OPA PROTEINS
Neisserial Opa proteins were originally identified because their expression changes the color and opacity of gonococcal colonies [167, 168]. This effect may be due to an increased interbacterial aggregation that results from the lectin-like ability of Opa proteins to bind to LOS on adjacent bacteria [ 169], The Opa proteins were, however, subsequently shown to constitute a family of closely related but size-variable [170] integral outer membrane proteins that are predicted to span the membrane eight times to expose four surface loops [171, 172]. An essential role for Opa proteins in neisserial pathogenesis is suggested by the finding that gonococci recovered after urogenital, cervical, or rectal infections typically express at least one Opa protein, as do bacteria recovered after the inoculation of human volunteers with transparent (Opa~) bacteria [173, 174]. The exception to this in vivo selection is that Opa~ bacteria predominate in the cervix early in the menstrual cycle [168], thus implying that their expression is detrimental under these conditions. A single strain can possess as many as 3 ^ (in meningococci [175]) or 11 (in gonococci; see Table I [171, 176]) unlinked chromosomal alleles that encode distinct Opa variants. Although these sequences are approximately 70% identical, their well-described antigenic variability [177, 178] results from exposure of one semi variable and two hypervariable domains at the cell surface [171, 172, 176]. Expression from each opa allele is phase variable due to RecA-independent changes that alter the number of pentanucleotide (CTCTT) coding repeat (CR) units in the hydrophobic core of the leader sequence, and thereby influence the reading frame of these constitutively transcribed genes (Fig. 3B) [179, 180]. This seems to occur via the slipped-strand mispairing of coding repeat units during DNA-dependent DNA polymerase-mediated replication of the bacterial chromosome [181]. Support for such a hypothesis comes from the fact that phase variation can also occur in E. coli [180], implying that a specific system of adding or deleting these units is unlikely. The frequency of phase variation at each opa allele depends on the number of CR units present. The CR-repeat sequence has a tendency to form H-DNA, a triple helical conformation that contains stretches of single stranded DNA. The rate of Opa variation may therefore also be influenced by environmental stimuli, since such H-DNA structures are sensitive to intracellular pH and the degree of chromosomal supercoiling [182]. This phenomenon of phase variation maintains a heterogenous population of bacteria that expresses no, one, or multiple Opa proteins. The complexity of this situation is further increased because both intergenic and interstrain opa recombination can occur, although at a low rate compared to pilin variation. A vast array of alleles has thereby been generated that may be differentially distributed between strains [60, 183-185]. This has led Achtman and coworkers [172] (http://novell-ti.rz-berlin.mpg.de) to systematize all alleles described to date using a nomenclature scheme based on that of Kupsch et al (1993) [176].
12.
577
NEISSERIA
Table I
Cellular Receptors for Opa Proteins Expressed by N. gonorrhoeae MS 11
Opa allele"
Opa protein"
C30/C50^ B51 G52 A53 154 E55 F56 K57 J58 D59 H60
30 (50)^ 51 52 53 54 55 56 57 58 59 60
Cellular receptor HSPG CEACAM5 CE AC AM 1/3/5/6 CEACAMl CE AC AM 1/5 CEACAM5 CEACAM5 CEAC AM 1/3/5/6 CEAC AM 1/3/5/6 CEAC AM 1/5 CEAC AM 1/3/5/6
References [186, 187] [199,201] [199,201] [199,201] [199, 201] [199, 201] [199,201] [199, 201] [199, 201] [199,201] [199,201]
"Nomenclature used is as described in Malorny et al. (1998) [172], and was provided by M. Achtman of the Max-Planck-Institut fiir Molekulare Genetik, Berlin, Germany. A list of nomenclature for all currently described opa alleles can be found at http://novell-ti.rz-berlin.mpg.de. '^opaCT^o/OpsiT^Q and opaC^o/Opa^Q refer to chromosomal and recombinant forms of the same allele, respectively.
2.
OPA-MEDIATED INTERACTIONS WITH CELLULAR HEPARAN SULFATE PROTEOGLYCAN RECEPTORS
A subset of Opa protein variants expressed by the pathogenic Neisseria bind to the host cell surface-associated heparan sulfate proteoglycans (HSPGs) [106 186, 187]. HSPGs consist of long repeating sulfated glycoconjugates known as the heparan sulfate glycosaminoglycans (HS-GAGs), which can be linked to various core proteins [188]. Among the 11 variant Opa proteins encoded by N. gonorrhoeae strain MSll, several appear to bind cellular HS-GAGs of HSPGs [176, 186, 187], while only one, Opa5(), mediates intimate adherence sufficient to trigger uptake into most cultured epithelial cell lines [176, 189, 190]. This process of binding and internalization, which is mediated by HSPG receptors, has been studied in most detail for this neisserial Opa variant. a. HSPG-Dependent Uptake of Opa^g-expressing Neisseria into Epithelial Cells, HSPG-dependent internalization of Opa5()-expressing gonococci into cultured epithelial cells has been shown to occur by at least three alternative mechanisms. Invasion into some epithelial cell lines (i.e., Chang human conjunctiva and Me-180 human cervical carcinoma cells), depends on the activation of phosphatidylcholine-dependent phospholipase C (PC-PLC) and acidic sphingomyelinase (ASM), which results in generation of the second messengers: diacylglycerol and
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SCOTT D. GRAY-OWEN ETAL
-t
I t "H-J °-A
H8PCS
%>ingomy«iiii
Cytoskeletai rearrangements I'hogocytosis
DAG
Chong
A| A
Helo/CHO HEp-2
Fig. 4 Intracellular signaling events that follow neisserial contact. (A) Heparan sulfate proteoglycan-dependent invasion into cultured epithelial cell lines. Depending on the cultured epithelial cell type being used, Opaso-expressing gonococci can be engulfed by at least three different processes, (a) HSPG-mediated invasion into Chang conjunctiva epithelial cells involves activation of phosphatidylcholine-dependent phospholipase C (PC-PLC), which generates the second messenger diacylglycerol (DAG) from phosphatidylcholine (PC). DAG activates the acidic sphingomyelinase (ASM), which then generates ceramide from sphingomyelin. By an uncharacterized process, ceramide seems to mediate cytoskeletal rearrangements that result in bacterial uptake via a mechanism that resembles conventional phagocytosis, (b) Efficient bacterial uptake into HeLa cervical carcinoma and Chinese hamster ovary (CHO) cells relies on the ability of Opaso to bind both the cellular HSPG receptors and the extracellular matrix protein vitronectin (VN). The subsequent binding of VN to its a^ integrincontaining receptors coligates the HSPG and VN receptor, resulting in bacterial engulfment by a process that requires the activity of protein kinase C (PKC). (c) In Hep-2 larynx carcinoma cells, the efficient uptake of gonococci instead requires Opaso binding to the extracellular matrix protein fibronectin (FN). Presumably, this would result in bacterial uptake being mediated by the coligation of the HSPG and integrin-containing FN receptors in a manner reminiscent to that outlined in (b).
ceramide, respectively (Fig. 4A) [191]. In many other epithelial cell lines (i.e., HeLa human cervical carcinoma and Chinese hamster ovary (CHO) cells), this signaling pathway appears to be less prominent and bacterial entry is poor. In these cell lines, however, an alternative pathway of HSPG-dependent invasion is triggered in the presence of serum. One serum-derived factor that stimulates this phenomenon is the extracellular matrix protein vitronectin (VN) [192, 193]. VN binds specifically to Opaso-expressing gonococci and stimulates bacterial uptake into HeLa cells in an ttvPs and avp5 integrin-dependent manner.
12.
579
NEISSERIA
fl^E CEACAM1 CEACAM6 CEACAM3
Hcr/par--^
« « '
P "lyr
-frr^o'ittU
PAK j JNK i TranscriptioR
1 Phagoi;ytosls
JOSK-M/PMN F/g. 4 (B) CEACAM-dependent invasion into phagocytic cells. The opsonin-independent phagocytosis of Neisseria by polymorphonuclear neutrophils and by the myelomonocytic cell line JOSK-M is mediated by Opa binding to the cellular CEACAMl (biliary glycoprotein, BGP), CEACAM6 (nonspecific crossreacting antigen, NCA), and/or CEACAM3 (CEAgene family member 1, CGMla) receptors. Interactions mediated by the CEACAM-specific Opa proteins (e.g., Opa52) result in activation of the Src-family nonreceptor protein tyrosine kinases Hck and Fgr. This results in an increased level of cellular protein tyrosine phosphorylation and activation of the small GTP-binding protein Racl, which has been implicated in the cytoskeletal rearrangements that ultimately lead to the phagocytic uptake of bound bacteria. Rac 1 also activates p21 -activated protein kinase (PAK) and Jun N-terminal kinase (JNK), probably leading to subsequent activation of nuclear transcription of stress response mediators.
'"*. ?
w . GTPoses
t
PAK
Cytoskeletal rearrangemefits
MKKKs
Epitiieliaiceli
t MKK4 JNL
-'
^
Cytokines/ chem«kliies
Nucleus
1 AP-IJ|NF-KB| X
/
Fig. 4 (C). Opa-independent induction of a cellular cytokine response. Gonococcal binding to epithelial cells also triggers a signaling cascade that is independent of Opa expression or cellular invasion, as evidenced by the fact that it is also seen following infection by nonopaque, noninvasive gonococci. The response involves activation of Rho-family GTPases Rac 1 and/or Cdc42, followed by the activation of a PAK kinase. Subsequent activation of MAP kinase kinase kinase (MKKK), MAP kinase kinase 4 (MKK4), and c-Jun N-terminal kinase (JNK) ultimately results in induction of the immediate early response: basic leucine zipper transcription factor activator protein 1 (AP-1). Whether the resulting synthesis and release of immune response mediators, including some inflammatory cytokines, benefits the host or the infecting microbe is still uncertain.
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SCOTT D. GRAY-OWEN ETAL
This signaling process also appears to depend on the activity of protein kinase C (PKC) (Fig. 4A) [194]. A specific role for HSPG ligation in these two distinct Opa5o-dependent bacterial uptake mechanisms has been confirmed using latex beads coated with antibodies directed against the HS-GAG sidechains of HSPGs, thereby mimicking the binding activity of Opa5(). Consistent with bacterial uptake, these beads were efficiently internalized by Chang cells in the absence of any additional factor, while serum or purified VN (which nonspecifically associates with the beads) was necessary to stimulate efficient uptake into HeLa cells [195]. Aside from VN, the functionally related extracellular matrix protein fibronectin (FN) is also bound by OpasQ-expressing gonococci. This interaction triggers bacterial uptake into HEp-2 cells by coligating HSPGs and the FN-integrin receptor a5 (31 (Fig.4A) [196]. The core protein of different HSPGs may either possess a transmembrane and intracellular domain, composing the syndecan receptor family, or may be glycosylphosphatidylinositol (GPI)-anchored to the membrane, composing the glypican family of HSPGs [197]. Syndecan-4 is widely expressed by many epithelial cell lines. Interestingly, the overexpression of this receptor by HeLa cells increases both the VN-triggered uptake of Opa^o-expressing gonococci and the basal level of uptake in the absence of VN. Overexpression of a mutant form of syndecan-4 that carries a deletion of the cytoplasmic domain instead reduces bacterial uptake [197a]. Syndecan-4 thus seems to play a major role in mediating bacterial uptake, and the cytoplasmic domain appears to be critical for this process. The finding that PKC activity is necessary for bacterial uptake into HeLa cells [194] is consistent with this premise, since PKC binds direcUy to the Syndecan-4 cytoplasmic domain resulting in an increase in its kinase activity [197b]. The roles of other syndecans (e.g., Syndecan-1) and the glypicans in mediating gonococcal interaction with epithelial cells still remain to be investigated. b. HSPG-Mediated Uptake of Opa^Q-expressing Neisseria into other Cell Types. Owing to the ubiquitous expression of HSPGs on eukaryotic cells, Opamediated interactions with HSPGs are not limited to epithelial cells. Opa5o-expressing bacteria bind strongly to endothelial cells, but uptake is inefficient in the absence of additional factors. Recruitment of either VN or FN to the surface of bacteria triggers efficient bacterial internalization in an integrin-dependent manner [197c]. Such a process could contribute to neisserial entry into the bloodstream and/or extravascularization of the bacteria, leading to colonization of nonmucosal tissues during disseminated disease. Fibroblasts, being potential targets in the submucosal tissue, are also bound and invaded by Opa5o-expressing gonococci, albeit with lower efficiency [191]. Although Opa5o expression has little effect on gonococcal interactions with polymorphonuclear leukocytes, it does mediate the efficient phagocytosis of these bacteria by monocytes. Moreover, measurements of luminol-enhanced chemiluminescence also demonstrated that
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the phagocytosis of Opa5()-expressing gonococci by monocytes was accompanied by a release of oxygen-reactive metabohtes [155].
3.
OPA-MEDIATED INTERACTIONS WITH THE CELLULAR
CEACAM
RECEPTORS
Recently, several groups have clearly shown that most neisserial Opa variants specifically bind to the CEACAM receptors that are differentially expressed on multiple tissues within the human host. CEACAM 1 (biliary glycoprotein, BGP), CEACAM6 (nonspecific crossreacting antigen, NCA), CEACAM3 (CEA gene family member 1, CGMl) and CEACAM5 (carcinoembryonic antigen, CEA) can all function as receptors for N. gonorrhoeae and N. meningitidis, while CEACAM8 (CGM6) is not recognized by any Opa protein tested to date. As shown in Table I using the well-characterized Opa variants of N. gonorrhoeae strain MS 11 as an example, individual gonococcal and meningococcal Opa proteins may display various patterns of reactivity with one or more of the CEACAM receptors [198-201]. Although most Opa proteins bind to either CEACAM or HSPG glycoproteins, a few appear to interact with both types of these cellular receptors [106]. The gonococcal Opa variants that do bind to both receptors, however, are only able to mediate cellular invasion via CEACAM [176, 189]. CEACAM proteins represent a subset of the CEA receptor family, which itself belongs to the immunoglobulin superfamily. Each receptor consists of a single, highly conserved amino-terminal immunoglobulin variable region- (Igy-) like domain, followed by a variable number of immunoglobulin Igc2-like constant domains exposed at the cell surface. The carboxy-terminal domains of CEACAM 1 and CEACAM3 contain transmembrane and cytoplasmic domains, while anchorage of CEACAM8, CEACAM6, and CEACAM5 occurs via a glycosylphosphatidylinositol (GPI) anchor attached to the protein's carboxyl terminus [202]. Although the primary role of CEACAM receptors in vivo is still unclear, they appear to mediate intercellular adhesion via both homotypic (CEACAM 1, CEACAM6, and CEACAM5) and heterotypic (CEACAM8-CEACAM6 and CEACAM6-CEACAM5) interactions [203, 204]. This function may or may not be related to their apparent influence on cell cycle control and cellular differentiation [205, 206]. CEACAM 1 and CEACAM6 have also been shown to present the sialyl Lewis^ (sLe^) blood group antigen to E-selectin, and CEACAM6 can stimulate the upregulation of CDl 8-integrins on Huvecs. Both of these processes should facilitate the adherence of PMNs to inflammatory cytokine-stimulated endothelial cells in vivo [207], perhaps targeting the phagocyte to loci of infection. Interestingly, carbohydrate structures expressed by CEACAM 1, CEACAM6, and CEACAM5 have also been shown to function as a cellular receptor for the type 1 fimbriae of E. coli and Salmonella strains. This interaction has been proposed to faciUtate colonic colonization by these species [208, 209].
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SCOTT D. GRAY-OWEN ETAL.
More than 95% of Opa-expressing mucosal and disease isolates of N. gonorrhoeae and A^. meningitidis have been shown to bind CEACAMl [198], thus implying the importance of these interactions for neisserial infection. It is also significant that meningococcal strains expressing both capsule and sialylated lipopolysaccharide are still able to bind CEACAMl [198], since these structures had previously been thought likely to sterically hinder any potential Opa-mediated interactions with a cellular receptor. Although the individual CEACAM proteins are highly glycosylated, these sugar structures do not influence binding by the Opa proteins [210]. Due to their close relationship to immunoglobulin, a three-dimensional structure for the CEACAM protein backbone has been proposed [211]. Neisserial binding studies using transfected epithelial cells that express chimeras of CEACAM8 and CEACAM6 indicate that Opa proteins bind to protein sequences located on a surface composed of four (3-strands (CC'FG) in the amino-terminal domain of CEACAM [201a, 201b, 211a]. The differential specificity of Opa variants to some CEACAM receptors is determined by a divergent tripeptide sequence within this region [211a]. The CC'FG p-sheet has also been proposed to function as the ligand-binding site of other members of the immunoglobulin superfamily, including the closely related membrane-bound CD2 [212] and CD4 [213] receptor proteins. It is thus interesting to speculate that the natural ligand for CEACAM receptors may also bind here. a. CEACAM'Dependent Interactions with Epithelial and Endothelial Cells. Individual CEACAM receptors are differentially distributed on human tissues [202, 214, 215], suggesting that the distinct specificities of individual Opa variants may influence both tissue tropism and the cellular response to bacterial binding. Characterization of Opa binding specificities has been achieved using stably transfected cell lines that express each CEACAM receptor in isolation. In each case, adherence leads to the subsequent engulfment of bound bacteria, thus demonstrating that each family member is itself capable of facilitating cellular invasion [199-201]. The singular importance of Opa proteins in this process is indicated by the fact that E. coli strains expressing recombinant Opa proteins are also efficiently internalized [200, 201]. Several cell lines that naturally express CEACAM receptor proteins have also been identified, and some of these have been employed in infection assays to determine whether the Opa-expressing bacteria are also capable of invading into cells in a somewhat less artificial system. Consistent with what was seen with the transfected cell lines, CEACAM receptors expressed by human colonic (HT29) and lung (A549) epithelial cell lines can mediate meningococcal binding and engulfment [107], and Opa-expressing gonococci have been shown to be taken up by the CEACAM5-expressing LS174T colonic adenocarcinoma cells [200]. We have shown that bacteria expressing CEACAM-specific Opa proteins are capable of passing from the apical to the basolateral surface of polarized T84
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epithelial cell line monolayers without disrupting its transepithelial barrier function [216]. Electron microscopy of infected monolayers clearly shows that this transmigration occurs via the transcytotic route, since gonococci were evident within a tightly adherent phagocytic vacuole in the cell cytoplasm. This process depends on one or more of the CEACAMl, CEACAM6, and CEACAM5 receptors that were seen to be expressed exclusively on the apical surface of the monolayers, since CEACAM-specific antisera can block both bacterial binding and cellular invasion [201, 216, 217]. This remarkable process suggests that the presence of CEACAM on epithelia of the cervix and uterus [215] may mediate an analogous penetration of the epithelia to allow gonococcal dissemination into submucosal tissues. Although primary endothelial cells (Huvecs) grown in culture typically express only very low amounts of CEACAMl, there is a substantial upregulation of its expression following stimulation of these cells with the proinflammatory cytokine tumor necrosis factor alpha (TNF-a) [201, 217]. The upregulated expression of CEACAMl results in a marked increase in binding by recombinant A^. gonorrhoeae strains that express CEACAMl-specific Opa proteins [201], and our preliminary results indicate that this adherence may lead to bacterial uptake by the Huvecs [217a]. b. CEACAM'Dependent Interactions with Phagocytic Cells. Clinical specimens from patients with gonorrhea typically display human polymorphonuclear neutrophils (PMNs) containing intracellular gonococci. In vitro, neisserial binding to PMNs results in opsonin-independent phagocytosis of these bacteria [218220]. CEACAMl, CEACAM8, CEACAM6, and CEACAM3 receptors are thought to be constitutively expressed at low levels on the surface of PMNs, and activation of these cells results in further release of large amounts of CEACAM proteins from primary and secondary granules [221, 222]. Consistent with this, neisserial binding to PMNs increases significandy following their stimulation either by adherence to glass or treatment with phorbol myristate acetate [223, 224]. Opa-mediated binding to CEACAM receptors on PMNs correlates with generation of an enhanced respiratory burst in comparison to that which is stimulated by nonopaque or piliated gonococci, however, one that is reduced when compared to that seen with commensal Neisseria spp. [219, 225, 226]. The presence of Opa-specific F(ab')2 antibody fragments prevents bacterial binding and the subsequent oxidative response, confirming a role for these adhesins in both of these events [219]. The fact that purified Opa protein can also abrogate this response suggests that the induction of an oxidative response requires multiple cellular CEACAM receptors to be ligated by the high density of Opa proteins expressed on the bacterial surface [227]. Whether, and in which way, Opa-directed phagocytosis via CEACAM receptors could provide a survival
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SCOTT D. GRAY-OWEN ETAL.
advantage to the bacterial population is currently still a matter for intriguing speculation. c. CEACAM-Dependent Intracellular Signaling. The cytoplasmic domains of CEACAMl and CEACAM3 contain sequences that have homology to the immunoreceptor tyrosine-based inhibitory (ITIM) and activation (ITAM) motifs, respectively [228, 229]. The presence of these structures on other receptors has been shown to modulate the response of various immune cells. For example, the clustering of ITAM-containing T-cell receptor complexes results in Src kinasemediated activation of T cells, whereas the coligation of an ITIM-containing receptor abrogates this response [230, 231]. Similarly, the stimulation of B cells can be blocked by the inhibitory action of FcyRIIb receptors [232]. Consistent with this [233], CEACAMl has been shown to associate with the Src-family kinases Lyn and Hck [234], and the Opa-mediated binding of A^. gonorrhoeae to neutrophils does result in activation of Hck and Fgr in a process that is essential for bacterial phagocytosis [235]. The Src homology 2-containing tyrosine phosphatase 1 (SHP-1) also interacts with CEACAMl [229] and appears to be inactivated by receptor crosslinking [235a]. Subsequent to these events, the GTPase Racl, the p21-activated protein kinase (PAK), and Jun N-terminal kinase (JNK) have been shown to be stimulated. Bacterial uptake can be reduced either by the presence of Src kinase inhibitors or by the preincubation of cells with anti-sense oligonucleotides, which downregulate the expression of Rac 1, indicating that both activities are essential for this process [235]. This is consistent with the role of the small G-protein Racl in regulating cytoskeletal rearrangements [236], such as those that might be required for neisserial phagocytosis. The stress-activated protein kinase JNK has previously been implicated in the induction of activator protein 1 (AP-l)-regulated transcription [237], suggesting that neisserial binding to CEACAM receptors may trigger a long-term adaptive response. Figure 4B summarizes the signaling cascade mediated by the interaction of Opa and cellular CEACAM receptors in phagocytic cells. It is important to note that the JNK/AP-1 pathway may also be activated in epithelial cells by a mechanism that is independent of Opa expression by the bacteria. This alternate cascade has been shown to involve the Rho family of GTPases and the cellular kinases PAK, MKKK and MKK4 [238] (Fig. 4C; see §VIII.A.2). Whether costimulation of Opa-dependent and Opa-independent signal cascades differentially influences the cellular response to neisserial binding has not yet been determined.
4.
OPA-MEDIATED BINDING TO INTRACELLULAR PYRUVATE KINASE
Although intracellular gonococci are generally considered to remain inside a phagosomal compartment, occasional reports indicate that they may have the
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capacity to escape into the cytoplasm [239]. Recently, gonococcal Opa proteins were reported to bind human pyruvate kinase (PK) subtype M2 in vitro, and this cytoplasmic enzyme appears to associate with intracellular Opa-expressing gonococci [240]. PK catalyzes the irreversible conversion of phosphoenolpyruvate to pyruvate with the resulting generation of ATP. Interestingly, a A^. gonorrhoeae mutant that is unable to use pyruvate or lactate is unaffected in its uptake into epithelial cells, but does not survive intracellularly [240]. It is thus possible that intracellular gonococci bind PK to gain an ample source of pyruvate, one of only three carbon sources known to be used by N. gonorrhoeae. Whether pyruvate does actually play a role in intracellular survival of Neisseria spp. in vivo, however, is still unknown. C. Opc-Mediated Interactions Ope is an outer membrane adhesin of pathogenic Neisseriae that is similar in size to the Opa proteins, but is structurally and antigenically distinct from them [241, 242]. Phase-variable expression of the meningococcal Ope is mediated by slip-strand errors in a homopolymeric cytidine tract residing within the promoter of the ope gene. The resulting changes in spacing between the -10 and -35 promoter elements affects the strength of transcription, resulting in either strong, low, or no expression of Ope (Fig. 3B) [243]. Although Ope has previously been considered to be a meningococci-specific protein, a gonococcal homolog has now been identified [242]. In contrast to the meningococcal ope gene, DNA sequence analysis suggests that expression of the gonococcal homolog is not to be subject to phase variation, but rather to a conventional transcriptional regulation. The gonococcal Ope protein is, therefore, a possible candidate adhesin for the contact-inducible invasion of HeelB cells [244]. However, the functional characterization of Ope has so far been reported only for the meningococcal homolog. Opc-producing meningococci interact with the serum glycoprotein vitronectin, and appear to use this molecule to attach to avP3 integrins that are present on the apical pole of endothelial cells [245]. This molecular bridging may result in bacterial uptake into endothelial cells [92]. Ope expression also promotes meningococcal binding to and entry into certain epithelial cell lines (i.e., Chang conjunctiva cells) in the absence of additional factors [91, 246]. The host-cell receptor for this process has recendy been identified as being syndecan-like heparan sulfate proteoglycans (HSPGs) [106]. The apparently analogous functions of the HSPGA^N-binding Opa proteins (e.g., Opa5(); see §IV.B.2) and Ope seen in vitro makes the reason for neisserial maintenance of these two otherwise unrelated adhesins an intriguing question. D. Interactions Mediated by a Novel Multiple Adhesin Family Screening of known host cell surface components for their capacity to bind gonococci has lead to the description of several lacto- and ganglio-series
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SCOTT D. GRAY-OWEN ETAL
glycolipids as being putative adhesion receptors for A^. gonorrhoeae [247, 248]. For one of these putative receptors (asialo-Gmi-Gral(pl-3)GalNAc(Pl-4)Gal(Pl4)Glc(pl-l)Cer), a corresponding bacterial Hgand has been identified. The heterologous expression of a plasmid encoding a 36-kDa neisserial protein in E. coli was suggested to confer bacterial adhesion to the immobilized glycolipid [249]. More recent experiments suggest that the 36-kDa lipoprotein exhibits a different as-yet-undefined binding function, and that a separate genetically linked adhesin instead possesses the above-cited glycolipid specificity [249a]. These adhesins are both encoded by multiple genes in the neisserial genome, and as members of a multiple adhesin family have now been termed MafA and MafB, respectively. P36/MafA is clearly different from the sialic acid-specific 27-kDa adhesin Sia-1, which is expressed by the commensal species Neisseria flava and recognizes the structure NeuAc(a2-3)Gal(pl-4)Glc on erythrocytes [250]. Although the ubiquitous expression of such glycolipids could potentially contribute to neisserial interactions with many host tissues, the current lack of described function for MafA and Maffi makes their role in neisserial infection uncertain.
V. PorB Porins are a major protein constituent of the neisserial outer membrane. They resemble a hydrophilic pore that allows the passage of small nutrients and waste products across the outer membrane of these Gram-negative bacteria. In other bacteria, such pores are typically formed by a trimeric arrangement of the porin monomer [251, 252]. The three-dimensional structure of neisserial porin protein has not yet been resolved; however, various topological membrane models have been proposed. Each of these predicts 16 membrane-spanning p-strands that come together to form a p-barrel with eight loops exposed at the bacterial surface [253, 254]. A^. meningitidis possesses two different porins: ForA (also called Class 1) and ForB (of either Class 2 or Class 3 serotype). While A^. gonorrhoeae does not express its ForA homolog [255], ForB can be antigenically separated into two serotypes (F.IA and FIB), reminiscent of the mutually exclusive class 2 and class 3 ForB proteins in meningococci [256]. Several unusual features of the gonococcal porin indicate that ForB may have additional functions aside from acting as a simple pore in the outer membrane. The ForB porins of pathogenic Neisseria species have been reported to translocate from live intact bacteria into artificial membranes, a property not observed with porins of commensal Neisseria species [257]. Furthermore, electron microscopic examination of epithelial cells that have been infected with N. gonorrhoeae has revealed that the neisserial ForB is present in the eukaryotic cytoplasmic and phagosomal membranes [190]. It is currently unknown how these integral outer membrane proteins translocate from the bacterial envelope into artificial membranes or into the membrane of target cells. It is, however, possible that differences in membrane fluidity might be the driving force, resulting in
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movement of porin from the higher energy state of the relatively rigid bacterial membrane to the lower energy state of the more fluid cellular membrane [258]. Alternatively, the PorB-containing blebs that are released from the outer membrane of pathogenic Neisseria may fuse with the target cell, or there may be receptor-mediated integration of porin into the cellular membrane. This latter model is similar to that which described for the Staphylococcus spp. a-toxin. After binding to its receptor, multimers of the soluble a-toxin integrate into the target cell membrane to form amphipathic (i-sheet structures that are reminiscent of the porins [259]. Once inserted into the eukaryotic membrane, the neisserial porin forms a channel that is regulated by the cell [260]. This was demonstrated by patch-clamp experiments that showed the porin channel to remain closed in intact living cells but open immediately if the membrane fragment (patch) with inserted porin is excised from the cells. This approach also revealed that PorB has channel characteristics that are very similar to those of a class of eukaryotic porins known as the voltage-dependent anion channels (VDACs). Like neisserial PorB, the opening and closing of these channels is dependent on the applied voltage, with higher voltages (i.e., in the range of 40 to 80 mV), resulting in channel closure. In addition, PorB and VDACs both possess a binding site for nucleotides, which, once bound to the porin, also downregulate pore size and dramatically influence voltage dependence. Even at very low voltages (-10 mV), nucleotides bind to porin and close the channel. Both of these features are likely important for regulation of porin channels by the host cell, since high nucleotide concentrations and a significant membrane potential are present in the eukaryotic cell [260]. In bacteria, these properties appear to be restricted to the PorB porins of pathogenic Neisseria species, since, with the exception of Neisseria lactamica, they were not found in commensal neisserial strains or in other bacterial pathogens [260].
A. Influence of PorB on Cellular Interactions 1.
EFFECTS OF PORB ON THE PHAGOCYTIC RESPONSE
During the infection process, N. gonorrhoeae encounter the bactericidal action of PMNs. Purified gonococcal porin selectively interferes with the signaling machinery of PMNs, inhibiting degranulation, downregulating both actin polymerization and opsonin receptor expression, and reducing the level of bacterial phagocytosis [261, 262]. Consistent with this, recombinant gonococci in which PorB has been replaced by either the porin of the commensal species N. lactamica or by a PorB mutant lacking its amino-terminal extracellular loop are phagocytosed more efficiently by polymorphonuclear neutrophils (PMNs) than are the wild-type controls [263]. Preincubation of PMNs with purified porin also inhibits subsequent phagocytosis of wild-type A^. gonorrhoeae [262]. However, urethral
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SCOTT D. GRAY-OWEN ETAL
exudates from gonorrhea patients contain PMNs associated with both intracellular and extracellular gonococci, indicating that the phagocytic response of neutrophils is not completely abolished by PorB in vivo. Consistent with this, the activity of NADPH-oxidase, an enzyme necessary for the oxidative killing response of phagocytes, is unchanged by the presence of porin [261]. PorB does, however, also influence the maturation of phagosomes within the cell. This was most clearly demonstrated by comparing the protein profile of latex bead-containing phagosomes in cells incubated in the presence or absence of porin, since the composition and amount of proteins present within the phagosome do differ [264]. Several of the proteins enriched in PorB-containing phagosomes were identified as being typical markers of the early endosome (e.g., the Rab 5 protein). Consistent with this, phagosomes containing N. gonorrhoeae do not contain cathepsin G, a marker of mature phagosomes, while this protein is present in phagosomes that harbor phagocytosed E. coli (C. R. Hauck and T. F Meyer, unpublished observations). This is consistent with the fact that degranulation, the release of bactericidal enzymes at the cell surface or into the phagosome during its maturation into a lysosome, is strongly reduced in treated cells (Fig. 5) [261]. Taken together, there is a clear line of evidence that suggests a function of porin in helping A^. gonorrhoeae to survive the encounter with a hostile phagocyte by interfering with phagocytosis and/or processing of ingested bacteria.
2.
EFFECT OF PORB ON NEISSERIAL INTERACTIONS WITH EPITHELIAAND ENDOTHELIA
Mutations in the gonococcal PorB also reduce the level of bacterial invasion into epithelial cells following Opa-mediated binding to the HSPG receptors expressed by epithelial and endothelial cells (see §IV.B.2). This is consistent with the previous observation that the expression of Opa5() in E. coli confers attachment to, but not uptake by, Chang epithelial cells [176, 265]. Thus, HSPG receptor-mediated invasion may need a factor in addition to the Opa^o adhesin to trigger uptake. An interesting finding in this context was that the PorBj^ allele, one of the two major PorB isoforms found in N. gonorrhoeae, mediates invasion into Chang epithelial cells even in the absence of any Opa proteins. However, invasion in this case was strictly dependent on an absence of phosphate in the medium, which apparently bind to PorBj^ porin in a manner similar to nucleotides [266]. The reason for phosphate's inhibitory effect on PorBi^-mediated invasion is not yet clear, but it may close the pore, a function that was previously thought to be restricted to nucleotides [266]. If true, then an open, regulated channel may be the trigger for neisserial uptake into Chang cells. In fact, PorBi^ was recently shown to allow a Ca^"^ influx into target cells [267], an effect that could potentially enhance bacterial uptake by its influence on the activation state of protein kinase C (PKC) [197c].
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589
Porin
-Ik
1 ^ ^ --- ™^
ATP T
Cellular
••
signals
\
y^
••
^ i , ^ i ^ ^
>-r^
±
•
151
\
t
HighCa^*
/
Azurophilic granules
>^: M Active ^ '^^ Active caspases^ coipain
Phogosome Apciptosis
F/g. 5 Model for the function of PorB in cell invasion, intracellular survival, and induction of apoptosis. A direct role of PorB for invasion of Neisseria into host cells has been shown under two experimental conditions: in the presence of phosphate, where invasion of epithelial cells requires expression of Opa5o protein that binds to the HSPG receptors, and under special in vitro conditions in the absence of phosphate, where gonococci invade epithelial cells without expression of any Opa protein. PorB translocates into the host cell cytoplasmic membrane, where it forms channels. PorB channels are closed by noncovalent binding of ATP immediately after translocation. PorB also interferes with phagosome maturation, probably by preventing fusion of the phagosome with azurophilic granules. Furthermore, by an unknown cellular signal (e.g., drop in the ATP level) an opening of the inserted PorB channels and an influx of Ca~^ into the host cell may occur. The increase in intracellular Ca~^, probably together with other signals, induces apoptotic death by active Ca^'^-dependent proteases like calpain and the central executioner proteases, the caspases.
B.
PorB Induction of Apoptosis
A^. gonorrhoeae induces apoptotic cell death of epithelial cells and phagocytes in vitro. Neisserial PorB is itself sufficient to induce this suicide program (Fig. 5) [267], which is present in all cells [268]. Cells treated with purified PorB respond with an immediate, transient uptake of extracellular Ca^^. Within 30 min, they start to form extensive membrane blebs, a hallmark of cells that have entered the apoptotic program. Then, the cells round up, detach from the monolayer, and eventually disintegrate into small particles known as apoptotic bodies. Interestingly, preincubation of porin with ATP closes the channel, thereby preventing both influx of Cd?^ and the subsequent apoptotic death of these cells [267]. This is consistent with the influx of Ca^"^ and the induction of apoptosis being linear processes. Normally, the process of apoptosis is controlled by a class of cysteinyl aspartate-specific proteinases (caspases). These enzymes are also activated in Neisseria- and porin-mediated apoptosis; however, the activity of the cysteine protease calpain is also elevated. Interestingly, the activity of calpain strictly
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SCOTT D. GRAY-OWEN ETAL.
depends on Ca^"^, the level of which is significantly increased in porin-treated cells. Pretreatment of cells with inhibitors of caspases and calpain reduces porin-induced apoptotic cell death more efficiently than does inhibition of either caspases or calpain alone. This result suggests that porin induces cell death by at least two independent pathways. Dual activation of caspases and calpain is rarely seen during control of apoptotic cell death, making neisserial induction of this novel pathway a particularly intriguing topic of future study.
W. IgA 7 Protease There is a strict correlation between the virulence of neisserial species and their ability to synthesize and secrete a serine protease that was originally shown to display a high specificity for human IgAl antibodies. Because of this, the IgAl protease is presumed to have an important role in neisserial disease [269]. Numerous serologically distinct IgAl proteases exist in A^. gonorrhoeae and A^. meningitidis due to the horizontal exchange of genetic sequences [55, 270, 271]. In each case, the iga sequences encode an approximately 170-kDa precursor protein consisting of five distinct domains [272]. The amino-terminal signal peptide and carboxy-terminal IgAp domain are responsible for transporting the IgAl protease (IgAp) and its associated domains to the bacterial surface (Fig. 6). Ultimately, the soluble IgAp^^y preproteins are cleaved from the transporter domains, and are then cleaved in a stepwise fashion to generate mature IgAl protease, a-protein, and y-peptide products. Proper secretion of neisserial IgAl protease expressed in E, coli [273] implies that this "autotransporter" process is intrinsic in the precursor protein. The contribution of IgAl protease to the infection process is still unclear, since, for example, the levels of bacterial attachment, invasion, and damage seen during infection of human fallopian tube organ cultures are indistinguishable between an IgAl protease-deficient mutant and its parental strain [274]. Mucosal secretory immunity is primarily mediated by IgA antibodies. It is interesting that the IgAl subclass predominates in secretions at the primary surfaces colonized by the Neisseria (i.e., respiratory and urogenital mucosa), while IgA2, which is not cleaved by the neisserial protease, is present only in relatively small amounts. Cleavage of IgAl antibodies by the neisserial protease leads to separation of the F(ab) fragment, which is involved in antigen binding, from the Fc domain, which is involved in effector function. This should result in the pathogen becoming decorated by IgA F(ab) fragments, thus masking the immunodominant epitopes that could otherwise be targets for IgG-mediated complement activation. Although IgAl protease has a high cleavage specificity for IgAl antibodies, the elucidation of its cleavage consensus sequence prompted a search for other potential targets that may be encountered during infection. The human lyso-
12.
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NEISSERIA
IgA proteose Igopra
.Slgnol peptide
Cytopiosm
Fig. 6 IgAl protease secretion. Following the N-terminal signal peptide-directed transport of the IgA 1 protease polyprotein (Iga) through the cytoplasmic membrane, the signal peptide is cleaved off the polyprotein. The C-terminal Igap domain then integrates into the outer membrane, forming an amphipathic P-barrel with a putative internal pore through which the remainder of the protein can translocate to the bacterial cell surface. This translocation step may also be necessary for correct folding of the IgAl protease into an enzymatically active conformation. Autoproteolytic cleavage at sites a, b, and c then leads to generation of mature IgAl protease, a-protein, and the small y-peptide. The functional significance of a-protein and y-peptide are still uncertain.
some/late endosome associated membrane protein 1 (h-lamp-1) contains an IgAl protease cleavage consensus sequence, and purified h-lamp-1 is cleaved in an in vitro reaction under conditions that are similar to those found in phagosomal compartments [275, 277]. h-lamp-1 is a major component of the lysosomal membrane, and is thought to protect the lysosomal membrane against the degradative enzymes present within the lysosome. This function is likely facilitated by the protein's high level of glycosylation, which should form a carbohydrate coat lining the luminal face of the lysosome [278, 279]. Consistent with the in vitro cleavage results, one report suggested a decrease of the intracellular levels of h-lamp-1 during neisserial infection of A431 endocervical epithehal cells, and the survival and growth of IgAl protease-deficient gonococcal mutants associated with these cells has been reported to be significantly reduced as compared to that of the isogenic parental strain [277]. A parallel study did not, however, show a similar effect of IgAl protease mutations on intracellular survival in other human epithelial cell types [275], suggesting that such a phenomenon may be cell-type dependent. This conclusion is consistent with the finding that h-lamp-1 produced
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by phagocytic cells is not cleaved by the neisserial protease, likely due to the higher glycosylation of the phagocyte-expressed form of this protein [275]. Whether the potential effect of IgAl protease on phagosomal integrity might play a role in the suggested phagosomal escape of gonococci [239, 240] is an intriguing hypothesis. The release of the IgAl protease precursor into the cytosol of an infected cell would probably lead to targeting of IgAl protease and its associated a-protein to the nucleus owing to a functional nuclear location signal in the precursor sequence [276].
VIL Iron Acquisition in Vivo Iron is essential for life, and the sequestration of iron from invading microbes is an important mechanism of a host's defence against the establishment and propagation of bacterial infection. More than 99.9% of iron is held intracellularly in the storage protein ferritin or as a component of heme compounds. The rest is carried by the host's extracellular iron transport and sequestration proteins transferrin and lactoferrin, which are present in serum and mucosal secretions, respectively. In order to circumvent this normally effective means of nutritional immunity, the pathogenic Neisseria are capable of obtaining iron from transferrin and lactoferrin. Iron removal from transferrin and lactoferrin is mediated by two transferrin (TbpA and TbpB) or lactoferrin (LbpA and LbpB) binding proteins expressed at the bacterial surface (Fig. 7) [280]. TbpB and LbpB likely function in the initial binding of their respective host glycoproteins, while the integral outer membrane proteins TbpA and LbpA strengthen this interaction and function as gated pores through which iron can pass into the periplasmic space. This is an energy-dependent process that is thought to be powered by interactions between TbpA and the cytoplasmic membrane-bound TonB protein complex [281]. Once in the periplasm, the free iron is bound by a soluble ferric binding protein (FbpA), which then shutdes the iron to an ATP-driven cytoplasmic membrane permease complex for its delivery into the bacterial cytoplasm [282]. The importance of this process in vivo has been clearly demonstrated using experimental infection of human male volunteers, since Tbp mutants are greatly reduced in their ability to colonize the urogenital tract and are completely unable to cause urethritis [283]. Neisseria are also capable of acquiring iron from free heme, hemoglobin, or hemoglobin bound to its serum carrier protein haptoglobin [284]. Each of these molecules is bound by a different receptor [285], and each process likely leads to bacterial internalization of the intact haem molecule, which is comprised of a protoporphyrin ring complexed with iron. Uptake from hemoglobin or hemoglobin-haptoglobin complexes also requires TonB function, while the binding and uptake of free haem does not [286]. These processes have the obvious benefit that
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they result in assimilation of both iron and porphyrin, a prosthetic group used by enzymes including those involved in respiration and oxygen metabolism. Due to the high-affinity interaction between heme and oxygen, heme accumulation at the bacterial surface has also been proposed to be a mechanism that could provide some resistance to toxic oxidative compounds encountered during the respiratory response of phagocytic cells [285]. Whether this protective effect actually occurs, however, remains to be determined.
W//. Immune Response Neisserial disease characteristically results from a massive inflammatory response to bacteria that have already established an infection. Therefore, it is the immune system that generates the pathology associated with symptomatic neisserial disease, leading to possible outcomes that include tissue destruction, septic shock, and/or death of the infected individual. The fact that only some people colonized with Neisseria present with any clinical manifestations suggests that other factors also contribute to the onset of disease. Several avenues of research have begun to shed light on the infectious process in vivo, and to suggest mechanisms by which to prevent a detrimental outcome of established neisserial infections.
A.
Cellular Response to Neisserial Infections 1.
CYTOKINE RESPONSE TO THE PATHOGENIC NEISSERIA
Several human male volunteer infection studies have found that, immediately following gonococcal infection of the urethra, there is a period during which bacteria are liberated in the urine. After this, a lag phase occurs during which few bacteria can be recovered before the symptomatic infection ensues [88, 173, 174]. This "lag" is not a period of inactivity by the host, since it is accompanied by an inflammatory response that results in increased levels of interleukin 6 (IL-6) and 8 (IL-8) and tumor necrosis factor alpha (TNF-a) being seen. In plasma, IL-lp, IL-6, IL-8, and TNF-a are also found to be elevated by the time symptoms arise. Consistent with the chemotactic function of IL-8, increased numbers of neutrophils are found in urine soon after infection [287], while the elevation of TNF-a, IL-1, and IL-6 levels should activate both T and B cells [288]. The recruitment of neutrophils and the lymphocytic release of serum factors thereby induced likely play a protective role that is at least partially effective at clearing the organisms that have already colonized the mucosa. The massive inflammation that occurs during symptomatic gonorrhea can also destroy the integrity of infected epithelia, allowing the bacteria to gain better access to deeper tissues [289]. Interestingly, TNF-a, IL-6, and IL-8 transcript levels are not elevated in peripheral blood mononuclear cells during gonococcal infection [287]. Although
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cytokine production was initially thought to be restricted to professional immune cells, it is now clear that many nonimmune cells, including epithelial and endothelial cells, can also produce some of these mediators [290]. Consistent with certain cytokines being produced at the site of mucosal colonization, the in vitro infection of epithelial cells by N. gonorrhoeae has demonstrated that synthesis of several cytokine mRNAs (i.e., GM-CSF, TNF-a, IL-8, MCP-1, TGF-p, and IL-lb) occurs within 15 min of infection. The levels of GM-CSF, IL-8, and TNF-a were then seen to steadily increase in the culture supernatant [291]. In contrast, other cytokines are induced with a somewhat slower time course (e.g., IL-6 was detected by 45 min). A similar pattern of proinflammatory cytokines is also induced on gonococcal infection of primary human umbilical vein endothelial cells (Huvecs) in vitro (P. Muenzner, M. Naumann, T. F. Meyer, and S. D. Gray-Owen, unpublished observations), suggesting that similar immune mechanisms may be responsible for combatting both localized and disseminated gonococcal diseases. Cumulatively, the production of these proinflammatory cytokines in vivo should generate a strong inflammatory response, resulting in activation and recruitment of granulocytes, lymphocytes and macrophage/monocytes to the locus of infection. Cytochalasin D treatment (which disrupts the actin cytoskeleton) blocks gonococcal invasion into the epithelial cells but does not influence cytokine mRNA levels, indicating that bacterial attachment is itself sufficient to induce the proinflammatory response [291]. LPS does not appear to be the inducer of this cytokine synthesis, since epithelial cells showed relatively little response to purified LPS [291]. This could be explained because epithelial cells do not normally express the CD 14 LPS receptor; however, the soluble CD 14 that is present in serum should provide a similar function in vivo [292]. Infection of epithelial cells by other bacterial pathogens can induce a different spectrum of cytokines [293, 294], suggesting that the cytokine pattern induced by A^. gonorrhoeae is not a general response to bacterial infection. TNF-a and IL-lb also play a pivotal role in triggering meningococcal-induced inflammation, and IL-6, which is elevated in patients with bacterial meningitis, has been implicated in the pathology of this infection [295]. The cytokines are generally recruited from serum, thus necessitating their passage across the blood-brain barrier and release into the subarachnoid space [295]. They may, however, also be produced by glial cells and brain capillary endothelial cells [296]. The inflammatory response that results from these cytokine signals leads to disruption of the blood-brain barrier and alteration of cerebral metabolism. Therapeutic strategies to intervene in this process have been undertaken. For example, administration of polyclonal anti-TNF-a antiserum in an infant rat model of meningococcal infection reduces mortality relative to animals pretreated with control serum but does not alter bacterial invasion into the cerebrospinal fluid [297]. Whether similar effects will be seen in humans is still unknown.
596 2.
SCOTT D. GRAY-OWEN ETAL INTRACELLULAR SIGNALS THAT LEAD TO THE CYTOKINE RESPONSE
Gonococcal contact with epithelial cells is sufficient to induce a cytokine response [291]. Infection of HeLa cells with piliated nonopaque gonococci, which are adherent but noninvasive, induces the immediate early response transcription factors nuclear factor kappa B (NF-KB) and the dimeric sequence-specific transcription factor activator protein 1 (AP-1). An analysis of transiently transfected IL-6 promoter deletion constructs confirms the importance of both NF-KB and AP-1 enhancer elements for transcriptional activation of the IL-6 promoter on neisserial infection [238]. The activation of NF-KB by N. gonorrhoeae occurs at a multiplicity of infection of 5, indicating a highly active response to this pathogen. The NF-KB complex is activated and translocates into the nucleus within 10 minutes after infection, at which point it is composed of a p50/p65 heterodimer [291]. The level of this NF-KB transactivation depends on the epithelial cell line being used in vitro [291]. It is therefore possible that target cells respond in a tissue-specific manner, although differences at the level of bacterial association to these cell lines cannot be ruled out. Gonococcal activation of AP-1 and the subsequent inflammatory cytokine gene expression are mediated by the c-Jun N-terminal kinase (JNK) pathway in HeLa epithelial cells (Fig. 4C) [238, 291]. The specificity of this response is illustrated by the fact that, except for AP-1, the basic leucine zipper transcription factors, including those that bind to the cAMP responsive element (CRE) or CAAT/enhancer binding protein (C/EBP) binding sites, are not induced by the gonococci. Although the initial trigger by which Neisseria induces this pathway is still unknown, this stress-response cascade has previously been shown to be induced by stimuli including various growth factors and cytokines, UV light, and protein synthesis inhibitors [298]. Consistent with JNK pathway involvement in the cellular response to gonococci, the Rho family of small GTPases appear to be involved in cytoskeletal rearrangements that follow neisserial binding [235, 265]. Studies have also shown that pretreatment of epithelial cells with enterotoxin B from Clostridium difficile, which glucosylates and inactivates the Cdc42, Racl, and Rho small GTPases [299], and blocks both N. gonorrhoeae-inducQd AP-1 and JNK activation [238]. Downstream of the GTPases, the p21-activated kinases (PAKl and PAK2) are also essential for the cytokine response, since the transient transfection of dominant negative mutants into HeLa cells blocks AP-1 transactivation, while overexpression of PAK2 instead enhances AP-1-dependent transcription in response to N. gonorrhoeae. Consistent with this, the kinase activity of endogenous PAK2 was also seen to be elevated following gonococcal infection [238]. It thus appears that the cellular response to N. gonorrhoeae attachment involves induction of proinflammatory cytokine secretion via a cascade of cellular stress response kinases. This pathway involves Rho family of GTPases, PAK, MAP kinase kinase kinase (MKKK), MKK4, JNK, and AP-1, ultimately inducing
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a coordinated response of cytoskeletal changes and transcriptional activity that yields the release of the immune response mediators.
B. Humoral Response to Neisserial Infection 1.
HUMORAL IMMUNITY
Clearly, humoral immunity is of fundamental importance in protection against neisserial infection. Individuals with a deficiency in one or more components of the complement cascade have a significantly increased susceptibility to neisserial disease [300]. Immunity generally correlates with bactericidal antibodies [301, 302], which induce the complement-mediated lysis of these bacteria [303-305]. Such antibodies can be directed against various surface components of the bacteria, including porin (see §V) [306], the transferrin receptor (see §VII) [307], LOS (see §III.A) [308], and the meningococcal capsule (see §III.B). These are, however, often highly strain specific due to the antigenic diversity of Neisseria, and are thus frequently not crossprotective against heterologous strains of the same species. In normal human serum, bactericidal immunoglobulin M (IgM) antibodies that recognize epitopes on certain neisserial lipooligosaccharide (LOS) molecules are present [308]. However, N. gonorrhoeae taken from urethral exudates are generally resistant to the killing effects of serum [309] due to sialylation of their LOS (see §III.A.l) [310]. Bacteria that express PorBjA have also been reported to be more serum resistant than those expressing PorBjg [306]. This association, however, is more likely to result from factors that are genetically linked to the por genes than from a direct effect of porin itself (see §I.B.l). The "reduction-modifiable protein" (Rmp) is another determinant of stable serum resistance. Antibodies generated against this surface-expressed protein compete with bactericidal antibodies for binding to A^. gonorrhoeae and interfere with proper insertion of the complement membrane attack complexes into its membrane [311]. In the case of A^. meningitidis, a deficiency of circulating antibodies has been shown to be associated with establishment of meningococcemia [301, 302]. The prevalence of bactericidal antibodies in a given population varies for different serogroups, but 67% of individuals have been reported to possess bactericidal activity against N. meningitidis serogroup A versus 86% for group B [301, 302]. It is therefore interesting that the meningococcal capsular polysaccharide antigens, which can protect the bacteria by inhibiting neutrophil phagocytosis and by preventing the bactericidal activity of complement, also plays a prominent role in the development of natural immunity and, ultimately, in immunologic protection of the host against colonization by the meningococci [312, 313]. Due to the maternal transfer of antibodies in the placenta and in milk, bactericidal antibodies against the meningococci can be detected in approximately 50% of infants at birth.
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The antibody titer then decreases after birth, reaching its lowest level when the child is between 6 and 24 months of age before increasing again until the age of 12 years. The asymptomatic carriage oiN. meningitidis (see §I.B.2) also appears to be an immunizing process, as evidenced by the fact that, while new military recruits have a high frequency of meningococcal carriage and disease, much lower rates are seen in seasoned veterans [301, 302, 314]. Natural immunity to some meningococcal strains may also develop by exposure to some strains of the commensal species Neisseria lactamica [308, 315]. Such observations help to explain why older adults with meningococcal disease often also have some other predisposing immunocompromising condition [48], since healthy individuals would normally have been exposed to protective crossreactive antigens by this time.
2.
POTENTIAL VACCINE TARGETS
Despite the importance of complement in protection against neisserial infection, serum that is deficient in terminal components of the complement cascade can trigger opsonin-mediated engulfment of Neisseria [316]. This is critical because it suggests that vaccination of susceptible individuals may also be protective by shifting immunity from serum bactericidal activity to antibody-dependent phagocytosis. The development of a protective vaccine against the pathogenic Neisseria, however, is complicated by the high level of intra- and interstrain antigenic variation that occurs in most surface-exposed antigens. Because of this, an effective vaccine that is broadly crossprotective against all neisserial strains is still unavailable, despite the years of research that has strived toward this important goal. Currently, commercial vaccines containing the meningococcal serogroups A, C, Y, and W-135 polysaccharide capsules are available. These preparations are safe and immunogenic in adults, but since carbohydrates are T-cell-independent antigens, they induce only a weak, predominandy IgM response with no immunological memory in infants. This problem may be overcome by preparation of protein conjugates of these capsular carbohydrates, since this has proven to be a highly effective mechanism of generating a T-cell-dependent memory response against the Haemophilus influenzae type b capsular polysaccharide [317]. Unfortunately, there is currently no carbohydrate vaccine available against the capsule of type B N. meningitidis, a serogroup that causes much of the endemic meningococcal disease in the Northern Hemisphere [318]. The lack of immunogenicity to this a-2,8 linked homopolymer of sialic acid may be due to its antigenic crossreactivity with human fetal or other tissues, a characteristic that would make it inappropriate for use as a target vaccine antigen. In order to circumvent the problems associated with carbohydrate antigens, an outer membrane vesicle (OMV) vaccine from group B meningococci is currently being assessed. This preparation, which contains the major outer membrane
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proteins (e.g., PorB, Opa, Ope), small amounts of less well-characterized proteins, and about 8% LPS, has been tested in volunteers. Intramuscular injection and nasal inhalation of the OMV vaccine have both been found to generate significant antibody responses, indicating that such preparations must continue to be explored. Obviously, purified and well-characterized antigens are preferable for use as vaccine constituents since they have a lower likelihood of causing adverse side effects following immunization. One of the most obvious antigens to consider for such purposes is pilin, the major constituent of neisserial pili (see §IV.A). Unfortunately, the incredible antigenic variation of this protein prevents generation of significant interstrain crossreaction of the generated immune responses. Other potential candidates are the neisserial porin proteins, since these are the most abundant proteins expressed in the outer membrane, are constitutively expressed and do not undergo high-frequency antigenic variation (see §V). Although some interstrain antigenic differences in the gonococcal PorB proteins do exist [8], monoclonal antibodies that crossreact against all isolates from the PorBiA or the PorBjg can be generated. This suggests that mixing only a few serotypes of porin could potentially produce a crossprotective vaccine. Some anti-porin monoclonal antibodies have also been shown to be bactericidal; they stimulate an oxidative burst by neutrophils and toxicity for cultured epithelial cells [319, 320], indicating that they may also provide protection via several different mechanisms. Despite these potential benefits, the bactericidal effect of these anti-porin antibodies is abolished by neisserial sialylation of LOS or by the meningococcal expression of capsule, both of which mask the surface exposure of the porin epitopes [310, 321]. Perhaps the most promising candidates as components of a purified protein vaccine are the transferrin-binding proteins (TbpA and TbpB; see §VII). They are essential for the establishment of infection [283] and by definition must be exposed at the bacterial surface in order to interact with the large (i.e., 80 kDa) host serum protein transferrin. Recent analysis of meningococcal tbpB genes suggests that horizontal genetic exchange may generate highly divergent alleles [322]. However, TbpB proteins are protective in animal models of infection [323, 324], and immunization with TbpB from only a few different strains appears to be sufficient to generate broadly crossreactive bactericidal antibodies against all clinical isolates tested to date [325]. The sequences of Tbp proteins from various gonococcal and meningococcal strains also indicate that they are highly conserved [326, 327], suggesting that such a vaccine may also be crossprotective against both pathogenic species.
IX. Summary The pathogenic Neisseria display a remarkable capacity for antigenic and phenotypic variation. This characteristic appears to be critical for the persistence
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of these important human pathogens within both a single host and in the human population, since it allows the bacteria to evade the powerful immune response that can be triggered by its presence. Unfortunately, it is this immune response that causes the pathology associated with neisserial diseases, since these bacteria do not appear to secrete enzymes or toxins with an aim to directly insult the host. It is also significant in this regard that a high proportion of infections caused by these organisms are asymptomatic, since this feature maintains a constant pool of unsuspecting carriers that can further disseminate the microbe. Although significant insights have been gained into the mechanisms of neisserial interactions with host cells in vitro, much is still unknown with regard to the means by which bacteria affect the host response. Hopefully, the future will allow us to see further into the host-microbe interactions that contribute to the success of Neisseria as a parasite, and to gain an understanding and ability to better control the immune mechanisms that are evaded during neisserial disease.
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239. Shaw, J. H., and Falkow, S. (1988). Model for invasion of human tissue culture cells by Neisseria gonorrhoeae. Infect. Immun. 56, 1625-1632. 240. Williams, J. M., Chen, G. C , Zhu, L., and Rest, R. F. (1998). Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: Intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth. Mol Microbiol. 27, 171-186. 241. Achtman, M., Wall, R. A., Bopp, M., Kusecek, B., Morelli, G., Saken, E., and Hassan-King, M. (1991). Variation in class 5 protein expression by serogroup A meningococci during a meningitis epidemic. J. Infect. Dis. 164, 375-382. 242. Merker, R, Tommassen, J., Kusecek, B., Virji, M., Sesardic, D., and Achtman, M. (1997). Two-dimensional structure of the Ope invasin from Neisseria meningitidis. Mol. Microbiol. 23, 281-293. 243. Sarkari, J., Pandit, N., Moxon, E. R., and Achtman, M. (1994). Variable expression of the Ope outer membrane protein in Neisseria meningitidis is caused by size variation of a promoter containing poly-cytidine. Mol. Microbiol. 13, 207-217. 244. Spence, J. M., Chen, J. C , and Clark, V. L. (1997). A proposed role for the lutropin receptor in contact-inducible gonococcal invasion of HeelB cells. Infect. Immun. 65, 3736-3742. 245. Virji, M., Makepeace, K., and Moxon, E. R. (1994). Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions. Mol. Microbiol. 14, 173-174. 246. Virji, M., Makepeace, K., Ferguson, D. J. R, Achtman, M., Sarkari, J., and Moxon, E. R. (1992). Expression of the Ope protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis. Mol. Microbiol. 6, 2785-2795. 247. Stromberg, N., Deal, C , Nyberg, G., Normark, S., So, M., and Karlsson, K. (1988). Identification of carbohydrate structures that are possible receptors for Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. U.S.A. 85, 4902^906. 248. Deal, C. D., and Krivan, H. C. (1990). Lacto- and ganglio-series glycolipids are adhesion receptors for Neisseria gonorrhoeae. J. Biol. Chem. 265, 12774-12777. 249. Purachuri, D. K., Seifert, H. S., Ajioka, R. S., Karlsson, K. A., and So, M. (1990). Identification and characterization of a Neisseria gonorrhoeae gene encoding a glycolipid-binding adhesin. Proc. Natl. Acad. Sci. U.S.A. 87, 333-337. 249a. Eickemjager, S., Meyer, T. F, Fischer, E., Maier, J., Rudel, R., Scheuerpflug, I., and Schwan, T. Manuscript in preparation. 250. Nyberg, G., Stromberg, N., Jonsson, A., Karsson, K. A., and Normark, S. (1990). Erythrocyte gangliosides act as receptors for Neisseria flava: Identification of the Sia-1 adhesin. Infect. Immun. 58, 2555-2563. 251. Weiss, M. S., Abele, U., Weckesser, J., Welte, W., Schiltz, E., and Schulz, G. E. (1991). Molecular architecture and electrostatic properties of a bacterial porin. Science 254, 1627-1630. 252. Cowan, S. W., Schirmer, T., Rummel, G., Steiert, M., Ghosh, R., Pauptit, R. A., Jansonius, J. N., and Rosenbusch, J. P. (1992). Crystal structures explain functional properties of two E. coli porins. Nature 358, 727-733. 253. Jeanteur, D., Lakey, J. H., and Pattus, F (1991). The bacterial porin superfamily: Sequence alignment and structure prediction. Mol. Microbiol. 5, 2153-2164. 254. van der Ley, P., van der Biezen, J., Sutmuller, R., Hoogerhout, P., Poolman and J. T. (1996). Sequence variability of FrpB, a major iron-regulated outer-membrane protein in the pathogenic Neisseriae. Microbiology 142, 3269-3274.
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255. Feavers, I. M., and Maiden, M. C. (1998). A gonococcal porA pseudogene: Implications for understanding the evolution and pathogenicity of Neisseria gonorrhoeae. Mol. Microbiol. 30, 647-656. 256. Hitchcock, P. J. (1989). Unified nomenclature for pathogenic Neisseria species, Clin. Microbiol. Rev. 2, 64-65. 257. Lynch, E. C , Blake, M. S., Gotschlich, E. C , and Mauro, A. (1984). Studies on porins: Spontaneously transferred from whole cells and from proteins of Neisseria gonorrhoeae and Neisseria meningitidis. Biophys. J. 45, 104-107. 258. Blake, M. S., and Gotschlich, E. C. (1987). Functional and immunological properties of pathogenic Neisseria surface proteins. In "Bacterial Outer Membranes as Model Systems" (M. Inouye, ed.), pp. 377-400. Wiley, New York. 259. Song, L., Hobaugh, M. R., Shustak, C , Cheley, S., Bayley, H., and Gouaux, J. E. (1996). Structure of staphylococcal alpha-hemolysin, a heptameric transmembrane pore. Science 11^, 1859-1866. 260. Rudel, T., Schmid, A., Benz, R., Kolb, H. A., Lang, F, and Meyer, T. F (1996). Modulation of Neisseria porin (PorB) by cytosolic ATP/GTP of target cells: Parallels between pathogen accommodation and mitochondrial endosymbiosis. Cell 85, 391-402. 261. Haines, K. A., Yeh, L., Blake, M. S., Cristello, P, Korchak, H., and Weissmann, G. (1988). Protein I, a translocatable ion channel from Neisseria gonorrhoeae, selectively inhibits exocytosis from human neutrophils without inhibiting O2 generation. J. Biol. Chem. 263, 945-951. 262. Bjerknes, R., Guttormsen, H. K., Solberg, C. O., and Wetzler, L. M. (1995). Neisserial porins inhibit human neutrophil actin polymerization, degranulation, opsonin receptor expression, and phagocytosis but prime the neutrophils to increase their oxidative burst. Infect. Immun. 63, 160-167. 263. Bauer, F. J., Rudel, T., Stein, M., and Meyer, T. F (1999). Mutagenesis of the Neisseria gonorrhoeae porin reduces invasion in epithelial cells and enhances phagocyte responsiveness. Mol. Microbiol. 31, 903-913. 264. Mosleh, I. M., Huber, L. A., Steinlein, P, Pasquali, C., Gunther, D., and Meyer, T. F (1999). Neisseria gonorrhoeae porin modulates phagosome maturation. J. Biol. Chem. 274, 3533235338. 265. Grassme, H. U., Ireland, R. M., and van Putten, J. P. (1996). Gonococcal opacity protein promotes bacterial entry-associated rearrangements of the epithelial cell actin cytoskeleton. Infect. Immun. 64, 1621-1630. 266. van Putten, J. P., and Duensing, T. D. (1998). Gonococcal invasion of epithelial cells driven by PI A, a bacterial ion channel with GTP binding properties. J. Exp. Med 188, 941-952. 267. Muller, A., Gunther, D., Dux, F, Naumann, M., Meyer, T. F, and Rudel, T. (1999). Neisserial porin (PorB) causes rapid calcium influx in target cells and induces apoptosis by the activation of cysteine proteases. EMBO J. 18, 339-352. 268. Martin, S. J., and Green, D. R. (1995). Protease activation during apoptosis: Death by a thousand cuts? Cell 82, 349-352. 269. Kilian, M., Reinholdt, J., Lomholt, H., Poulsen, K., and Frandsen, E. V. (1996). Biological significance of IgAl proteases in bacterial colonization and pathogenesis: Critical evaluation of experimental evidence. APMIS 104, 321-338. 270. Lomholt, H., Poulsen, K., Caugant, D. A., and Kilian, M. (1992). Molecular polymorphism and epidemiology of Neisseria meningitidis immunoglobulin Al protease. Proc. Nad. Acad. Sci. U.S.A. 89,2120-2124.
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291. Naumann, M., Wessler, S., Bartsch, C , Wieland, B., and Meyer, T. F. (1997). Neisseria gonorrhoeae epithelial cell interaction leads to the activation of the transcription factors NF-KB and API and the induction of inflammatory cytokines. J. Exp. Med. 186, 247-258. 292. Pugin, J., Schurer-Maly, C. C , Leturcq, D., Moriarty, A., Ulevitch, R. J., and Tobias, P S. (1993). Lipopolysaccharide activation of human endothelial and epithelial cells is mediated by lipopolysaccharide-binding protein and soluble CD 14. Proc. Natl. Acad. Sci. U.S.A. 90, 2744-2748. 293. Agace, W., Hedges, S., Andersson, U., Andersson, J., Ceska, M., and Svanborg, C. (1993). Selective cytokine production by epithelial cells following exposure to Escherichia coli. Infect. Immiin. 61, 602-609. 294. Hedges, S., Agace, W., Svensson, M., Sjogren, A. C , Ceska, M., and Svanborg, C. (1994). Uroepithelial cells are part of a mucosal cytokine network. Infect. Imnum. 62, 2315-2321. 295. Beutler, B., Krochin, N., Milsark, I. W., Luedke, C , and Cerami, A. (1986). Control of cachectin (tumor necrosis factor) synthesis: Mechanisms of endotoxin resistance. Science 232, 977-980. 296. Moller, B., Mogensen, S. C, Wendelboe, P, Bendtzen, K., and Petersen, C. M. (1991). Bioactive and inactive forms of tumor necrosis factor-alpha in spinal fluid from patients with meningitis. J. Infect. Dis. 163, 886-889. 297. Nassif, X., Mathison, J. C, Wolfson, E., Koziol, J. A., Ulevitch, R. J., and So, M. (1992). Tumour necrosis factor alpha antibody protects against lethal meningococcaemia. Mol. Microbiol. 6,591-597. 298. Karin, M., Liu, Z., and Zandi, E. (1997). AP-1 function and regulation. Curr Opin. Cell Biol. 9, 240-246. 299. Aktories, K. (1997). Rho proteins: Targets for bacterial toxins. Trends Microbiol. 5, 282-288. 300. Figueroa, J. E., and Densen, P. (1991). Infectious diseases associated with complement deficiencies. Clin. Microbiol. Rev. 4, 359-395. 301. Goldschneider, I., Gotschlich, E. C , and Artenstein, M. S. (1969). Human immunity to the meningococcus, II: Development of natural immunity. J. Exp. Med. 129, 1327-1348. 302. Goldschneider, I., Gotschlich, E. C , and Artenstein, M. S. (1969). Human immunity to the meningococcus, I: The role of humoral antibodies. J. Exp. Med. 129, 1307-1326. 303. Nicholson, A., and Lepow, I. H. (1979). Host defense against Neisseria meningitidis requires a complement-dependent bactericidal activity. Science 205, 298-299. 304. Griffiss, J. M. (1982). Epidemic meningococcal disease: Synthesis of a hypothetical immunoepidemiologic model. Rev. Infect. Dis. 4, 159-172. 305. Ross, S. C , and Densen, P. (1984). Complement deficiency states and infection: Epidemiology, pathogenesis and consequences of Neisserial and other infections in an immune deficiency. Medicine 63, 243-273. 306. Rice, P. A. (1989). Molecular basis for serum resistance in Neisseria gonorrhoeae. Clin. Microbiol. Rev. 2, 112-117. 307. Rokbi, B., Mignon, M., Maitre-Wilmotte, G., Lissolo, L., Danve, B., Caugant, D. A., and Quentin-Millet, M. J. P. (1997). Evaluation of recombinant transferrin-binding protein B variants from Neisseria meningitidis for their ability to induce cross-reactive and bactericidal antibodies against a genetically diverse collection of serogroup B strains. Infect. Immun. 65, 55-63. 308. Griffiss, J. M., Yamasaki, R., Estabrook, M., and Kim, J. J. (1991). Meningococcal molecular mimicry and the search for an ideal vaccine. Trans. Roy. Soc. Trop. Med. Hyg. 85 (Suppl.), 6. 309. Ward, M. E., Watt, P. J., and Glynn, A. A. (1970). Gonococci in urethral exudates possess a virulence factor lost on subculture. Nature 227. 382-384.
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CHAPTER 13
Bordetella PEGGY A. COTTER JEFF R MILLER
I. Introduction II. Respiratory Infections byfit-Wf/W/rtSpecies A. B. pertussis Infections of Humans B. B. parapertussis Infections of Humans and Sheep C. B. bronchiseptica Infections of Mammals III. Evolutionary Relationships among Bordetella Subspecies IV. Bordetella Virulence Factors A. LPS B. TCT C. Pertactin and Other Auto-Exporters D. Fimbriae E.
V. VI. VII.
VIII.
IX.
FHA
F. Dermonecrotic Toxin G. Adenylate Cyclase H. Pertussis Toxin I. The Bordetella Type III Secretion System The Bordetella-Host Interaction The BvgAS Sensory Transduction System Phenotypic Modulation A. The Bvg" Phase B. The Bvg'Phase Transcriptional Control of Bvg-Regulated Genes A. BvgAS-Mediated Activation of Virulence Gene Expression B. BvgAS-Mediated Repression of Gene Expression C. A Model for the Global Regulation of Gene Expression by BvgAS The Role of Bvg-Mediated Signal Transduction in the Bordetella Life Cycle References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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/. Introduction The Bordetella genus can broadly be divided into two groups of Gram-negative bacilli. The first includes B. pertussis, B. parapertussis, and B. bronchiseptica, each of which colonizes the respiratory tracts of mammals. On the basis of extensive phylogenetic analysis, members of this group have appropriately been classified as subspecies [1]. These bacteria share a nearly identical virulence control system encoded by the bvgAS locus. They also express a common set of surface and secreted molecules involved in colonization and virulence. They differ, however, in a variety of characteristics, including host range specificity, severity of disease, the ability to establish persistent infection, and perhaps even pathways for transmission. Thus far, major phenotypic differences have not been shown to result from the presence or absence of pathogenicity islands, bacteriophage genomes, transposable elements, or plasmids. Instead, several Bvg-regulated loci found in the genomes of all three subspecies are differentially expressed by B. pertussis, B. parapertussis, and B. bronchiseptica. Examples include the genes and operons that encode a type III secretion system [2], a motility apparatus [3], and pertussis toxin [4-7]. Differential gene expression, as well as polymorphisms within expressed genes, may therefore contribute to complex phenotypic differences. As a result of their extremely high degree of genetic relatedness, comparative studies of the similarities and differences in the infectious cycles of Bordetella subspecies offer an opportunity to use "experiments of nature" as a guide to understanding fundamental features of bacterial-host interactions. The second group of Bordetellae, which are distantly related to the subspecies described above, include B. avium and three recently identified species, B. hinzii, B. holmesii, and B. trematum. B. avium infects the respiratory epithelium of domestic fowl and is responsible for rhinotracheitis in chicken and turkey poults [8]. Since its initial description in 1994, B. hinzii has been isolated from the blood of a patient with AIDS, the sputum of an adult with cystic fibrosis, and respiratory aspirates of healthy chickens and turkeys [9-11]. B. hinzii is phenotypically similar to B. avium, and both species may share the same natural habitat. In either case, human disease is presumably an anomaly associated with compromised immunity. B. holmesii has been isolated from blood cultures of patients with septicemia or endocarditis and sputum from an adult in severe respiratory distress [12, 13]. Finally, B. trematum is most commonly associated with human wound infections and chronic otitis media [14]. The natural reservoirs of these organisms have not been identified. In comparison to B. pertussis and related subspecies, considerably less is known about B. avium, and almost nothing has been reported regarding the colonization and virulence mechanisms of B. hinzii, B. holmesii,or B. trematum. This review therefore focuses on a comparative analysis of Bordetella subspecies that have clearly adapted to colonize the mammalian respiratory tract.
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//. Respiratory Infections by Bordetella Species A. B, pertussis Infections of Humans B. pertussis has exclusively adapted to the human host, and there is no evidence for the existence of an animal or environmental reservoir. Transmission occurs by respiratory droplets and, possibly, by environmental contamination with respiratory secretions. The most commonly recognized clinical manifestation of infection, alternatively known as pertussis or whooping cough, is a highly contagious acute childhood disease [15]. Infection of a susceptible host begins with colonization of ciliated respiratory epithelia. During the initial catarrhal phase, mild cold-like symptoms increase in severity as multiplying bacteria synthesize toxins that cause both local and systemic effects. Progression to the paroxysmal phase is characterized by intense coughing spasms. Repetitive series of forceful coughs during a single expiration are sometimes followed by a massive inspiratory effort that produces a characteristic "whoop" as inhaled air is forced through a narrowed glottis. Post-tussive vomiting, cyanosis, and apnea may also occur. Although fever is rare, systemic manifestations include lymphocytosis, which appears to be due to pertussis toxin. Paroxysms decrease in severity and frequency during the recovery period, which can last for several weeks. Although complications are rare, they are serious in infants and include pneumonia, seizures, encephalopathy, and death. Classic childhood illness usually lasts 6-8 weeks, although B. pertussis can be cultured from the upper respiratory tract only during the initial stages of disease. It is somewhat paradoxical that the ability to recover B. pertussis from the nasopharynx decreases as the severity of disease increases. Classic illness as described above occurs as a primary infection of unimmunized children between 1 and 10 years of age. Mild, nonclassical illness is relatively common, occurring in vaccinated as well as nonvaccinated children [16]. Clinical manifestations of B. pertussis infection can vary dramatically depending on age, vaccination status, previous infection, and other unidentified factors. Although asymptomatic carriage does not seem to occur, mild, atypical infections are quite common [17]. Since the late 1940s, the incidence of pertussis has decreased dramatically in most developed countries as a result of widespread immunization. Initial vaccine formulations, which are still in use, consist of killed but otherwise intact B. pertussis cells. Concerns regarding documented and perceived adverse side effects accompanying whole cell vaccination prompted the development of acellular vaccines based on a subset of highly purified components of the organism [15, 18]. Several acellular vaccines are now licensed for use in the United States beginning at 6 weeks of age, and efficacy studies indicate good levels of protection. All current acellular vaccines contain chemically or genetically inactivated pertussis toxin. Also included in some formulations are filamentous hemagglutinin, pertactin, and fimbriae, each of which has been implicated
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in adherence. While one would predict that acellular vaccines function by inducing neutralizing and/or bactericidal antibodies, only recently has a correlation between antibody levels, particularly anti-pertactin and anti-fimbriae, and protection against pertussis been shown [ 19, 20]. As discussed later in this chapter, cell-mediated immunity may also be important. The relative contributions of humoral and cell-mediated immune responses to protection from disease and protection from colonization are currently unknown. Widespread vaccination of infants and children has resulted in several interesting changes in the epidemiology of B. pertussis. Although the frequency of disease has certainly declined, pertussis has by no means been eliminated. Instead, the age-related incidence of disease has shifted. In the prevaccine era, approximately 85% of cases in the United States were noted in children aged 1-9 years [15]. In contrast, from 1992 to 1994, 41% of cases occurred in infants and 27% in persons >10 years of age [21]. Disease in infants, which can be life-threatening and often requires hospitalization, usually results from exposure before sufficient levels of protection have been achieved through immunization. In contrast, postchildhood disease results from waning immunity after the standard course of immunizations have been completed. A 1998 report suggests that widespread vaccination may have influenced the evolution of B. pertussis populations as well. Mooi and colleagues surveyed a large collection of Dutch B. pertussis isolates and found a decrease in the predominance of "vaccine" pertactin and pertussis toxin S1 subunit alleles, and a concomitant increase in the predominance of "nonvaccine" alleles, in strains collected after introduction of the whole-cell pertussis vaccine to The Netherlands during the 1950s [22]. Determining if this correlation reflects a true causal relationship will obviously be important for continued development of efficacious vaccines. Although clinical vigilance and vaccination efforts have traditionally been targeted at infants and young children, it has recently been proposed that adolescent and adult pertussis is far more common than previously suspected. Studies of prolonged coughing illness in adults have implicated B. pertussis as the etiologic agent in 12 to 32% of cases [21]. Adult pertussis is infrequently recognized and often misdiagnosed. It is epidemiologically significant, however, because it provides a reservoir for infection of unprotected individuals. Routine booster vaccination of adolescents and adults may therefore provide the key to eliminating pertussis in infants and young children. B. B, parapertussis Infections of Humans and Sheep B. parapertussis has historically been considered to be a human-adapted pathogen that causes a pertussis-like syndrome. A controlled comparison of cHnical characteristics conducted in conjunction with an acellular pertussis vaccine trial concluded that illness caused by B. parapertussis is remarkably similar to that caused by B. pertussis [23]. Paroxysmal coughing, for example, was noted at
13.
BORDETELIA
623
approximately the same frequency following infection by either organism. Two significant differences in clinical presentation were observed. In comparison with B. pertussis, infection with B. parapertussis did not result in lymphocytosis, and illness was less severe [23]. The lack of lymphocytosis is most likely due to the lack of pertussis toxin expression by B. parapertussis. Although pertussis toxin may contribute to disease severity, the similarities between illness caused by B, pertussis and B. parapertussis provide evidence against the hypothesis [24] that pertussis toxin is qualitatively responsible for the major symptoms of disease. Given its longstanding position as a host-restricted human pathogen, the isolation of B. parapertussis from sheep in New Zealand [25] and Scodand [26] came as a considerable surprise. In both cases, the organism was found in the respiratory tracts of animals with chronic nonprogressive pneumonia as well as animals that were apparently healthy. Although epidemiological data are scarce, ovine B. parapertussis infections appear to be relatively common, they can be symptomatic or asymptomatic, and infection with B. parapertussis may predispose animals to pneumonia resulting from secondary infection by other pathogens such as Pasteurella hemolytica. The observation that B. parapertussis strains are capable of causing infections in both sheep and humans raises several interesting questions. Do sheep provide a reservoir from which transmission to humans (or vice versa) can occur? Alternatively, if human and ovine strains are genetically distinct, can their high degree of relatedness be used to identify determinants that specify host range? Answers to these and other questions are beginning to unfold as phylogenetic relationships become apparent. Molecular evolution and host adaptation within the Bordetella genus are discussed in more detail below. C. B. bronchiseptica Infections of Mammals Although B. pertussis and B. parapertussis are apparently confined to humans or sheep, B. bronchiseptica causes respiratory infections in a wide variety of mammals. This organism has been isolated from mice, rats, guinea pigs, skunks, opossums, rabbits, raccoons, cats, dogs, ferrets, foxes, pigs, hedgehogs, sheep, koala bears, leopards, horses, and lesser bushbabies [27]. Although human infections are well documented, they are rare and are usually associated with a severely compromised host. As would be expected, most reports involving B. bronchiseptica depict the organism as a pathogen. Indeed, infection is associated with a variety of respiratory diseases, several of which extract a significant economic toll. Atrophic rhinitis in pigs, kennel cough (rhinotracheatis) in dogs, and bronchopneumonia in rabbits and other laboratory animals are commonly associated with infection. Although B. bronchiseptica can certainly function as a pathogen, from the perspective of the subspecies as a whole this may be the exception rather than the rule. From epidemiological [28, 29] and laboratory studies [30, 31], it is apparent that many, and possibly most, infections by B. bronchiseptica result in asympto-
624
PEGGY A. COTTER AND JEFF F. MILLER
matic colonization of the upper respiratory tract. Nonetheless, the possibility that specific strains differ in virulence is supported by data from several comparative studies [28, 32, 33]. As with other Bordetella species, B. bronchiseptica initiates infection by colonizing the ciliated respiratory epithelium of the nasal cavity, trachea, and in some cases the large airways in the lungs. Although ciliated cells are usually considered to be the primary targets for attachment, it is not known whether Bordetella species can also adhere to other cell types present in the upper respiratory tract. This issue may be particularly relevant to the nasal epithelium, which includes olfactory cells, sustentacular cells, goblet cells, and squamous cells in addition to ciliated cells. In contrast to human infection by B. pertussis and B. parapertussis, B. bronchiseptica is easily isolated from the nares of infected animals throughout the course of infection. Although host immune responses include antibody production [30, 31] and CD4 T-cell priming [34], colonization commonly persists for the life of the animal. Disease is rare in immunocompetent hosts; however, a variety of perturbations can markedly alter the outcome of infection by B. bronchiseptica. In livestock and domesticated animals, these include stresses associated with confinement and suboptimal rearing conditions and, most notably, secondary respiratory infections [27]. In swine, for example, it is well documented that primary infection by B. bronchiseptica predisposes animals to secondary infection by a variety of viruses and bacteria. Pasteurella hemolytica and Pasteurella multocida are commonly associated with atrophic rhinitis in swine following initial infection with B. bronchiseptica [35, 36]. The naturally occurring association between B. bronchiseptica and laboratory animals, the availability of genetic techniques for manipulating the B. bronchiseptica genome, and the close similarities among B. bronchiseptica, B. pertussis, and B. parapertussis have been exploited for studies of pathogenic mechanisms [30, 31, 37]. The ability to use natural hosts for experimental infections eliminates many of the problems associated with models based on laboratory animals and pathogens that have exclusively adapted to humans. The use of chimeric strains in which B. bronchiseptica loci are substituted with their B, pertussis counterparts further increases the utility of this approach [38].
///. Evolutionary Relationships among Bordetella Subspecies The population structure of the Bordetella genus has been the subject of numerous studies employing a variety of techniques [1, 39-44]. A recent report by van der Zee et al. [45] attempted to reconstruct the evolutionary history of Bordetella
13.
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subspecies using a combination of multilocus enzyme electrophoresis (MEE) and the identification of insertion sequence polymorphisms. In MEE, strains are differentiated by comparing the electrophoretic mobilities of a collection of metabolic enzymes [46]. Strains displaying identical mobilities for the entire set of enzymes are assigned to the same electrophoretic type (ET), whereas differences in mobility indicate different alleles at the corresponding structural genes. The number of polymorphisms between different ETs is roughly proportional to the time of divergence from a common ancestor. Hence, "genetic distance" between pairs of ETs can be calculated from the proportion of loci at which different alleles are represented. Dendrograms can then be constructed from a matrix of genetic distances by the average linkage method [46]. A variation of this technique, called multilocus sequence typing (MLST), is based on direct sequencing of portions of "housekeeping" genes [47]. Although traditional MEE analysis has been applied to numerous genera of pathogenic bacteria, MLST is a simpler technique capable of generating data that can be directly compared between different laboratories. It is therefore expected that MLST will be the method of choice for future epidemiological and phylogenetic studies. The dendrogram shown in Figure 1 [45] displays relationships between ETs along with the distribution of three insertion sequences—IS 1001, IS 1002, and IS481—which are polymorphic in copy number and chromosomal location. In comparison with many other pathogenic bacteria, the genetic diversity displayed by B. pertussis, B. bronchiseptica, and B. parapertussis isolates is highly restricted [1, 45]. This supports their classification as subspecies, rather than species, and suggests a very recent evolutionary origin. At a genetic distance of approximately 0.6, two clusters of strains, designated A and B, can be differentiated in Figure 1. Both clusters contain isolates that are phenotypically classified as B. bronchiseptica. At a distance of approximately 0.3, cluster D, containing human-adapted B. pertussis strains, separates from the other Bordetella isolates. B. parapertussis strains are all found in cluster G, and their pattern of divergence is particularly intriguing. B. parapertussis isolates from sheep {B. parapertussis^^^) group together in cluster I, and these strains show a moderate level of divergence. In striking contrast, B. parapertussis isolates from humans {B. parapertussis hu) are genetically identical by this and other analyses [42], regardless of when and where they were obtained. For example, ET 28 (Fig. 1) includes strains from The Netherlands, Finland, the United States, Germany, and New Zealand. Furthermore, and somewhat unexpectedly, B. parapertussis hu isolates are more closely related to the B, bronchiseptica strains in cluster J than to the B. parapertussisQ^ strains in cluster I. ISlOOl is found in nearly all members of cluster G but not in the other lineages (Fig. 1). This insertion sequence may therefore have been acquired by the common ancestor of cluster G. Similarly, IS481 is predominantly associated with strains of B. pertussis. IS 1002 has an interesting pattern of distribution. It is found almost exclusively in Bordetella isolates from humans, regardless of the subspe-
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cies. A possible explanation for this observation is the occurrence of horizontal transfer from B. pertussis to a coinfecting human B. parapertussis strain that subsequently emerged as a highly successful clone. Although insertion sequences can be acquired by horizontal or vertical transmission, the pattern of distribution of IS elements within clusters of related multilocus genotypes is consistent with a predominantly vertical descent. In general, the distribution of ISIOOl, IS1002, and IS481 supports assignment of genetic relationships based on MEE. Several hypothesis can be advanced based on the data summarized in Figure 1 [45]. Although B. pertussis and B. parapertussis l^^^ both infect humans, they do not appear to have a recent common ancestor and may instead have evolved from different lineages at different times. It therefore appears that there have been two independent host-range adaptations to humans, the earliest by B. pertussis (cluster D) and the most recent by B. parapertussis l^^^ (ET 28 in Fig. 1). In both cases, B. bronchiseptica is likely to be the evolutionary progenitor, suggesting that B. pertussis and B. parapertussis ^u strains may be considered as host-adapted lineages of B. bronchiseptica. Sequence comparisons of genes encoding fimbrial subunits [48], adenylate cyclase toxin [49], pertactin [50], pertussis toxin [51], and BvgAS [52] confirm the very close relationship between B. parapertussis ^u and B. bronchiseptica and the more distant relationship between these subspecies and B. pertussis. The lack of diversity among B. parapertussis hu strains is also consistent with the hypothesis that human adaptation is a very recent event. Since human and ovine B. parapertussis isolates comprise genetically distinct populations [42, 45] (Fig. 1), it is unlikely that sheep provide a reservoir from which transmission to humans commonly occurs. Although speculative in nature, the framework advanced by van der Zee et al. [45] provides a valuable tool for investigating mechanisms of pathogenesis and host-range adaptation. For example, the close relationship between B. parapertussis hu strains and the B. bronchiseptica isolates in cluster J could potentially be exploited to identify genetic correlates of human adaptation. The complete genomic sequences of B. pertussis, B. parapertussis hu, and B. bronchiseptica, which are presently being determined, will undoubtedly facilitate this comparative analysis. There are several examples of differential expression of surface and secreted factors with potential roles in the infectious cycles of Bordetella subspecies. It is now possible to ''hang" various phenotypes on the evolutionary tree and determine if the presence or absence of a particular factor correlates with the proposed phylogenetic lineage and/or parameters of infection. For example, it is likely that the progenitor in Figure 1 expressed both the pertussis toxin operon (PT"^), which is activated by the BvgAS control system, as well as genes for motility (Mof^), which are repressed by BvgAS. Since B. bronchiseptica and B. parapertussis strains are uniformly PT~ [51], the ability to express the pertussis toxin operon must have been lost twice, and independently, early in the formation of clusters B and C. In contrast, the Mot"^ phenotype is observed exclusively and uniformly in B. bronchiseptica isolates [3, 53]. Motility expression might therefore have
628
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been lost in three independent events, associated with the evolution ofB. pertussis (cluster D), B. parapertussisQ^, (cluster I) and B. parapertussis^^ (ET 28). This predicts that the specific lesion(s) responsible for the lack of motility gene expression should be conserved within these lineages, but divergent between them. It is also interesting to consider that loss of motility correlates with restricted adaptation to specific mammalian hosts. Finally, preliminary data suggest that the type III secretion system is functional in B. bronchiseptica and B. parapertussisQ^, but not in B. parapertussis \^^ or B. pertussis [2]. The lack of type III secretion, therefore, appears to correlate with human adaptation as well as with acute disease versus chronic infection. The simplest alternative hypothesis for differential expression of specific factors is that pertussis toxin genes, motility genes, or type III secretion loci were acquired at various points in evolution by horizontal gene transfer. This is inconsistent with the observation that these genes are present, but not necessarily expressed, in all of the subspecies under consideration. The challenge in understanding the evolution of Bordetella subspecies, as well as other bacterial pathogens is to make the transition from correlation to causation.
IV. Bordetella Virulence Factors Bordetellae express a large number of surface-exposed and secreted factors with postulated roles in pathogenesis (Fig. 2). Many of these were identified and characterized biochemically before genetic and molecular biological tools were available. However, even recent advances in genetic and molecular methodologies have in many cases not been sufficient to overcome problems stemming from the lack of a natural-host animal model for B. pertussis. Thus, while extensive in vitro characterization has greatly increased our appreciation of what many identified factors can do, we still know relatively litde about what they actually do during the course of respiratory infection.
A.
LPS
The lipopolysaccharides (LPSs) contained in the outer membranes of Bordetella species, like endotoxins from other Gram-negative bacteria, are pyrogenic, mitogenic, toxic, and can activate and induce tumor necrosis factor production in macrophages [54-56]. Bordetella LPS molecules differ in chemical structure from the well-known smooth-type LPS expressed by Enterobacteriaceae. B. pertussis LPS lacks a repetitive O-antigenic structure and is therefore more similar to
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rough-type LPS. It resolves as two distinct bands (A and B) on silver-stained sodium dodecyl sulfate-polyacrylamide gels [57]. The faster migrating moiety, band B, consists of a Hpid A molecule linked via a single ketodeoxyoctulosonic acid (KDO) residue to a branched oligosaccharide core structure containing heptose, glucose, glucuronic acid, glucosamine, and galactosaminuronic acid (GalNAcA) [58-61]. The charged sugars—GalNAcA, glucuronic acid, and glucosamine—are not commonly found as core constituents in other LPS molecules. The slower-migrating moiety (band A) consists of band B plus a trisaccharide consisting of A^-acetyl-A^-methyl-fucosamine (FucNAcMe), 2,3dideoxy-2,3-di-A^-acetylmannosaminuronic acid (2,3-diNAcManA), and N-acetylglucosamine (GlcNAc) [58-61]. B. bwnchiseptica LPS is composed of band A and band B plus an 0-antigen structure consisting of a polymer of the single sugar 2,3-dideoxy-di-A^-acetyl-galactosaminuronic acid [62]. Human isolates of B. parapertussis contain LPS that lacks band A, has a truncated band B, and contains an 0-antigen that appears to consist of the same sugar polymer as in the B. bronchiseptica 0-antigen [62]. LPS from ovine B. parapertussis isolates appear to lack 0-antigen and contain band A- and B-like molecules that are distinct from those of the other Bordetella species [63]. Although a distinct role for LPS in Bordetella pathogenesis has not yet been demonstrated, monoclonal antibodies against band A can adoptively transfer immunity to B. pertussis in an infant mouse lung infection model [64]. The importance of LPS in pathogenesis is further suggested by the observation that changes in LPS structure in B. bronchiseptica are controlled by the BvgAS virulence regulatory system [63]. The LPS structures of ^. parapertussis^^ and B. parapertussis hu also vary in response to the same environmental signals to which BvgAS responds, suggesting they are controlled by BvgAS as well [63]. Allen and Maskell have recently identified, cloned, and sequenced genetic loci required for LPS biosynthesis in B. pertussis, B. parapertussis hu, and B. bronchiseptica, and have constructed strains with various mutant LPS phenotypes [65-67]. Compared with their wild-type parental strains, B. pertussis, B. parapertussis hu, and B. bronchiseptica strains that synthesize only band-B LPS show decreased colonization in a mouse model of respiratory infection [68]. For B. bronchiseptica and B. parapertussis hu, this difference may be attributable to differences in sensitivity to antibody-dependent serum killing [68]. Further characterization of these and other mutants with defined mutations affecting LPS structure will greatly facilitate deciphering the precise role(s) of LPS in Bordetella pathogenesis. B.
TCT
Of the various toxins and virulence-associated factors synthesized by Bordetellae, only tracheal cytotoxin (TCT) has been shown to reproduce the specific epithelial cytopathology characteristic of pertussis. TCT corresponds to a disaccharide-
13.
BORDETELIA
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tetrapeptide monomer of peptidoglycan. Its structure is A^-acetylglucosaminyll,6-anhydro-A^-acetylmuramyl-(L)-alanyl-Y-(D)-glutamyl-m^5odiaminopimelyl-(D )-alanine [69]. Although this peptidoglycan fragment is produced by all Gramnegative bacteria as they break down and rebuild their cell wall during growth, only Bordetella spp. [70] and Neisseria gonorrhoeae [71] have been shown to release it into the environment. Other bacteria, such as E. coli, recycle this peptidoglycan fragment by transporting it back into the cytoplasm via an integral cytoplasmic membrane protein called AmpG [72]. It appears that Bordetella do not express a functional AmpG, although sequences resembling ampG can be identified in the recendy released B. pertussis genome database (R. Lyon and W. E. Goldman, personal communication) [73]. Expression of £". coli ampG in B. pertussis results in a substantial decrease in the amount of TCT produced (R. Lyon and W. E. Goldman, personal communication). The activities of TCT have been studied in vitro using hamster tracheal organ culture and cultured hamster tracheal epithelial (HTE) cells. TCT causes mitochondrial bloating, disruption of tight junctions, and extrusion of ciliated cells, with little or no damage to nonciliated cells, in hamster tracheal ring cultures and a dose-dependent inhibition of DNA synthesis in HTE cells [74, 75]. TCT has also been shown to cause loss of ciliated cells, cell blebbing and mitochondrial damage in human nasal epithelial biopsies [76]. There is strong evidence that this cytopathology is due to a TCT-dependent increase in nitric oxide (NO). Inducible nitric oxide synthase (iNOS) expression is positively controlled by interleukin-la (IL-la), and TCT has been shown to trigger IL-1 a production in HTE cells [77]. Both TCT and IL-la result in increased NO production when added to HTE cells [78]. It is hypothesized that TCT stimulates IL-la production in noncihated mucus-secreting cells, which activates expression of iNOS, resulting in the formation of large amounts of NO. This NO then diffuses to neighboring ciliated cells, which are much more susceptible to its damaging effects [79]. The ability to construct TCT-deficient mutants by expressing a heterologous ampG gene in Bordetella will allow this hypothesis to be tested using in vivo models. C. Pertactin and Other Auto-Exporters Bordetellae express a number of related surface-associated proteins that appear to direct their own export to the outer membrane, where they undergo autoproteolytic processing of their C termini in a manner similar to the IgA proteases of Neisseria gonorrhoeae [80] and Haemophilus influenzae [81] and the elastase of Pseudomonas aeruginosa [82]. The first member of this family to be identified in Bordetella, and the best characterized, is pertactin. Mature pertactin is a 68-kDa protein in B. bronchiseptica, the subspecies in which it was first discovered [83], a 69-kDa protein in B. pertussis [84], and a 70-kDa protein in B. parapertussis i^^^ [85]. All three pertactin proteins contain an Arg-Gly-Asp (RGD) tripeptide motif as well as several proline-rich regions and leucine-rich repeats, motifs commonly
632
PEGGY A. COTTER AND JEFF F. MILLER
present in molecules that form protein-protein interactions involved in eukaryotic cell binding [86]. The B. pertussis, B. bronchiseptica, and B. parapertussis pertactins differ primarily in the number of proline-rich regions they contain [50]. The X-ray crystal structure of B. pertussis pertactin was recently determined. It consists of a 16-stranded parallel p-helix with a V-shaped cross-section and is the largest (i-helix known to date [87]. Other Bordetella proteins with predicted autoexport ability include TcfA [88], BrkA [89], and Vag8 [90]. All of these proteins show significant amino-acid sequence similarity in their C termini and contain one or more RGD tripeptide motifs. Based on predicted amino-acid sequence similarity with all of these proteins, the B. pertussis genome appears to encode at least three additional members of this autoexporter family. It is hypothesized that the C termini of the autoexporting precursor proteins form a pore in the outer membrane through which the N-terminal portions are threaded and then cleaved, but which remain cell-associated by an uncharacterized mechanism. In support of this model, Charles et al have shown that deletion of the y region of prn^^ prevents surface exposure of the molecule [91]. In the case of Vag8, however, cleavage may not occur since the predicted size of the entire protein encoded by vagS corresponds to the size seen by SDS-PAGE [90]. The presence of RGD and other sequence motifs suggests that pertactin and the other autoexporters may function as adhesins. In vitro studies demonstrated that purified pertactin could promote binding of CHO cells to tissue culture wells and that expression of prn in Salmonella or E. coli could increase the adherence and/or invasiveness of these bacteria to various mammalian cell lines [92]. In contrast, a Pm~ strain of B. pertussis did not differ from its wild-type parent in its ability to adhere to or invade HEp2 cells in vitro or to colonize the respiratory tracts of mice in vivo [93]. Similarly, we have constructed a B. bronchiseptica strain with an in-frame deletion mutation in prn and found it to be indistinguishable from wild-type B. bronchiseptica in its ability to establish a persistent respiratory tract infection in rats (our unpublished data). Thus, although pertactin appears to be a strong and potentially protective immunogen [83, 84, 94], its role in pathogenesis remains unknown. Potential adhesive functions for TcfA, BrkA, and Vag8 have not been investigated directly, although TcfA" B. pertussis show a decreased ability to colonize the murine trachea compared to wild-type B. pertussis [88]. BrkA has been proposed to play a role in serum resistance [89]. D.
Fimbriae
Like most Gram-negative pathogenic bacteria, Bordetellae express fimbriae. The major fimbrial subunits that form the two predominant Bordetella fimbrial serotypes—Fim2 and Fim3—are encoded by unlinked chromosomal loci fim2 and fimS, respectively [95, 96]. A third unlinked locus,//mX, is expressed only at very low levels, if at all [97]. In addition to positive regulation by BvgAS, the//m genes are subject to fimbrial phase variation by a mechanism that appears to
13.
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involve slipped-strand mispairing within a stretch of ctyosine residues located between the -10 and -35 elements of ihefim2,fim3, and fimX promoters [98]. Since transcription of the individual genes is affected independently by this mechanism, bacteria may express Fim2, Fim3, and FimX, or any combination at any given time. In all cases, the minor fimbrial subunit that most likely forms the tip adhesin appears to be FimD [99]. The/zmD gene is located within the fimbrial biogenesis operon adjacent to fimB and fimC [100, 101], Interestingly, this operon is positioned between///(^^ and fhaC, genes required for synthesis and processing of another putative adhesin, filamentous hemagglutinin (FHA). The proposed functions of FimB and FimC as chaperone and usher, respectively, are based on the similarity of their predicted amino-acid sequences with those of the E. coli PapD and PapC proteins [100, 101]. Mutation of any one of the genes in the fimBCD locus results in a complete lack of fimbrial structures on the bacterial cell surface, suggesting/FmBCD is the only functional fimbrial biogenesis locus on the Bordetella chromosome [102]. In B. pertussis, a truncated major fimbrial subunit gene,/?mA, is located at the 5' end of the fimBCD gene cluster [101]. It was recently shown that in B. bronchiseptica and B. parapertussis, fimA is intact and capable of encoding a fourth fimbrial subunit type, FimA [103]. The putative promoter region of fimA does not contain a "C stretch" and therefore probably does not undergo phase variation. As a critical early step in bacterial pathogenesis, attachment to host epithelium is often mediated by fimbriae. Although it is likely that Bordetella fimbriae function as adhesins in establishment of respiratory tract colonization, definitive evidence that they in fact perform this function has been difficult to obtain for several reasons. First, the ability to construct strains devoid of fimbriae, but unaltered with respect to other putative adhesins, has been hampered by the unlinked locations of multiple major fimbrial subunits encoding genes as well as the complex genomic organization and transcriptional and translational couphng of the fimbrial biogenesis operon with the flia operon. Second, the presence of several other putative adhesins with potentially redundant functions has, in many cases, obscured the ability to detect clear phenotypes for Fim~ mutants. Finally, since the interactions between bacterial adhesins and host receptor molecules are expected to be highly specific, the use of heterologous hosts for studies with B. pertussis could severely limit the ability to detect in vivo roles for putative adhesins. Nonetheless, a number of studies by van Furth, Mooi, and colleagues (e.g., [105, 106, 108]) suggest fimbriae may mediate binding of Bordetella to respiratory epithelium via the major fimbrial subunits and to monocytes via FimD. Sulfated sugars are ubiquitous in the mammalian respiratory tract and Geuijen et al. have shown that purified B. pertussis fimbria, with or without FimD, were able to bind to heparan sulfate, chondroitin sulfate, and dextran sulfate [104]. Heparin-binding domains within the Fim2 subunit were identified and found to be similar to those of the eukaryotic extracellular matrix protein, fibronectin [104]. FimD-mediated adherence to monocytes/macrophages was suggested by a
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series of in vitro studies by Hazenbos et al. The resulting model suggests FimD mediates binding of nonopsonized B. pertussis to the very late antigen 5 (VLA-5) on the surface of monocytes, which then causes activation of complement receptor type 3 (CR3), which enhances its ability to bind FHA [105, 106]. As discussed below, there is evidence that FHA-mediated binding to monocytes, via the leukocyte response integrin/integrin associated protein (LRI/IAP) complex, also stimulates CR3 binding activity [107]. In vivo studies have shown that Fim~ B. pertussis strains are defective in their ability to multiply in the nasopharynx and trachea of mice [99, 108]. Using a B. bronchiseptica strain devoid of fimbriae but unaltered in its expression of FHA and other putative adhesins, we have recently shown that fimbriae contribute to the efficiency of establishment of tracheal colonization and are absolutely required for persistence in the trachea using both rat and mouse models [108a]. Moreover, the serum antibody profiles of animals infected with Fim~ bacteria differ qualitatively and quantitatively from those of animals infected with wild-type B. bronchiseptica. Taken together, these results suggest that Fim-mediated interactions with epithelial cells and/or monocyte/macrophages may play important roles, not only in adherence, but also in the nature and magnitude of the host immune response to Bordetella infection.
E. FHA
FHA is an unusually large and highly immunogenic hairpin-shaped molecule that has been included as a primary component in acellular pertussis vaccines [109]. It is synthesized as a 367-kDa precursor, FhaB, which is modified at its N terminus [110] and cleaved at its C terminus [111] to form the mature 220-kDa FHA protein. Although efficiently secreted via a process requiring the outer membrane protein FhaC, a significant amount of FHA remains associated with the cell surface by an unknown mechanism [111]. In vitro studies using a variety of mammalian cell types suggest FHA possesses at least four distinct attachment activities, and four separate FHA-binding domains have been proposed. The Arg-Gly-Asp (RGD) triplet [112], situated in the middle of FHA and locaHzed to one end of the proposed hairpin structure [113], stimulates adherence to monocyte/macrophages and possibly other leukocytes via LRI/IAP and CR3 [107, 112,114]. The CR3-recognition domain in FHA has yet to be identified. FHA also possesses a carbohydrate-recognition domain (CRD), which mediates attachment to ciliated respiratory epithelial cells as well as to macrophages in vitro [114,115]. Finally, a lectin-like activity for heparin and other sulfated carbohydrates, which can mediate adherence to nonciliated epithelial cell lines, has been identified [116]. This heparin-binding site is distinct from the CRD and RGD sites and is required for FHA-mediated hemagglutination [116].
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Evidence for FHA-dependent phenotypes in vivo has been more difficult to obtain. Using a rabbit model, Saukkonen et al found fewer FHA mutants than wild-type B. pertussis in the lungs 24 hours after intratracheal inoculation [114]. Based on in v/rro-determined binding characteristics of the various mutants used in their study, they inferred that wild-type B. pertussis were adhering both to ciliated epithelial cells and macrophages, and competition experiments with lactose and anti-CR3 antibody suggested both CRD- and RGD-dependent binding was involved [114]. Using mouse models, however, others have found FHA mutants to be indistinguishable from wild-type B. pertussis in their ability to persist in the lungs, but defective for tracheal colonization [99, 108, 117]. Still others [118-122], also using mouse models, have observed no difference between FHA mutants and wild-type B. pertussis. The difficulty in achieving a complete and detailed understanding of the role of FHA in the anatomic localization of B. pertussis during infection probably reflects the absence of a natural animal host (other than humans), as well as the complexity of this molecule and its associated biological activities. We have recently explored the role of FHA in pathogenesis by constructing two types of FHA mutant derivatives oiB. bronchiseptica: one containing an in-frame deletion in jhaB, the FHA structural gene, and one in which FHA is expressed ectopically in the Bvg~ phase, in the absence of the array of Bvg"^ phase virulence factors with which it is normally expressed [123]. Comparison of these mutants with wild-type B. bronchiseptica showed that FHA is both necessary and sufficient to mediate adherence to rat lung epithelial cells in vitro. Using a rat model of respiratory infection, we showed that FHA is absolutely required, but not sufficient, for tracheal colonization in healthy, unanesthetized animals. FHA was not required for initial tracheal colonization in anesthetized animals, however, suggesting its role in establishment may be dedicated to overcoming the clearance activity of the mucociliary escalator. The use of anesthetized rats also revealed a role for FHA in persistence. B. pertussis has recently been shown to inhibit T-cell proliferation to exogenous antigens in vitro in an FHA-dependent manner [124]. Taken together, these data suggest FHA may also perform immunomodulatory functions in vivo.
F. Dermonecrotic Toxin Although initially misidentified as endotoxin, dermonecrotic toxin (Dnt) was one of the first B. pertussis virulence factors to be described [125]. This heatlabile toxin induces locaUzed necrotic lesions in mice and other laboratory animals when injected intradermally, and is lethal for mice at low doses when administered intravenously [125-128]. The Dnts ofB. pertussis, B. parapertussis, and B. bronchiseptica are much more highly related genetically and biologically to each other than to the Dnt of B. avium [128a]. The Dnts of B. pertussis and B.
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bwnchiseptica are cytoplasmically located single polypeptide chains of about 140 kDa [129-132]. In vitro studies have shown that purified Dnt from B. bwnchiseptica induces dramatic morphological changes, stimulates DNA replication, and impairs differentiation and proliferation in osteoblastic clone MC3T3 cells [133, 134]. Recent evidence indicates that these effects are due to Dnt-mediated activation of the small GTP-binding protein rho [135], which results in tyrosine phosphorylation of focal adhesion kinase (pi25^''^) and paxillin [136]. pi25^"^ and paxillin are involved in embryonic development and cell locomotion [137], and their activation leads to profound alterations in the actin cytoskeleton and assembly of focal adhesions [138-143]. Lacerda et al also showed that Dnt stimulates DNA synthesis without activation of p42'""/'^ and p44'"^'^'^, providing evidence for a novel p2P^'^^-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3 cells [136]. If and how these effects of Dnt contribute to Bordetella pathogenesis is not known. Although B. bwnchiseptica strains with decreased dermonecrotic toxin activity have been associated with decreased turbinate atrophy in infected pigs [144, 145], transposon mutants of B. pertussis lacking dermonecrotic toxin are no less virulent than wild-type bacteria in mice [121]. G. Adenylate Cyclase All of the Bordetella species that infect mammals secrete CyaA, a bifunctional calmodulin-sensitive adenylate cyclase/hemolysin. CyaA is synthesized as a protoxin monomer of 1706 amino acids. Its adenylate cyclase catalytic activity is located within the N-terminal 400 amino acids [146, 147]. The 1300-aa C-terminal domain mediates delivery of the catalytic domain into the cytoplasm of eukaryotic cells and possesses low but detectable hemolytic activity for sheep red blood cells [147-149]. Amino-acid sequence similarity among the C-terminal domain of CyaA, the hemolysins of Escherichia coli (HlyA) and Actinobacillus pleuropneumoniae (HppA), and the leukotoxins of Pasteurella hemolytica (LktA) 2ind Actinobacillus actinomycetemcomitans (AaLtA), places CyaA within a family of calcium-dependent pore-forming cytotoxins known as RTX (repeats in toxin) toxins [150]. Each of these toxins contains a tandem array of a 9-aa repeat (L-X-G-G-X-G-(N/D)-D-X) that is thought to be involved in calcium binding [150]. Before the CyaA protoxin can intoxicate host cells, it must be activated by the product of the cyaC gene, which is located adjacent to, and transcribed divergendy from, the cyaABDE operon [151]. CyaC activates the CyaA protoxin by catalyzing the palmitoylation of an internal lysine residue (Lys-983) [152], The E. coli HlyA protoxin is also activated by fatty acyl group modification [153155]. CyaA can enter a variety of eukaryotic cell types [156]. Once inside, CyaA is activated by calmodulin [157] and catalyzes the production of supraphysiologic
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amounts of adenosine 3',5'-monophosphate (cAMP) from adenosine triphosphate (ATP) [158-161]. Purified CyaA inhibits chemiluminescence, chemotaxis, and superoxide anion generation by peripheral blood monocytes [ 162] and PMNs in vitro with little effect on phagocytic activity [163]. CyaA has also been shown to induce apoptosis in cultured murine macrophages [119]. In vivo studies have shown that, compared with wild-type B. pertussis, CyaA~ mutants are defective in their ability to cause lethal infections in infant mice [121, 164] and grow in the lungs of older mice [118, 164]. Moreover, significandy fewer inflammatory cells, particularly PMNs, are recruited to the lungs in response to murine respiratory infection with CyaA" mutants compared with wild-type B. pertussis [165]. Taken together, these results suggest CyaA functions primarily as an antiinflammatory factor during infection. By comparing wild-type and CyaA~ B. bronchiseptica strains in wild-type and immunodeficient mice, we have recendy confirmed this suggestion and shown that phagocytic cells are indeed a primary in vivo target of the Bordetella adenylate cyclase toxin [166].
H. Pertussis Toxin
Among the Bordetella species identified so far, only B. pertussis synthesizes and secretes the ADP-ribosylating toxin known as pertussis toxin (Ptx). Ptx is composed of six polypeptides, designated S1-S5, which are present in a ratio of 1:1:1:2:1. Each subunit is synthesized with an N-terminal signal sequence, suggesting that transport into the periplasmic space occurs via a general export pathway analogous to the sec system of E. coli. Secretion across the outer membrane requires a specialized transport apparatus composed of nine Pd (pertussis toxin Hberadon) proteins [167, 168]. Extensive similarity between the ptl locus and the Agrobacterium tumefaciens virB operon, which encodes a secretion system involved in exporting single-stranded "T-DNA," suggests these systems function by a common mechanism [ 169-171 ]. Both appear to be involved in transporting large protein complexes since T-DNA is exported as a proteincoated DNA complex [172]. Furthermore, there is evidence that only the fully assembled Ptx holotoxin is efficiendy secreted [173, 174]. The ptl genes are located direcdy 3' to, and within the same transcripdonal unit as, \htptxA-E genes that encode the Ptx subunits [168, 175]. While the chromosomes of 5. parapertussis and B. bronchiseptica also contain ptx-ptl loci that encode functional polypeptides, these genes are transcriptionally silent due to nucleodde differences in the promoter regions [4-7]. In both B. parapertussis and B. bronchiseptica, replacement of native ptx-ptl promoter sequences with those of B. pertussis results in secredon of biologically acdve Ptx [176]. The biological relevance of differential Ptx expression among Bordetellae is not known.
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The Ptx subunits are held together by noncovalent interactions and arranged in an A-B architecture typical of many bacterial toxins. The A protomer, consisting of the enzymatically active S1 subunit, sits atop a ring, the B oligomer, formed by the remaining S2-S5 subunits [177-179]. The B oligomer binds to eukaryotic cell membranes and dramatically increases the efficiency with which the SI subunit gains entry into host cells [178]. Unlike diphtheria toxin, Ptx does not require an acidic environment for entry into eukaryotic cells [180]. It has therefore been suggested that Ptx may traverse the membrane directly without need for endocytosis. Within the host cell cytosol, binding of ATP to the B oligomer that has intercalated into the cytoplasmic membrane causes release of SI subunit, which becomes active upon reduction of its disulfide bond [181]. The biochemical properties and biological effects of Ptx have been extensively characterized in vitro. In its reduced form, the SI subunit catalyzes the transfer of ADP-ribose from NAD to the a subunit of guanine nucleotide-binding proteins (G proteins) in eukaryotic cells [178, 182, 183]. G proteins that Ptx ADP-ribosylates, and hence inactivates, include Gj, G^ (transducin), and GQ. When active, Gj inhibits adenylyl cyclase and activates K"^ channels, G^ activates cyclic GMP phosphodiesterase in specific photoreceptors, and GQ activates K+ channels, inactivates Ca^"^ channels, and activates phospholipase C-p [184]. Biological effects attributed to disruption of these signaling pathways include histamine sensitization, enhancement of insulin secretion in response to regulatory signals, and both suppressive and stimulatory immunologic effects [24, 185]. Using in vitro assays, Ptx has been shown to inhibit chemotaxis, oxidative responses, and lysosomal enzyme release in neutrophils and macrophages [182, 186-193]. Using mouse and rat models, Ptx has been shown to inhibit chemotaxis and migration of neutrophils, monocyte/macrophages, and lymphocytes [194-196]. Ptx has also been suggested to function as an adhesin involved in adherence of B. pertussis to human macrophages and ciliated respiratory epithelial cells [112, 197]. Ptx is commonly cited as the major virulence factor expressed by B. pertussis, and pertussis has been proposed to be a toxin-mediated disease, with Ptx being responsible for many, if not all, of the disease's typical symptoms [24]. However, despite a plethora of experimental evidence demonstrating what Ptx can do in vitro and in animal models, clear evidence for an in vivo role for Ptx in human disease is lacking. One approach has been to compare symptomatology in children infected with either B. pertussis or B. parapertussis hu since these organisms differ primarily in the absence of Ptx expression by B. parapertussis hy. Such studies have indicated that the only significant difference between the two is increased leukocytosis in B. pertussis-mftciQd children [23, 198]. These observations suggest Ptx may not play a decisive role in causing the paroxysmal coughing, whooping, and vomiting characteristic of pertussis. The exact role of Ptx in establishment of infection, disease, and/or transmission of pertussis remains a mystery.
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I. The Bordetella Type III Secretion System A functional type III secretion system has recently been discovered in B. bronchiseptica [2]. Type III, or contact-dependent, secretion systems have been identified in several pathogenic Gram-negative bacteria (for a review see [199]). A common theme is the use of these systems to deliver bacterial effector proteins directly into the cytoplasm of host cells, leading to induction of signal transduction cascades [200]. While it has not yet been determined if any of the proteins secreted by the B. bronchiseptica type III system enter host cells, B. bronchiseptica can induce cytotoxicity in several cultured cell lines [2, 201], dephosphorylation of specific host cell proteins [2], apoptosis in fibroblast and macrophage cell lines, and nuclear translocation of NFKB [201a]. All of these phenotypes require a functional type III system [2, 201a]. In vivo, the B. bronchiseptica type III system contributes to persistent colonization of the trachea in both rat and mouse models of respiratory infection [2, 201a]. Additionally, animals infected with a mutant defective in type III secretion are completely protected against superinfection by wild-type B. bronchiseptica (P. A. Cotter and J. F. Miller, unpublished data). Taken together, these data suggest the B. bronchiseptica type III secretion system may be involved in modulating the host immune response and hence may contribute to the typically chronic nature of B. bronchiseptica infections. Interestingly, while the chromosomes of all Bordetella subspecies shown in Figure 1 contain bsc {Bordetella secretion) loci, only B. parapertussis^y isolates, an atypical B. pertussis strain (18323), and B. bronchiseptica strains express these genes in vitro [2]. Although it is possible that, for B. parapertussis hu and most B. pertussis strains, the requirements for induction of the type III system in vitro are more stringent than for B. bronchiseptica. Western blot analysis using sera from children recovering from pertussis suggests the type III system of B. pertussis is not expressed in vivo either [201a]. Additional phylogenetic analyses will help clarify the relationship between expression of bsc loci, host range, and/or disease.
V. The Bordetella-Hosf Interaction Bordetella species interact with their mammalian hosts primarily, and perhaps exclusively, at respiratory surfaces. Several scanning-electron-micrographic studies have demonstrated the predilection of these organisms for the cilia of respiratory epithelia [202-205]. In the nasal cavity, requirements for colonization appear to be few; B. bronchiseptica strains multiply deficient in the expression of
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FHA, Fim, Pm, and CyaA are capable of persisting in the nasal cavities of rats for at least 60 days (S. Mattoo, P. A. Cotter, and J. F. Miller, unpublished data). Establishment of infection in the trachea, however, requires that bacteria be able to resist or overcome the clearance action of the mucociliary escalator as well as the killing effects of defensins, complement, and other antimicrobial factors. FHA, presumably by functioning as an adhesin, appears to be essential for overcoming mucociliary clearance [123], and LPS may be important for resistance to complement [205a]. TCT, released by Bordetella growing among the cilia, is proposed to stimulate IL-la production in neighboring nonciliated mucus-secreting cells, inducing expression of iNOS. The large amount of NO that is produced then diffuses into the ciliated epithelial cells, causing mitochondrial bloating, membrane blebbing, and extrusion from the mucosal surface. Damage and loss of tracheal epithelial cells containing adherent bacteria probably contributes to respiratory disease symptoms and possibly also to transmission by the aerosol route. Damage to respiratory epithelia undoubtedly also results in release of inflammatory cytokines. Although the specificity and magnitude of cytokine release has not been measured, recruitment of inflammatory cells, predominantly neutrophils, into the lungs of mice within 3 days following intranasal inoculation with either B, pertussis or B. bronchiseptica has been demonstrated [34, 165, 166]. This inflammatory response is significantly decreased in animals infected with ptx or cyaA mutants [34, 165, 166]. Both Ptx and CyaA have been shown to inhibit the microbicidal activities of neutrophils and macrophages in vitro. By doing so in vivo, these toxins may serve as anti-defense mechanisms, allowing Bordetella to resist the killing action of phagocytic cells. Without CyaA and Ptx, Bordetella mutants are efficiently eliminated by fewer numbers of neutrophils and macrophages, and hence less inflammation occurs. The importance of CyaA in resisting constitutive host defense mechanisms was recently demonstrated using mice that lack the ability to mount an adaptive immune response. SCID/Beige mice, defective in the development of T and B cells and NK-cell activity, are dependent on constitutive nonadaptive defense mechanisms for protection against microbial pathogens. Wild-type B. bronchiseptica killed these immunodeficient mice within about 50 days, while CyaA-deficient mutants did not even cause noticeable disease in these animals [166]. SCID mice are also killed by B. bronchiseptica, but with a slightly longer time to death [166]. This result indicates a role for CyaA in overcoming the defense mechanisms that are retained in SCID and SCID/Beige mice, which includes neutrophils. Mice rendered neutropenic, either by administration of cyclophosphamide or by a homozygous null mutation of the gene encoding granulocyte colonystimulating factor (G-CSF), were killed by both wild-type and cyaA mutant B. bronchiseptica, demonstrating that in the absence of neutrophils cyaA is not
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required [166]. The conclusion drawn from these experiments is that neutrophils are in vivo targets for the action of CyaA. Experiments using immunodeficient mice also revealed the importance of adaptive immunity in controlling Bordetella infections. B. bronchiseptica is confined to the upper respiratory tract in immunocompetent hosts but causes a lethal systemic infection in SCID or SCID/Beige mice. For B. pertussis, the ability of mice to mount an adaptive immune response determines whether the infection persists indefinitely in the lungs or is cleared entirely [205a]. While these observations demonstrate a crucial role for adaptive immunity, they do not reveal the nature of the immune response that is involved. It has been assumed that B. pertussis, as a noninvasive respiratory pathogen, is controlled primarily by a humoral immune response. The generation of ^nii-Bordetella antibodies in response to Bordetella infection is not only well documented but is considered diagnostic for pertussis in the absence of positive nasopharyngeal swab cultures. Using mice that do not express the interferon-y receptor, interleukin-4, or immunoglobulin heavy chain genes, Mills et al have demonstrated an absolute requirement for B cells, or their products, in clearance of B. pertussis infection in mice [206] and we have demonstrated that serum containing dinii-Bordetella antibodies can rescue SCID/Beige mice from lethal infection by B. bronchiseptica [206a]. However, there is increasing evidence that cell-mediated immunity also plays a significant role in controlling Bordetella infections. CD4"^ T cell clones that proliferate when stimulated with Ptx or FHA have been identified in peripheral blood mononuclear cells from children and adults following recovery from pertussis [207-210]. Secretion of interferon-y by these cells suggests they are Th-1 T cells [211], which are crucial for activation of phagocytic cells such as macrophages. Spleens of mice recovering from respiratory infection with B. pertussis were also shown to contain T cells that proliferate in response to heat-killed B. pertussis and secrete cytokines indicative of a Th-1 response [212, 213]. These spleen cells, upon adoptive transfer, were capable of inducing clearance of ^. pertussis from the lungs of nu/nu mice that lack functional T cells [214]. It therefore appears that both humoral and cell-mediated immune responses are important for the control and/or clearance of Bordetella infections. The relative contributions of each, and the mechanisms involved, await further investigation. Perhaps even less is known about the relative contributions of humoral and cell-mediated immunity in protection against reinfection. Recovery from pertussis in humans corresponds to the development of long-lasting protection against subsequent disease [215]. It has been assumed that this immunity is antibody-mediated, and vaccination strategies have been focused on the induction of anii-Bordetella antibodies. More recent evidence suggests, however, that, while vaccination against B. pertussis in humans and B. bronchiseptica in lower animals confers protection against disease, it is less effective at preventing colonization [216218]. The ability to detect Bordetella-spec'ific CD4"^ T cell clones in humans
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recovering from pertussis suggests the possibility that, while antibody may suffice to neutralize the effects of secreted toxins, activation of phagocytic cells may be required for eliminating bacteria from the respiratory tract.
VL The BvgAS Sensory Transduction System The genes and operons encoding all of the protein virulence factors described above are positively regulated by the products of bvgAS (originally called vir). This locus was first identified by Weiss and Falkow as the site of a Tn5 insertion in B. pertussis, which had simultaneously lost the ability to synthesize Ptx, CyaA, and FHA [219]. The locus was proposed, and subsequently proven, to encode trans-aciing regulatory factors required for coordinate activation of virulence gene expression. Inactivation of bvgAS by mutation, or of its products (BvgAS) by the presence of modulating signals, results in loss of virulence gene expression. Modulating signals that inactivate BvgAS in the laboratory include millimolar concentrations of nicotinic acid or MgS04, or growth at low temperatures (<26°C). BvgA and BvgS are members of a large family of signal-transducing proteins, known as two-component regulatory systems, that use phosphorylation reactions to regulate stimulus/response pathways (for reviews see [220-222]). Prototypical two-component systems contain a sensor kinase, which is usually a cytoplasmic membrane protein with a periplasmic signal input domain and a cytoplasmic transmitter domain, and a response regulator, which is typically a cytoplasmic protein containing a receiver domain and an output domain. Signal recognition by the input domain of the sensor kinase results in autophosphorylation of a conserved histidine (His) residue in the transmitter. This phosphoryl group is then transferred to a conserved aspartic acid (Asp) residue in the receiver of the response regulator, rendering the response regulator competent to modify cellular behavior via its output domain, which is usually, but not always, involved in activating and/or repressing gene transcription. The subfamily of two-component systems to which BvgAS belongs use a four-step His-Asp-His-Asp phosphotransfer signaling mechanism involving a transmitter, a receiver, a more recently discovered histidine phosphotransfer domain (HPD), and a second (response regulator) receiver domain. These complex systems are not necessarily limited to "two components" [223]. For example, in the phosphorelay that governs sporulation in B. subtilis, the first member of this subfamily to be described, the signaling domains are contained on four separate proteins: Kin(A, B, or C), SpoOF, SpoOB, and SpoOA [224]. In BvgAS, the first three phosphotransfer domains (transmitter, receiver, and HPD) are contained on a single, integral cytoplasmic membrane sensor kinase, BvgS,
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and the last (receiver) is contained on BvgA, a typical cytoplasmic response regulator (Fig. 3). Mutational studies have shown that His-729 of the transmitter, Asp-1023 of the BvgS receiver, His-1172 of the HPD, and Asp-54 of the BvgA receiver are essential for the phosphorylation cascade and virulence gene activation [225, 226]. The phosphorelay modeled in Figure 3 was deciphered using a biochemical approach in which the BvgS-signaling domains, expressed and purified alone and in combination, were analyzed in in vitro phosphorylation assays with purified BvgA [226, 227]. In this model, signal inputs detected by the periplasmic domain are relayed through the membrane to the transmitter, which autophosphorylates at His-729 (step 1) by a reaction that is reversible in vitro. His-729 then donates the phosphoryl group to Asp-1023 of the receiver (step 2). Asp-1023 can donate the phosphoryl group to His-1172 of the HPD (step 3a) or to water to form inorganic phosphate (step 3b). The HPD can then transfer back to the BvgS receiver (step 3a) or it can phosphorylate, and thus activate, BvgA (step 4). An elegant study by Gross and colleagues, in which domains of the E. coli BvgAS homolog EvgAS were swapped with the corresponding domains of BvgAS, demonstrated that the specificity of BvgS for BvgA is mediated by the HPD [228]. Phosphorylated BvgA (BvgA-P) has increased affinity for Bvg-activated promoters and is competent in in vitro transcription assays [229-234]. For simplicity, the model in Figure 3 shows phosphoryl group transfer between the signaling domains of a single BvgS monomer. In vivo complementation of bvgS mutations indicate intermolecular phosphotransfer can occur [227, 235], and experiments using a X repressor based dimerization probe system suggest both the transmitter and the HPD can mediate dimerization [235]. Genetic and biochemical analyses indicate BvgA probably also functions as a dimer [229, 235, 236] and may form higher-order multimeric complexes when cooperatively binding DNA at specific promoters [231]. The question that arises from the elucidation of this phosphorelay is: Why is this pathway so complex? For the vast majority of two-component regulatory systems that have been identified, including many that control virulence gene expression in various pathogenic bacteria, a two-step phosphorelay appears to suffice. In the B. subtilis sporulation control system, the four-step phosphorelay appears to provide a mechanism for allowing signals other than those that affect the sensor kinases (KinA, B, C) to also regulate the signal transduction pathway. Phosphatases (RapA and RapB), which are controlled by peptide pheromones, have been discovered that dephosphorylate SpoOF~P, the second component in the sporulation phosphorelay [224, 237, 238]. It has been hypothesized that the Rap phosphatases may have been recruited to help regulate sporulation because the number of signals to which this process must respond exceeds the recognition capacity of the sensor kinases [239]. Although phosphatases that affect the BvgAS phosphorelay have not yet been discovered, their existence remains a distinct possibility. Alternatively, or additionally, the complexity of the BvgAS phosphorelay may reflect an ability to
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respond to signal intensity rather than signal diversity so that, rather than functioning like a switch that is responsive to many different signals, BvgAS may function like a rheostat that is adjusted in response to variations in intensity of a limited number of signals. As discussed below, there is evidence that BvgAS controls expression of a spectrum of phenotypic phases in response to subde yet distinct quantitative differences in environmental cues [37]. There is also evidence that the different patterns of gene expression required to produce these various phenotypic phases occur in response to variations in BvgA~P levels. The ability of the BvgS receiver to mediate phosphorylation and dephosphorylation of the HPD as well as dephosphorylation of the transmitter suggests it plays a pivotal role in the phosphorelay and may be responsible for regulating the flow of phosphate to the HPD and hence to BvgA [226]. Further genetic and biochemical analyses will be required to determine the validity of this hypothesized mechanism for adjusting BvgA~P levels. Although, as mentioned above and below, signals to which BvgS responds in the laboratory have been identified and extensively characterized, the true signals that are sensed in nature are unknown. The large BvgS periplasmic domain is thought to be involved in signal recognition since mutations in this region alter or abrogate signal sensitivity [38, 240]. Two regions with similarity to E. coli solute-binding proteins involved in glutamine and histidine transport are present in the BvgS periplasmic domain; however, their relevance is currently unknown. Also unknown is the relevance of PAS/PAC domains that are present in the BvgS linker, a region where many mutations that render BvgS insensitive to modulating signals occur. PAS/PAC domains were originally associated with light reception, light regulation, and clock proteins [241]. A broad, comprehensive search has revealed a large family of PAS/PAC-containing signal-transducing proteins, many of which mediate responses to changes in concentration of oxygen, redox carriers and carbon sources [242]. Although it has not been determined if BvgAS responds to oxygen concentration or redox potential, BvgAS reciprocally controls the expression of cytochrome d [243] and c oxidases (M. Liu, P. A. Cotter, and J. F. Miller, unpublished data), which presumably facilitate respiration under low and high oxygen environments, respectively. In addition to contributing to our understanding of how the BvgAS signal transduction system functions mechanistically, mutational analyses ofbvgAS have also produced a number of valuable tools for deciphering the structure of the BvgAS regulon and for investigating the role of Bvg-mediated signal transduction in vivo. Most notable are mutations that alter, rather than abrogate, the signal transduction pathway. One class, the bvgS constitutive mutations, is exemplified by the bvgS-C3 allele (Fig. 3) [240]. These mutations result in single amino-acid substitutions in the BvgS linker that lock BvgS in its active form, rendering it insensitive to modulating conditions. Strains that are isogenic with wild-type Bordetella except for these single nucleotide differences constitutively express all known Bvg-activated virulence factors [30, 240]. A second class, exemplified by
646
PEGGY A. COTTER AND JEFF F. MILLER
the bvgS-ll mutation (Fig. 3), appears to decrease the overall activity of the system [37]. The bvgS-ll mutation results in a methionine-to-threonine substitution four amino acids away from the primary site of phosphorylation in the BvgS transmitter. A third class, which includes the bvgAW60 and bvgA1056 mutations, maps to the extreme 3' end of bvgA [244]. These mutations abrogate the ability of BvgA to activate transcription of a subset of Bvg-activated genes and were instrumental in deciphering how BvgAS differentially controls virulence gene expression.
VIL Phenotypic Modulation The phenomenon of phenotypic modulation was first recognized early in the twentieth century [245] and was rigorously characterized by Lacey in 1960 [246]. It was originally defined as the reversible loss of virulence-associated phenotypes by Bordetella species that occurs in response to certain growth conditions [246]. Using colony morphology, hemolysin production, and antigenicity as indicators, Lacey described distinct phenotypic phases and designated them X mode (virulent phase), C mode (avirulent phase), and I mode (intermediate phase). High temperature (37°C), and certain ions such as sodium, potassium, halides, formate, and nitrate were shown to favor the virulent phase, while low temperature (25°C) and ions such as S04~^, and mono- and dicarboxylic acids favor avirulent phase growth. Further investigation showed that chlorate anions and nicotinic acid derivatives also resulted in downregulation of virulence factors [247]. It is now known that these changes are controlled by BvgAS. Lacey's X mode apparently corresponds to the Bvg"^ phase, C mode to the Bvg" phase, and I mode to the more recently characterized Bvg-intermediate (BvgO phase. A. The Bvg Phase Although Lacey's work provided evidence for C mode (Bvg~ phase)-specific antigens, BvgAS was traditionally considered to function merely as an ON/OFF switch for virulence gene expression. It was the discovery of Bvg-repressed genes, vrgs in B, pertussis [248], and genes required for motility in B. bronchiseptica [53] that proved that BvgAS also controls phenotypes expressed exclusively in the Bvg" phase. These discoveries necessitated a redefinition of phenotypic modulation to include the induction of Bvg~ phase factors and demonstrated that BvgAS actually functions to mediate a transition between at least two distinct phases, each with unique characteristics and unique patterns of gene expression (Fig. 4).
13.
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In B. bronchiseptica, the Bvg~ phase is distinguished by the prominent phenotype of motihty. Two motihty loci, flaA (the flagelUn structural gene) and frlAB (the motility master regulatory locus, similar io flhDC of E. coli), have been characterized [3, 53] (Fig. 2). Both are expressed only in the Bvg~ phase [3, 53]. Based on complementation studies, and by analogy with the E. coli motility regulon, iht frlAB gene products are proposed to function together as a transcriptional activator positioned at the top of a regulatory cascade required for synthesis and assembly of flagella as well as proteins required for chemotaxis [3]. Substitution of the frlAB promoter with the Bvg-activated/Txfl^ promoter, so that frlAB transcription is activated by BvgAS, results in ectopic synthesis of flagella and motility in the Bvg"*" phase [31]. This result confirmed that activation offrlAB is sufficient to induce the entire motility regulatory cascade and demonstrated that repression offrlAB expression is the point at which BvgAS controls motility./r/A5 represents the first intermediate regulatory locus in the BvgAS regulon to be identified. Although B. pertussis, B. parapertussis hu, and B. parapertussis^y strains are nonmotile, their chromosomes contain sequences that hybridize io flaA [53] (U. Heininger, P. A. Cotter, and J. F. Miller, unpublished data). B. pertussis also contains anfrlAB locus that is functional when expressed in B. hronchiseptica (B. J. Akerley and J. F. Miller, unpublished data); however, its role in B. pertussis is unknown. Urease activity has been used as a diagnostic feature to distinguish B. hronchiseptica and B. parapertussis (Urease^) from B. pertussis (Urease") for many years. It was shown to be a Bvg-repressed phenotype in B. hronchiseptica [249]. Interestingly, urease expression is also repressed by BvgAS in B. parapertussis hu, while B. parapertussis^^ strains appear to have lost BvgAS control of urease (Fig. 1) (U. Henninger, unpublished data). The nucleotide sequence of the urease gene cluster in B. hronchiseptica was recendy determined revealing putative structural and accessory genes (ureA-G, 7, /), as well as a postulated regulatory locus (hhuR) with potential to encode a LysR transcriptional activator homolog [250]. B. pertussis and B. hronchiseptica produce an iron-chelating siderophore called alcaligin [251], and it is likely that the siderophore produced by B. parapertussis strains is also alcaligin [252]. Genes required for alcaligin biosynthesis (alcABC) and a potentially Fur-responsive AraC-type transcriptional regulator (alcR) have been identified in B. pertussis and B. hronchiseptica [252, 253]. Alcaligin biosynthesis is negatively regulated by BvgAS in some B. hronchiseptica strains [254] but appears not to be regulated by BvgAS in B. pertussis [255]. BvgAS regulation of alcaligin production in B. parapertussis strains has not been examined. Using transposon mutagenesis, we have recently identified additional Bvg-repressed loci in B. hronchiseptica (M. Liu, P. A. Cotter, and J. F. Miller, unpublished data). These genes include ccoC, which encodes a cytochrome c oxidase subunit 3 homolog, and prpE, a propionyl-coA synthetase homolog. The discovery that genes involved in electron transport-mediated oxidative phosphorylation and carbon utilization are BvgAS regulated indicates that, in addition to controlling the expression of virulence genes and other "accessory" factors, BvgAS
PEGGY A. COTTER AND JEFF F. MILLER
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controls basic physiologic processes. This observation highlights the importance of this global regulatory system in the life cycle of the organism. In comparison to B. bronchiseptica, little is known about the Bvg" phase of B. pertussis. Although Knapp and Mekalanos discovered five Bvg~ phase-specific B. pertussis genes {vrg6, vrgl8, vrg24, vrg53, and vrg73) in 1988 [248], the functions of the gene products are still unknown. Their regulation, however, has been studied in some detail and involves bvgR, the second intermediate regulatory locus in the BvgAS regulon to be identified [256]. Additional Bvg-repressed factors were discovered using a biochemical approach. Using two-dimensional gel electrophoresis and a hybridoma bank of monoclonal antibodies, Stenson and Peppier demonstrated the existence of several Bvg~ phase-specific outer membrane proteins in B. pertussis [257]. Two that were further characterized, vra-a and vra-b, appear not to be encoded by any of the previously identified vrgs and are not expressed in B. bronchiseptica or B. parapertussis [257]. Roles for these factors have also not yet been determined. Repression of vra-a and vra-b has been shown to involve BvgR [258, 259].
Fig. 4 (opposite) The three phases of Bordetella. BvgAS control at least three distinct phenotypic phases in response to environmental conditions. (A) The Bvg^ phases of B. pertussis (B.p.) and B. bronchiseptica {B.b.) are nearly identical. Both subspecies express a variety of surface molecules (solid and dashed lines) and secreted factors (solid circles). Both subspecies appear to also express a similar Bvg' phase, characterized by the expression of a subset of Bvg-activated factors (dashed lines) as well as factors expressed maximally in this phase (shaded triangles). The Bvg" phases of B. pertussis and B. bronchiseptica differ, vrgs and vras (whose products are represented by solid ovals), are expressed only by B. pertussis, while motility (flagella are represented by curved lines) and other coregulated factors such as urease (represented by solid circles) are expressed by B. bronchiseptica but not by B. pertussis. The Bvg"^ phases of both B. bronchiseptica and B. pertussis have been shown to be necessary and sufficient for respiratory tract colonization, while the Bvg' phase is hypothesized to be involved in aerosol transmission. The Bvg~ phase of B. bronchiseptica is necessary and sufficient for survival under nutrient poor conditions and may contribute to transmission of this organism by allowing it to survive in an environmental reservoir. (B) BvgAS controls expression of at least four classes of genes. "Late" Bvg-activated genes (1), such as cyaA, are expressed only under Bvg"'" phase conditions (in the absence of modulators such as nicotinic acid), while "early" Bvg-activated genes (2), such asfhaB and bvgAS), are expressed under both Bvg' and Bvg^ phase conditions. Bvg' phase genes (3), such as bipl, are expressed maximally under Bvg' phase conditions (e.g., in the presence of low levels of nicotinic acid). Expression of Bvg-repressed genes (4) is maximal under Bvg~ phase (fully modulating) conditions. (C) Western blots showing three classes of Bvg-regulated antigens. Whole cell lysates of Bvg' or Bvg~ phase-locked B. bronchiseptica, or wild-type B. bronchiseptica grown in the presence of various concentrations of nicotinic acid (mM nic), were separated by SDS-PAGE, transferred to PVDF, then probed with serum from a rat infected with wild-type B. bronchiseptica (left panel) or a rat infected with a Bvg' mutant (right panel). Polypeptides encoded by "late" Bvg-activated genes (1) are present only in wild-type B. bronchiseptica grown in the presence of <0.2 mM nicotinic acid, while polypeptides encoded by "early" Bvg-activated genes (2) are present in the Bvg' mutant as well as wild-type B. bronchiseptica grown in the presence of <2.0 mM nicotinic acid. Bvg' phase antigens (3) are maximally expressed by wild-type B. bronchiseptica grown in the presence of 0.4 mM nicotinic acid.
650
PEGGY A. COTTER AND JEFF F. MILLER
B. The Bvgi Phase Experiments aimed at understanding the mechanism of action and consequences of BvgAS regulation have focused primarily on the Bvg"^ and Bvg" "poles," that is, the gene expression patterns and phenotypic phases that result when BvgAS is either fully active or inactive. The serendipitous finding of a B. bronchiseptica mutant locked "in the middle" led to the rediscovery of the Bvg-intermediate (BvgO phase. Molecular analysis of this Bvg' phase-locked mutant, and of wild-type B. bronchiseptica grown under semimodulating conditions (e.g. 0.4-1.6 mM nicotinic acid), showed that the Bvg^ phase is characterized by the presence of a subset of Bvg"^ phase factors, the absence of a different subset of Bvg"^ phase factors, and the presence of factors that are expressed exclusively in this phase. Bvg~ phase loci do not appear to be expressed in the Bvg' phase (Fig. 4) [37]. Bvg"^ phase genes expressed in the Bvg' phase include/Tza^ and bvgAS, and Bvg"^ phase genes that are not expressed in the Bvg' phase include cyaA [37]. We have recently discovered a candidate Bvg' phase-specific gene, designated bipl (K. E. Stockbauer, B. Fuchlocher, J. F. Miller, and P. A. Cotter, unpublished data; R. Deora, P. M. H. Cham, J. F. Miller, and P. A. Cotter, unpublished data). Antibodies that recognize Bvg' phase polypeptides in B. bronchiseptica (Fig. 4) also recognize Bvg' phase polypeptides of similar size in B. pertussis (G. Martinez de Tejada, J. F. Miller, and P. A. Cotter, unpublished data), demonstrating similarity between the Bvg' phases of these organisms. Characterization of the Bvg' phase demanded further evolution of our thinking about BvgAS. It now appears that, rather than mediating an alteration between two distinct phases, BvgAS may actually control an entire spectrum of phenotypic phases in response to a variety of subde but discernible changes in environmental conditions. The proposed relevance of this ability is discussed in a later section.
VIIL Transcriptional Controi of Bvg-Regulated Genes More than 50 years after the first description of phenotypic modulation in Bordetella [245], Weiss and Falkow provided a genetic basis for the phenomenon by identifying the bvgAS master regulatory locus [219]. Elucidation of the four-step BvgAS phosphorelay (described above) has provided considerable insight into how environmental signals are transduced. How these signal-transduction events contribute to the complex patterns of gene expression that produce the various phenotypic phases is discussed below. A. BvgAS-Mediated Activation of Virulence Gene Expression Initial experiments aimed at understanding how BvgAS controls gene expression focused on fhaB, which encodes FHA, and bvgAS. Both are activated by BvgAS
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at the level of transcription; hence, bvgAS is positively autoregulated [260, 261]. fhaB and bvgAS are adjacently located and oppositely oriented on the Bordetella chromosome [262]. Primer extension and SI nuclease analyses detected several transcription start sites within the 425-bp bvgAS-fhaB intergenic region [236, 261]. fhaB transcription is initiated 70 bp upstream of the fhaB translation initiation codon while bvgAS is transcribed primarily from two promoters [236]. Under Bvg"^ phase conditions, bvgAS transcription initiates at bvgAS-?\, located 93 bp upstream of the bvgAS structural genes. Under Bvg~ phase conditions bvgAS is transcribed at a low basal level from bvgAS-?2, located 143 bp upstream of bvgAS [236, 261]. Deletion analysis, gel mobility shift analysis, and DNase I protection studies identified TTT(C/G)NTA heptanucleotide repeats, located approximately 80 bp upstream of the fhaB and bvgAS-Fl transcription initiation sites, as BvgA-binding sites [263]. The location of the heptanucleotide repeat at the fhaB promoter suggests that bound BvgA (probably as a dimer) may directly facilitate binding and transcription initiation by RNA polymerase. The heptanucleotide repeat at the bvgAS promoters is located such that BvgA binding simultaneously activates transcription at bvgAS-Fl and blocks transcription from bvgAS-P2. BvgA therefore functions as both an activator and a repressor at this site. Consistent with the hypothesis that BvgA directly activates transcription of bvgAS Sind fhaB, bvgAS supplied in trans is sufficient to activate transcription of fhaB'-lacZ and bvgA'-lacZ fusions in E. coli [260, 261]. Analysis of the ptxA and cyaA promoters yielded different results, c/^-acting sequences required for BvgAS-dependent transcriptional activation of ptxA and cyaA were shown to be located much further upstream from the promoters of these genes than for bvgAS mdfhaB, and they did not contain TTT(C/G)NTA repeats [264-266]. Additionally, neither ptxA'-lacZ nor cyaA'-lacZ fusions could be activated by bvgAS in E. coli [267, 268], and initial attempts to demonstrate binding of purified BvgA to ptxA and cyaA promoter sequences in vitro failed [263, 269]. Studies by Scarlato et al. further distinguished transcriptional activation of bvgAS and fhaB from that of cyaA and ptxA. These authors demonstrated that, when B. pertussis cultures were shifted from modulating (Bvg~ phase) to nonmodulating (Bvg+ phase) conditions, bvgAS and fhaB transcripts could be detected within 10 minutes, while cyaA and ptxA transcripts did not begin to accumulate until 2 hours postshift [270]. Taken together, these data indicate that transcription of "late" genes (ptxA and cyaA) is not activated by the same straightforward mechanism by which transcriptional activation of "early" genes (bvgAS and fhaB) apparently occurs. A well-accepted hypothesis for this difference was that an accessory or intermediate regulatory factor(s) was required for transcriptional activation of late, but not early, genes. Several groups have tried to find accessory regulatory loci involved in induction of cyaA and ptxA expression, without success. Characterization of the mutants generated in these studies, however, contributed significantly to our
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PEGGY A. COTTER AND JEFF F. MILLER
current understanding of how BvgAS differentially controls expression of early and late Bvg-activated genes. One B. pertussis mutant that was unable to activate expression of ptxA and cyaA, but was relatively unaffected for bvgAS ^nidfliaB expression, was shown to contain a mutation just upstream of the translation start site of rpoA, which encodes the a subunit of RNA polymerase (RNAP) [271, 272]. This mutation results in overexpression of rpoA [272]. Many transcriptional regulators activate transcription by interacting with the C-terminal domain of the a subunit of RNAP [273, 274]. One explanation for how overexpression of RpoA could selectively prevent expression of cyaA and ptxA is that excess a subunits may bind to and thus titrate BvgA, decreasing the relative concentration of active BvgA in the cell to a level below that required for activation of ptxA and cyaA. Evidence that the ptxA and cyaA promoters do in fact require higher levels of active BvgA than the bvgAS and fhaB promoters is discussed below. Other B. pertussis mutants specifically defective for activation of cyaA Sind ptxA expression contained mutations in bvgA [244]. These mutations affect negatively charged residues at the extreme C terminus of BvgA. By analogy with characterized mutations in the Vibrio fischeri LuxR protein [275], these residues may be critical for interacting with the a subunit of RNAP, without affecting DNA binding. The specificity of these mutations for cyaA and ptxA suggests that BvgA-dependent transcriptional activation at the cyaA and ptxA promoters differs mechanistically from BvgA-dependent transcriptional activation at the bvgAS dind fhaB promoters. Together, these data support a model in which BvgA directly activates transcription of both early and late genes, but, because of differences in affinity and location of BvgA-binding sites, higher concentrations of active BvgA are required to activate transcription of cyaA and ptxA than fhaB and cyaA. Additional and more direct evidence now exists indicating that BvgAS alone is sufficient to directly control expression of Bvg-activated genes. In vitro transcription studies by three laboratories have shown that, although unphosphorylated BvgA is able to bind the heptanucleotide repeats at the bvgAS and fhaB promoters, BvgA must be phosphorylated to bind at the cyaA and ptxA promoters [231, 232, 234, 276]. Moreover, the concentration of BvgA~P required to bind ptxA promoter sequences in vitro is 10-fold higher than that required to bind the bvgAS and fhaB promoters [276]. These data suggest that the failure to detect BvgA binding to ptxA and cyaA promoter sequences in earlier studies was due to the use of unphosphorylated BvgA. Although not absolutely required for binding at the bvgAS and fhaB promoters, phosphorylation of BvgA enhances binding affinity and BvgA~P binds a larger region of DNA at these promoters than unphosphorylated BvgA, suggesting cooperative binding of multiple BvgA~P molecules [230, 276]. Boucher et al. also demonstrated that BvgA~P, in fact, and not BvgA, redirects RNA polymerase binding to a site appropriate for transcriptional activation of fhaB [231]. Cooperative binding of multiple BvgA~P dimers appears to also be important, if not essential, for transcriptional activation of cyaA
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and/?rjcA, as the region of DNA bound by BvgA~P at these promoters extends all the way from -139 (for cyaA) or -168 (for ptxA) to the -35 region [232, 276]. Closer examination of cyaA and ptxA promoter regions revealed several heptanucleotide sequences with less conformity to consensus and suboptimal spacing compared to the repeats present at fhaB and bvgAS, providing a basis for their apparent lower affinity for BvgA~P [232, 233]. These data suggest that BvgAS can differentially regulate virulence gene expression simply by controlling the intracellular concentration of BvgA~P; when BvgA~P levels are low, only bvgAS, fhaB, and other so-called early genes will be expressed. When BvgA~P levels are high, all Bvg-activated genes will be expressed. B. BvgAS-Mediated Repression of Gene Expression Very little mechanistic information is available regarding BvgAS-mediated repression of gene expression. As mentioned above, the frlAB locus encodes a transcriptional activator required for expression of the entire motility regulon in B. bronchiseptica. Genetic analyses indicate frlAB transcription is direcdy repressed by BvgA, and sequences with similarity to consensus BvgA-binding sites are present upstream of i\\t frlAB promoter [3]. Thus, BvgAS may negatively regulate motility directly by repressing expression of a transcriptional activator, frlAB. In contrast, BvgAS negatively regulates vrg expression in B. pertussis by activating expression of bvgR, a gene involved in repression. Although BvgR mediates repression of all vrgs and the outer membrane proteins vra-a and vra-b [256, 259], it has not yet been determined if BvgR acts directly as a transcriptional repressor or if additional regulatory factors are involved. Beattie et al identified a 32-bp consensus sequence within the coding region of four of the five vrgs [277]. Characterization of this element in vrg6 showed that it is involved in BvgAS-mediated repression and southwestern experiments identified a 34-kDa protein that binds to a DNA fragment containing the consensus sequence [277]. Since bvgR is predicted to encode a 23-kDa protein, the 34-kDa protein could represent an additional regulatory factor. C. A Model for the Global Regulation of Gene Expression by BvgAS It is now evident that BvgAS differentially regulates expression of at least four classes of genes resulting in expression of at least three distinct phenotypic phases (Fig. 4). The Bvg~ phase is characterized by the expression of Bvg-repressed genes that include vrgs and those encoding vra-a and vra-b in B. pertussis and those encoding motility, alcaligin production, urease, a cytochrome c oxidase, and a propionyl CoA synthetase in B. bronchiseptica (Fig. 2). These genes appear to be expressed only under fully modulating, or Bvg~ phase, conditions. The Bvg' phase is characterized by the expression of "early" Bvg-activated genes, such as
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PEGGY A. COTTER AND JEFF F. MILLER
fhaB and bvgAS, and Bvg^ phase-specific genes, but not "late" Bvg-activated genes, such as cyaA and ptxA, or Bvg-repressed genes. The Bvg"^ phase is characterized by expression of both early and late Bvg-activated genes and not Bvg^ or Bvg~ phase-specific genes. To mediate the expression of multiple distinct phenotypic phases, BvgAS itself must be capable of achieving multiple functional states, each with the ability to control the pattern of gene expression characteristic of that particular phenotypic phase. The simplest hypothesis is that altered functionality is a reflection of BvgA~P concentration, and that different classes of Bvg-regulated genes, because of differences in the locations and affinities of BvgA-binding sites at their promoters, require different critical threshold levels of BvgA~P for activation or repression. A model for how this could occur is as follows. Under fully modulating conditions, or when bvgS is inactivated by mutation, the low level of predominantly unphosphorylated BvgA in the cell is insufficient to activate Bvg-activated genes or to repress Bvg-repressed genes. Bvg-repressed loci are therefore expressed. Under semimodulating conditions, the level of BvgA~P in the cell is intermediate between the maximal level achieved in the Bvg^ phase and the very low level present in the Bvg~ phase. One could envision that this could occur either by submaximal induction of bvgAS transcription or by a submaximal rate of phosphotransfer to BvgA. Although bvgAS appears to be maximally transcribed under "semimodulating" conditions and in a Bvg' phase-locked mutant [37], it seems likely that both mechanisms contribute to control of BvgA~P levels. It is also not known whether the ratio of BvgA~P to BvgA, or only the absolute amount of BvgA~P, is important. However generated, the model predicts that, under semimodulating conditions, the resulting intermediate level of BvgA~P is sufficient to repress Bvg-repressed loci, and to activate early genes and Bvg^ phase-specific genes (predicted to contain high-affinity Bvg-activation sites), but not to activate late genes (containing only low-affinity Bvg-activation sites). When grown in the absence of modulating signals, BvgA~P levels are maximal and sufficient to activate both early and late Bvg-activated genes and to mediate repression of genes encoding Bvg' phase factors. This model therefore predicts that Bvg^ phase promoters will contain low-affinity Bvg-repression sites as well as high-affinity Bvg-activation sites.
IX, The Role of Byg-Mediated Signal Transduction in the Bordetella Life Cycle As is apparent from the preceding sections, BvgAS precisely and coordinately controls expression of a large number of genes, including those involved in virulence, motility, iron acquisition, oxygen utilization, and energy metabolism.
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The question that arises is why? What is the role of Bvg AS-mediated signal transduction in the Bordetella Hfe cycle? When and why are the various BvgAS controlled phenotypic phases expressed? Infection by Bordetella begins when the organism enters the respiratory tract of a susceptible host. Adherence to epithelial surfaces in the nasal cavity is an early critical step in the establishment of infection. Multiplication and spread to the lower respiratory tract may then follow. At some point, Bordetella must leave the infected host and be transmitted to a new host. For B. pertussis and B. parapertussis, transmission is thought to occur exclusively, and very efficiently, by aerosolized respiratory droplets. This assumption is based in part on the fastidious nature of these species, especially B. pertussis, and the failure to isolate them from environmental reservoirs or fomites. B. bronchiseptica, in contrast, is amazingly adaptive in its ability to survive adverse conditions and may be capable of transmission via an environmental reservoir in addition to transmission by the aerosol route. The life cycles of ^. pertussis, B. parapertussis, and B. bronchiseptica therefore appear to be similar during the time spent in the host but may differ during the time spent in transit from host to host. As discussed below, we hypothesize that differences in the Bvg" phases of these organisms may reflect differences in the environments they occupy between hosts. The bvgAS loci of B. pertussis, B. parapertussis, and B. bronchiseptica are highly conserved. DNA sequence analysis indicates that the BvgA proteins encoded by these organisms are identical [52, 278]. The BvgS proteins encoded by B. bronchiseptica and B. parapertussis differ at only 6 of 1238 amino acids, and both differ from BvgS encoded by B. pertussis at only 60 amino acids, with most of the differences (40) occurring in the periplasmic domain [52, 278]. We have constructed a B. bronchiseptica strain containing the bvgAS locus from B. pertussis. This chimeric strain is indistinguishable from wild-type B. bronchiseptica in its ability to efficiendy establish a persistent respiratory infection in rats [38]. The Bvg" phase expressed by the chimeric B. bronchiseptica strain was indistinguishable phenotypically from that of wild-type B. bronchiseptica [38]. The functional conservation of BvgAS within and across Bordetella species suggests it mediates a common and important adaptive response in these organisms. It is clear that the Bvg"^ phase, which is nearly identical among the Bordetella that infect mammals, is required for virulence and the importance of several Bvg"^ phase factors during infection has been established. Roles for the Bvg^ and Bvg" phases have not yet been determined, but since B. pertussis and B. parapertussis are thought to be unable to persist outside the host, it has been assumed that phenotypic modulation must occur in vivo. Results from a series of experiments using B. bronchiseptica and B. pertussis mutants specifically affected in signal transduction, however, indicate that this widely held assumption may be incorrect. Postulated roles for a switch to the Bvg~ phase in vivo include evasion of antibodies directed against Bvg"^ phase factors, tempering of damage to host
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tissues as a result of decreased toxin expression, increased transmission as a result of decreased adhesin expression, and a requirement for Bvg" phase factors for the initial interaction with the host or for surviving within host cells [246, 279-282]. To experimentally determine if the ability to alternate between the Bvg"^ and Bvg~ phases is important during infection, we constructed B. bronchiseptica mutants that were locked into either the Bvg^ or Bvg" phase and compared them with wild-type B. bronchiseptica in rabbit and rat models [30, 31]. To our surprise, the Bvg"^ phase-locked mutant was indistinguishable from wild-type B. bronchiseptica in its ability to efficiendy colonize the nasal cavities and tracheas of rabbits and rats. Neither strain caused disease, and both induced high titers of anti-^ordetella antibodies that were directed primarily against Bvg"^ phase factors. These results demonstrate that the Bvg"^ phase is both necessary and sufficient for establishment of respiratory infection and that the Bvg~ phase is not required in vivo. Furthermore, antibodies against Bvg~ phase factors could not be detected in sera from any animal, suggesting wild-type B. bronchiseptica do not switch to the Bvg" phase during infection. Since BvgAS simultaneously activates virulence gene expression and represses genes and operons required for motility in B. bronchiseptica, the Bvg"^ phase is characterized by the presence of Bvg"^ phase adhesins and toxins as well as the absence of Bvg~ phase factors such as a rotating flagellar filament. To determine if the ability of BvgAS to repress gene expression is important in vivo, we constructed a strain in which flagella are expressed ectopically in the Bvg"^ phase. This strain, called FrF (Frl-reversed), was constructed by replacing the native frlAB promoter with the Bvg-activated/Tza^ promoter. Although the FrF strain was able to colonize the nasal cavities of rats as well as wild-type B. bronchiseptica, it was severely defective in its ability to colonize the trachea [31]. This result demonstrates that inappropriate expression of Bvg~ phase factors, flagella in this case, can actually be detrimental to the development of infection, underscoring the importance of Bvg-mediated repression of gene expression in vivo. Furthermore, anti-flagellin antibodies were detected in sera of animals infected with the FrF strain, indicating that B. bronchiseptica flagella are indeed antigenic if expressed in vivo and supporting the hypothesis that wild-type B. bronchiseptica do not switch to the Bvg~ phase during infection. The results obtained with B. bronchiseptica have been confirmed with B. pertussis [258, 259]. Bvg"^ phase-locked mutants were indistinguishable from wild-type B. pertussis in their ability to colonize the nasal cavity, trachea, and lungs [259] or to establish lethal infections [258] in mouse models. Analogous to the B. bronchiseptica FrF strain, B. pertussis strains containing in-frame deletions in bvgR fail to repress specific Bvg~ phase genes under Bvg"^ phase conditions. Compared to wild-type B. pertussis, BvgR" strains showed decreased ability to colonize the trachea and lungs [259] and decreased lethality [258] in mice, demonstrating the importance of Bvg-mediated repression of gene expression in B. pertussis in vivo. Furthermore, in contrast to a previously published report
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[279], the Bvg-repressed vrg6 gene was shown to be dispensable for respiratory infection and the colonization defect previously attributed to vrg6 was shown to be due to a second, unlinked mutation present on the original mutant strain [259]. Together, these results provide strong evidence that the Bvg~ phase is not required and probably not even expressed in vivo. What, then, is the role of the Bvg~ phase? Porter et al. have shown that B. hronchiseptica can survive and multiply in phosphate-buffered saline (PBS), in reagent-grade water, and in filtered lake and pond waters without added nutrients [283, 284]. These nutrientpoor conditions could mimic those of a postulated environmental reservoir for B. hronchiseptica. Comparison of wild-type and Bvg phase-locked mutants indicated that only Bvg" phase bacteria, induced either by mutation or by the presence of modulating signals, could survive and multiply in PBS or in other nutrient-deplete media [30]. This result demonstrates a clear advantage for the Bvg~ phase under nutrient limiting conditions. Together with the apparent lack of an in vivo role for the Bvg~ phase, these data suggest that the Bvg~ phase may play a role in transmission by allowing B. hronchiseptica to survive in the environment during the time spent between mammalian hosts. As mentioned above, a spontaneous mutation, hvgS-W, led to the discovery of the Bvg' phase in B. hronchiseptica. The mutation was selected on the basis that it conferred upon a Bvg"^ phase-locked strain the ability to survive in PBS [37]. Concomitant with increased ability to withstand nutrient deprivation was decreased virulence, demonstrating the reciprocity of these phenotypes. Introduction of the hvgS-\\ mutation into the chromosome of B. pertussis resulted in a Bvg' mutant with similar Bvg' phase-specific phenotypes to those characterized for B. hronchiseptica, that is, lack of expression of a subset of Bvg"^ phase factors and increased expression of Bvg' phase antigens (G. Martinez de Tejada, P. A. Cotter, and J. F. Miller, unpublished data). Moreover, B. pertussis grown under semimodulating conditions displayed the same Bvg' phenotypes. These observations indicate that, in contrast to the Bvg~ phases, the Bvg' phases of B. pertussis and B. hronchiseptica might be quite similar. We hypothesized that the Bvg' phase might be involved in transmission by the aerosol route since this is also a feature shared by these organisms. Using a recently developed rabbit model of aerosol transmission, we have observed transmission by wild-type B. hronchiseptica but not Bvg"^ phase-locked mutants (P. A. Cotter, K. E. Stockbauer, and J. F. Miller, unpublished data). This result suggests that phenotypic modulation, to some extent, is required for transmission. To account for these observations, we propose the following hypothesis. The BvgAS sensory transduction system may have originally evolved to play the same role for all Bordetella species, that of sensing whether the organism is within or outside an animal host, with the Bvg~ phase functioning to allow extended survival in the environment. As B. hronchiseptica is relatively avirulent, the ability to multiply in an environmental reservoir may be crucial to the survival of this organism in nature. Pressure to maintain Bvg" phase phenotypes such as
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motility and the ability to survive nutrient limitation may therefore be strong. In contrast, B. pertussis and B. parapertussis hu, which typically cause more virulent, acute, and very contagious infections, may not need to survive for long periods of time outside the human host and may therefore be under no selective pressure to maintain Bvg~ phase phenotypes. In support of this hypothesis, B. pertussis and B. parapertussis have lost the ability to express their/r/A^ and flaA loci and are nonmotile. This hypothesis would in fact predict random loss of Bvg~ phase factors in B. pertussis and B. parapertussis. As mentioned above, B. pertussis, which diverged from B. bronchiseptica earlier than B. parapertussis, has apparently also lost the ability to synthesize urease. We further hypothesize that the Bvg' phase is important for transmission by the aerosol route. Serological evidence indicates that the Bvg' phase is expressed by B. bronchiseptica during infection of rabbits and possibly by B. pertussis during human infection (our unpublished data). We postulate that the Bvg' phase may facilitate release of the bacterium from the respiratory tract of the infected host, incorporation of the bacteria into respiratory droplets, the ability to survive within these droplets until a new host is encountered, and/or the initial interaction of the bacterium with a new, susceptible host. Since all Bordetella species can be transmitted by the aerosol route, and in fact B. pertussis and B. parapertussis appear to be dependent on transmission by this route, there should be strong selection to maintain Bvg^ phase phenotypes. In fact, the Bvg' phases of the various species appear to be quite well conserved. Both B. pertussis and B. bronchiseptica express the same subset of Bvg-activated genes (fhaB, bvgAS, and fim), as well as crossreactive Bvg* phase antigens, under semimodulating conditions.
Acknowledgments We are grateful to Frits Mooi, Anneke van der Zee, and Duncan Maskell, as well as members of our laboratory, for critical comments on this manuscript. We would also like to thank Bill Goldman for allowing us to cite unpublished data. Our laboratory is supported by grants from the NIH and the American Cancer Society.
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CHAPTER 14
Pathogenesis of Haemophilus influenzae Infections CHRISTOPH M . TANG DEREK W. HOOD E. RICHARD MOXON
I. Introduction A. Historical Perspective B. Microbiology C. Epidemiology and Global Public Health Importance II. Population Biology m. Molecular Determinants of Pathogenicity A. Capsular Polysaccharide B. Lipopolysaccharide C. Pili and Fimbriae D. Outer-Membrane Proteins E. IgA Proteases F. Iron Acquisition G. Peptidoglycan H. Phase Variation and Identification of Genes Involved in Pathogenesis IV. Pathogenesis A. Colonization B. Local Disease C. Bacteremia D. Localization V. Conclusions References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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/. Introduction A. Historical Perspective During the influenza pandemic toward the end of the nineteenth century, Gram-negative coccobacillary organisms were found to be prevalent in the respiratory tracts of affected individuals. At the time, it was thought that these bacteria were the causative agent of influenza, and were therefore named Haemophilus influenzae. Although it is now clear that influenza results primarily from a viral infection, this does not diminish the importance of H. influenzae in the morbidity and mortality of influenza pandemics through its role as a cause of secondary bacterial infection. The name of the genus Haemophilus (meaning blood loving) refers to the dependence of the organism on heme-related molecules for growth under aerobic conditions. During the early part of the twentieth century, it became clear that H. influenzae is an important pathogen in its own right as a major cause of purulent meningitis in infancy. Prior to the introduction of antimicrobials, this infection had a mortality rate approaching 100%, and even with appropriate antibiotics and supportive care Haemophilus meningitis still causes considerable morbidity [1]. Subsequendy, the wide spectrum of disease caused by Haemophilus spp. has become recognized. The bacterium has been shown to be responsible for disease resulting both from local spread in the upper respiratory tract (e.g., otitis media, pneumonia, conjunctivitis) [2] and from dissemination via the systemic circulation to distant sites (e.g., osteomyelitis, meningitis). H. influenzae has gained prominence not only as an important pathogenic bacterium but also as the subject of ground-breaking genetic and molecular research on several separate occasions. In the early 1950s, H. influenzae received much attention as the second example (after the pneumococcus) of a naturally transformable organism. Then in the late 1960s Hamilton Smith's pioneering investigation of homologous recombination in //. influenzae led directly to the discovery of type II restriction endonucleases that underlie much of the recombinant technology that has revolutionized biological science, and that resulted in the 1988 Nobel Prize. Over the last two decades, the importance of H. influenzae meningitis stimulated the development and implementation of a novel and highly successful strategy of vaccination. The first ever conjugate vaccine was designed to prevent diseases caused by type b encapsulated Haemophilus strains, and has been shown to eliminate virtually all invasive H. influenzae infections among individuals in both developed and developing countries [3-6]. The vaccine has served as the paradigm for further conjugate vaccines to prevent infections caused by other encapsulated bacteria such as pneumococcus and meningococcus [7-10]. Finally, in 1995, H. influenzae became the first bacterium to have its genome completely sequenced at The Institute for Genome Research [11]. This remarkable feat marked the beginning of widespread efforts to determine the whole genome
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OF HAEMOPHILUS
INFLUENZAE \NFECTKWS
677
sequences of bacteria, and initiated the application of genomics and bioinformatics in microbiological research. Therefore, studies on the biology and prevention of Haemophilus infections have provided significant landmarks in biological science during the latter part of the twentieth century.
B.
Microbiology
Haemophilus belongs to the family Pasteurellaceae, which contains two other genera, Actinobacillus and Pasteurella. Bacteria belonging to this family are small (1 X 0.3 |Lim), non-spore-forming coccobacilli that have fastidious growth requirements often needing supplemented media for isolation in vitro. Many members of the Pasteurellaceae are found only in certain specific hosts. H. influenzae, the species most often associated with systemic disease in man, is an obligate human commensal found principally in the upper respiratory tract. The morphology of H. influenzae in clinical specimens is variable, ranging from coccobacilli to long filaments. This inconsistent morphology and often variable staining (especially with saphronin in the Gram stain) may lead to diagnostic confusion in the clinical microbiology laboratory. Most Haemophilus spp. require two supplements—factors X (haemin) and V (nicotinamide adenine dinucleotide, NAD)—for aerobic growth on artificial culture media. Factor X can be supplied by iron-containing pigments that include protoporphyrin, and is essential for the catalases, peroxidases, and cytochromes involved in the electron transport chain of the bacterium. Factor V can be provided by NAD or NAD phosphate, or as a nicotinamide nucleoside. Both factor X and V are present in blood; however, because of the presence of heat-labile inhibitors of V factor, blood must be heated before being added to the media (resulting in chocolate agar) for successful isolation of Haemophilus. It should be noted that the viability of H. influenzae decreases rapidly when the bacterium is grown on laboratory media, becoming unculturable within 48 to 72 hours through unknown mechanisms. Therefore, if required, permanent stocks of Haemophilus strains should be made soon after they appear as discrete colonies on solid media. H. influenzae expresses one or none of six antigenically distinct polysaccharide capsules, designated a through f (Fig. 1). The synthesis, surface deposition, and elaboration of capsular polysaccharide are important for Haemophilus pathogenesis (see §III.A). Only a small percentage (on average <5%) of strains isolated from the respiratory tract of carriers possess a polysaccharide capsule, with the majority of isolates being non-typeable (ntHi). The presence of a capsule can be detected simply by examining colonies grown on translucent media for the characteristic iridescence using an indirect source of light. At the biochemical level, immunotyping and structural studies can be used to distinguish the relevant capsular structures. One proposed reason for the evolution of capsular polysaccharides is that they confer some resistance to desiccation as compared to capsule-deficient
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
type a
type b CH,OH
OH
^
HO
"
CHfH
type c
o HOCH2 ^ I U^^'-J > r -O OH
9H2
HOCH
HO
typed NHR
CH^OH
—o
I
GHgOH MO
HO-
AcNH
CO
NHAc
AcNH
typef
typee
HO
CHoOH
NHAc
OH I CH OH
COOH AcNH
HO
AcNH
I
CH^OH
AcO f}-0-fructose in type e'
Fig. 1 Structure of capsular polysaccharides of H. influenzae (serotypes a-f).
cells. This may provide a fitness benefit during host-to-host transmission, a crucial part of the bacterium's life cycle. H. influenzae is exquisitely adapted to the human host and has no other known natural reservoir of infection. Nonetheless, a variety of apparently closely related species are known (including Haemophilus parainfluenzae, Haemophilus galanarium, and Haemophilus avium) that are not restricted to humans. It is supposed that humans became consistently exposed to a novel range of bacteria following the transition from life as "hunter-gatherers" to existence within communities based on agriculture approximately 10,000 years ago. At that time, humans began to live in close proximity with livestock and may then have then acquired the common ancestor of Haemophilus, which was also the progenitor of several other
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PATHOGENESIS OF HAEMOPHILUS INFLUENZAE INFECTIONS
host-specific bacteria such as Actinobacillus and Pasteurella, now found in domestic animals such as cattle and pigs. The whole genome sequence of H. influenzae strain Rd has greatly increased our understanding of the biology of this bacterium [11]. Haemophilus appears to be metabolically adapted for existence in a nitrogen-rich, micro-anaerobic environment. Genes encoding three enzymes—aconitase, citrate synthetase, and isocitrate dehydrogenase—in the tricarboxylic acid (TCA) cycle are missing. This explains the requirement for glutamate that can enter the TCA cycle through a-ketoglutarate during growth in defined laboratory media. There is a paucity of two-component systems in Haemophilus (six in total) compared to Escherichia coli (>40); two-component systems transduce specific environmental signals such as changes in pH, osmolarity, and cation concentrations into programmed, coordinated responses within the bacterial cell. Apparently, the mechanisms by which Haemophilus adapts to environmental change are distinct from those in bacteria such as the Enterobacteriaceae, and include a role for contingency loci that undergo high-frequency on/off switching (see §III.H).
C. Epidemiology and Global Public Health Importance Epidemiological surveys based on culture of nasopharyngeal swabs indicate that //. influenzae is highly prevalent in human populations. Cross-sectional studies of carriage indicate that up to 80% of individuals are colonized with Haemophilus, with most isolates being nt/// [12]. Spread from one individual to another occurs as a result of intimate contact through airborne droplets or by direct contact with secretions. Exposure begins at birth and continues throughout infancy into adult life, and carriage of one or more strains of H. influenzae persists for days to months [13]. Colonization can be a dynamic process; resident strains are lost and new, genetically distinct strains are acquired [13]. The factors involved in this process are not fully understood. However, acquired immunity in the form of antibodies to surface antigens—including capsule, outer-membrane proteins, and lipopolysaccharides—are likely to be one of a number of important factors. Interand intrastrain heterogeneity of surface structures of H. influenzae offers, in part, a plausible explanation for the sequential colonization of individuals with different strains. The organism is a commensal of the upper, but not the lower, respiratory tract of humans, and, in general, carriers of//, influenzae remain healthy. It is supposed that there is selection for fitness both within hosts (duration and magnitude of carriage) and between hosts (efficiency of transmission), since these would be the major determinants of the organism's basic reproductive rate [14]. Neither places any absolute requirements on pathogenicity, but //. influenzae is nonetheless capable of highly virulent behavior. At the risk of simplicity, two patterns of //. influenzae disease are observed: invasive infection, and disease resulting from
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
local spread of the bacterium from the nasopharynx. The pathogenic potential of Haemophilus relates to its ability to disperse and multiply in the human host either by invasion into the systemic circulation or by contiguous spread. The capacity to evade host clearance mechanisms in the blood is a particular, though not exclusive, characteristic of encapsulated organisms. Disseminated infection can then lead to meningitis, septic arthritis, epiglottitis, and cellulitis. These infections are typically caused by encapsulated, particularly type b, strains. In contrast, the second category of diseases includes less serious but numerically much more common infections that occur as a result of contiguous spread of H. influenzae in the respiratory tract. This leads to otitis media, sinusitis, and conjunctivitis, and lower respiratory tract infections including pneumonia and bronchitis [15, 16]. These infections are typically caused by nt//i strains and occur in association with defects in host clearance mechanisms, often secondary to viral infections [17], which normally act to keep the relevant parts of the respiratory tract largely devoid of organisms, if not sterile. Prior to introduction of antimicrobials in the 1940s, invasive H. influenzae infections were associated with an almost 100% mortality rate. The introduction of the conjugate vaccine in developed countries has virtually eliminated the threat of invasive infections caused by type b organisms. However, nt/// continues to cause considerable morbidity, accounting for at least 20,000 episodes of otitis media per year in the United Kingdom alone, as well as being responsible for a significant proportion of cases of pneumonia in individuals with chronic bronchitis, bronchiectasis, and cystic fibrosis. However, the impact of H. influenzae is far greater in many parts of the world where neither antibiotics, vaccines, nor even rudimentary medical support is available. For example, in The Gambia, the incidence of invasive infections during the first year of life was found to be as high as 273/100,000 individuals, with a mortality rate of around 30% [18]. The overall global public health impact of H. influenzae infections is therefore enormous. Much work still needs to be done to prevent disease caused by nt///, and to develop interventions to protect high-risk populations from type b infections that are affordable for communities around the world.
//. Population Biology There is extensive variation among natural populations of bacteria, and //. influenzae is no exception. Understanding the extent of genetic variation is necessary to comprehend the influence of bacterial population diversity on pathogenesis [19]. One useful approach to determine genetic variation is multilocus enzyme electrophoresis (MLEE), in which the polymorphisms in essential metabolic enzymes are used to estimate genetic divergence from a presumed
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681
common ancestor. Over time, genes encoding essential metabolic enzymes accumulate mutual mutations that do not alter fitness and that are not therefore subject to selection. Using MLEE, 177 type b strains mosdy isolated from the blood or cerebrospinal fluid of North American children with invasive disease were grouped into several genetically distinct clusters (electrophoretic types, or ETs), each of which showed strong nonrandom associations of particular alleles [20]. Strains having identical or very similar ETs were isolated from geographically distinct regions over a period of up to 40 years. It was concluded that the population of type b strains was relatively clonal and that those causing most of the invasive disease episodes were a restricted subset of the genotypes of the species as a whole. Indeed, most of the North American isolates belonged to two closely related ETs and were distinct from a collection of isolates obtained from several European countries [21]. These findings suggested an epidemiological pattern in which "successful" type b clones sweep through the host population in a geographically distinct region and become hyperendemic over a period of years, analogous to the change of bacterial populations observed in Neisseria meningitidis [22, 23]. In a subsequent study, the same collection of type b strains was contrasted with 65 nt/// strains that had also been largely obtained from children with invasive and noninvasive infections [24]. All the serotype b isolates were represented by 29 ETs, while every one of the nt/// isolates had distinct ETs. This demonstrates that nt/// are not merely phenotypic variants of type b isolates, but represent a distinctive subset of extremely heterogeneous clones. MLEE analysis of type b and nt/// isolated during a survey of lower-respiratory-tract infections in Islamabad showed that nt/// isolates were clonally restricted with 9 clonal groups found among 34 isolates [25]. Most strains (82%) fell into just five clonal groups; however, 98% of the type b isolates were identical by MLEE. The substantial interclonal variants of pathogenic bacteria has led to the concept that a unit of pathogenicity is the clone. //. influenzae provides two examples in support of this. First, one particular clone of nt/// was found to be associated with cases of a rare, distinct clinical syndrome called Brazilian purpuric fever (BPF) that is characterized by conjunctivitis and life-threatening septicaemia. Although these strains were assigned to biogroup aegyptius, they were found to be quite distinct from other clones of aegyptius that possess far less pathogenic potential. The BPF-causing clone was related to Haemophilus strains producing type c polysaccharide capsule and found in pigs [26], even though the BPF clones are capsule deficient. Second, an analysis of //. influenzae strains resulting in maternal obstetric, urogenital, and neonatal sepsis identified a group of isolates with the unifying characteristic of being biotype IV [27]. These isolates were found to be highly divergent from other strains and seemed to represent a distinct species allied to //. influenzae and //. haemolyticus. These findings suggested that biotype IV
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
Strains belong to a cryptic species possessing unusual tropism for genital tract epithelium and displaying heightened virulence. From the epidemiological standpoint, it is difficult to draw firm conclusions about the population structure of H. influenzae from current information. This is because the data set is incomplete, with obvious potential for bias introduced by analyzing predominantly disease isolates. For example, apparent differences in type b compared with niHi might merely reflect differences in host selection rather than natural population structures. In bacteremic infections, typical of type b disease, strains might be subject to strong host selection, and only those clones that overcome the stringent conditions of evading systemic opsonophagocytic mechanisms of host clearance would be represented. The problem is further compounded by the finding that systemic disease often results from clonal proliferation [28], whereas colonization may involve a number of distinct strains. In contrast, disease caused by nt///, especially nonbacteremic infections, would include a greater proportion of strains manifesting breakdown of host clearance mechanisms, rather than intrinsic virulence. Furthermore, continued surveys are required to establish the effect of vaccination on populations of both type b and nt/// [29]. Thus, an accurate view of the population structure of Haemophilus requires further work in which particular attention is paid to choice of strains, with the ideal analysis including //. influenzae isolates from healthy hosts (representing carriage and commensalism) as well as those with disease
///. Molecular Determinants of Pattiogenicity A.
Capsular Polysaccharide
//. influenzae can express one or none of six antigenically distinct polysaccharide capsules (Fig. 1). Of these, bacteria expressing the type b capsule account for the overwhelming majority of invasive disease isolates. The genes responsible for the synthesis of the type b capsule polyribosyl ribitol phosphate (PRP) are located on a DNA segment of about 50 kb [30, 31]. This fragment, which contains DNA sequences that are unique to type b strains as well as sequences common to all encapsulated strains, is part of a larger, duplicated locus comprising two 18-kb segments [32]. This key finding provided a genetic explanation for the instability of the expression of the type b capsule that had been previously recognized [33]. The genotypic basis of spontaneous high-frequency loss of the capsular phenotype involves the loss of one of the duplicated DNA segments (Fig. 2). However, it was unclear why the reduction from two to a single copy would result in loss of encapsulation and accumulation of unexported PRP within the bacterial cells of
14.
PATHOGENESIS OF HAEMOPHILUS INFLUENZAE INFECTIONS
683
1.2 kb deletion
SI
Cap-
IJ S ^
cap^^
<\
m <—m
t Fig. 2 Phase variation in the expression of the type b capsule. Recombination between the repeats of IS1016 (gray) leads to expansion or reduction in cap gene (dotted boxes) number. The 1.2-kb deletion found in type b strains, and bexA (hatched boxes), which is essential for export of the capsule, are indicated. The level of capsule expression associated with each configuration is shown.
capsule-minus variants. Further dissection of the cap locus showed that the tandem duplication was imperfect [34]. One copy had lost approximately 1.2 kb of DNA, including a substantial part of a gene, bexA, required for PRP export. In the duplicated state, therefore, there was only one intact functional copy of bexA encoding a member of a family of ABC transporters that energize diverse membrane translocation systems. When the cap region underwent homologous recombination of its duplicated sequences, the single functional copy oibexA was lost. Under these circumstances, capsule is still synthesized but not exported. Secondary rescue mutations occur in PRP biosynthesis that alleviate potentially lethal consequences of the build-up of PRP within the cytoplasm [35]. The cap locus from the pathogenic type b strain Eagan can be divided into three distinct regions based on function. Region 1 comprises four genes, including bexA, encoding proteins involved in capsule export; the bexB gene product has the characteristics of an integral membrane protein and bexC encodes a candidate periplasmic component, while no function has been assigned to bexD. Region 2 contains genes involved in capsule biosynthesis, and this region is capsule-type specific. Region 3, by analogy with Neisseria meningitidis, comprises two genes involved in translocation of capsule through the cell wall envelope [36]. The organization of the cap locus with type-specific genes flanked by regions with general capsulation functions (i.e., assembly and transport) is further complicated by the finding that the entire region is part of a compound transposon bounded by an insertion sequence element, IS10J6 [37]. In addition to type b strains, the single capsulation loci of type a, c, and d H. influenzae also harbor
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
the same IS element. Two functional implications stem from this organization. First, the flanking copies of 18/076 mean that recombination in the cap locus can result in amplification as well as loss of capsule genes. In the case of type b strains, the deletion of 1.2 kb in bexA means that recombinational reduction results in the capsule-export deficient phenotype. Thus, the double copy state is required to maintain the Cap^ phenotype. However, such strains provide twice as much capsule and can undergo further amplification to up to five copies. This increased capsule production may be a factor in the heightened virulence of type b strains. This hypothesis is supported by the observation that this 1.2-kb deletion is characteristic of type b strains associated with invasive disease. Other type b strains have been identified with a single-copy cap locus lacking the 1.2-kb deletion, and appear to be distinct clones that show reduced virulence in infant rats. Second, common regions flanking the type-specific biosynthetic genes provide a substrate for recombination and genetic exchange. This could give rise to capsule switching between serologically distinct clones within natural populations. Convincing evidence that such events occur comes from analysis of a type a strain. This strain has the identical 1.2-kb deletion found in type b strains, but is genotypically and phenotypically type a [38]. An analysis of clinical isolates from diverse locations and spanning 40 years showed that the duplicated configuration of cap is characteristic of approximately 98% of type b isolates. A critical role for the type b capsule in virulence was long proposed because of its association with invasive disease, and was confirmed through a combination of molecular genetics and the infant rat model of H. influenzae type b bacteremia and meningitis. First, intranasal inoculation of infant rats with strain Rd, a spontaneous capsule-deficient strain, did not cause invasive infection. However, when strain Rd was transformed using DNA from a type b strain, a resulting transformant expressing a type b capsule was highly virulent. Second, when a series of isogenic single-copy encapsulated strains, each elaborating a different one of the six capsules, was tested, type b strains were substantially more virulent than the others in the model [39]. Indeed, aside from the transformants expressing type b capsule, only type a transformants showed any appreciable potential to cause invasive disease, and even this was substantially less than the type b transformants.
B.
Lipopolysaccharide
Lipopolysaccharide (LPS) is a critical, if not essential, component of the cell wall of Gram-negative bacteria. The membrane-anchoring lipid A portion is the part responsible for much of the pathological damage associated with disease, and the oligosaccharide portion can interact specifically with host molecules. These properties define a very important role for LPS in H. influenzae commensal behavior. The LPS of Haemophilus lacks a third hydrophilic region (0-antigen)
14.
PATHOGENESIS
OF HAEMOPHILUS
INFLUENZAE \KFECJ\ONS
685
distal to the core that is found in many bacteria. The more truncated LPS structures have been designated lipooHgosaccharides (LOSs) by some workers, but the term impHes a functional distinction from the LPS of other organisms. The absence of an 0-sidechain suggests that Haemophilus LPS is a simpler molecule than that from other bacteria. This is not so, since it exhibits a significant degree of interand intrastrain heterogeneity. Monoclonal antibodies (mAbs) specific to LPS have been used to detect variations in LPS structure and to characterize the extensive heterogeneity and crossreactivity of LPS structural epitopes from different isolates. Most H. influenzae strains show at least some crossreactivity of LPS epitopes and the potential for LPS antigenic variation. Spontaneous high-frequency loss and acquisition of reactivity of H. influenzae has been detected with specific mAbs [40]. These LPS changes correlate with altered serum resistance and virulence in the infant rat model of meningitis and provided a basis for grouping different strains by their reactivities with the mAbs [41]. Several genes related to LPS biosynthesis were characterized by molecular genetic analyses. Strain RdI69 expresses a deep rough LPS lacking all core sugars except a single phosphorylated Kdo [42]. This strain has a mutation in the isn gene [43] encoding the phosphoheptose isomerase catalyzing the first step in heptose biosynthesis [44]. Two further genes involved in heptose biosynthesis, tfaE [45] and rfaD [46], have been identified. In addition, there are three chromosomal loci, designated lid, lic2 and //ci, that are also involved in LPS biosynthesis [47]. lid comprises four ORFs (liclA-lidD) and is required for expression of two LPS epitopes, corresponding to mAb 6A2 and 12D9 reactivity, dependent on lidC and lidD, respectively [47]. lid is responsible for the addition of phosphorylcholine (PC) to LPS [48]. The liclA and liclB gene products have regions of homology to proteins involved in choline metabolism in other organisms, and mAb 12D9 reactivity on colony immunoblots mirrors that of a choline-specific mAb, TEPC-15. The first gene of this locus, lid A, mediates phase variation. Two of the identified start codons initiate translation in one reading frame while the third allows translation in a second frame. Three levels of expression of the phase-variable mAb 6A2 and 12D9 epitopes were reported [49] on colony immunoblotting of strain RM7004—strong (-I~I-I-H), weak (-I-), and undetectable (-), 97% being weak and 3% being either strong or undetectable. Similar phenotypes and frequencies have been observed for the PC-specific mAb TEPC-15. Analysis of the tetranucleotide repeat region of lid A from chromosomal DNA of colonies exhibiting each of the three phenotypes indicated that the most prevalent size of repeat DNA found in mAb 6A2 nonreactive colonies was 29 repeats. In mAb 6A2'^^'^"^ variants, the number of repeats was 31 copies, while most mAb 6A2'^ colonies had 30 repeats. These results correlate with the alternative reading frames: 6A2"^ variants have a single ATG in frame, while
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
6A2'^'^"^"*^ variants have either of two closely sited ATGs in frame; in 6A2" variants, lid A would be out of frame. Of the four ORFs in the lic2 locus, only lic2A, has a confirmed function in LPS biosynthesis [50]. lic2A contains multiple copies of 5'-CAAT-3' within its 5' end [50] and when mutated result in the loss of expression of oligosaccharide epitopes recognized by the mAbs 4C4, 5G8, and A1-12E-5G-8E (Al). The mAbs 4C4, 508, and Al have an element of their binding specificity dependent on the aOal(l^)pOal epitope. liclA contains two possible start codons upstream of the repeats in one frame and a third potential start codon closer to the repeats in another frame. Some correlation has been observed between the number of CAAT repeats (15-17 copies) in lic2A and mAb 4C4-specific LPS epitope expression [50]. A closer examination of the lic2 locus in another strain has implicated the third {lic2orf3) and fourth {lic2B) ORFs in LPS synthesis [51]. The lic3 locus contains four ORFs {lic3A through licSD). licSA contains CAAT tetranucleotide repeats but has no known function or homology with other database sequences. The second ORF, licSB, encodes a UDP-galactose-4-epimerase and is thus designated galE. Mutation of galE perturbs the balance of activated galactose for incorporation into LPS during synthesis, and a galE galK mutant of strain RM7004 elaborates LPS with no galactose. When a lacZ reporter fusion was constructed downstream of the repeats in licSA, three levels of P-galactosidase activity were noted, again generally correlating with repeat numbers (19-25 copies) in licSA [52]. A further locus, lex2, containing tetranucleotide repeats was identified as conferring mAb 5G8 reactivity to a type b strain following transformation [53]. The first of two contiguous ORFs has the tetranucleotide 5'-GCAA-3' repeated within the 5' end and would produce a protein of 101 amino acids. The second ORF encodes a larger product of 247 amino acids and when mutated removes expression of the mAb 5G8-reactive LPS epitope that has not as yet been structurally defined. A further locus comprising seven genes putatively involved in LPS biosynthesis was identified when cloned H. influenzae DNA directed assembly of a series of atypical saccharide components on the LPS in E. coli K-12 [54,55]. There is some evidence from homology comparisons that the locus is involved in LPS assembly and transport. The availability of the complete genome sequence of H. influenzae strain Rd facilitated a comprehensive study of LPS biosynthetic loci (Fig. 3; see color plate). More than 30 genes were identified by homology comparisons and investigated by mutational analysis. Gene functions and LPS structures have been assigned for all of the major biosynthetic steps for the saccharide portion of LPS in the type d strain, RM118 (E. R. Moxon et al, unpublished data), and in the type b strain Eagan [56]. Analysis of the genome sequence of strain Rd revealed that it contains all three lie loci, no lex2 locus, and a novel locus with multiple tandem tetranucleotide repeat that is a homolog of IgtC, a glycosyl transferase implicated in LPS biosynthesis in Neisseria [57]. IgtC is the third of three
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687
contiguous ORFs and contains multiple copies of the tetranucleotide 5'-GACA-3' [58]. Unlike the lie loci, only one translation start codon immediately upstream of the repeats appears to be involved in gene expression. Mass spectrometric analysis of the LPS from an IgtC mutant indicated that the terminal aGal(l4)pGal epitope was absent [56]. Genes for the synthesis of lipid A are essential and therefore not amenable to comparable genetic analysis. However, the genome sequence has permitted gene identification by homology comparisons alone. Homologs of the first seven genes characterized for lipid A synthesis in E. coli are all present in the H. influenzae genome sequence, indicating that the biosynthetic pathways are comparable. Determination of the detailed structure of Haemophilus LPS by mass spectrometric (MS) and nuclear magnetic resonance (NMR) structural analyses has been crucial to confirm functions of predicted biosynthetic genes and to understand the contribution of the molecule to bacterial virulence (Fig. 3B; see color plate). The lipid A anchor is an A^-acetylglucosamine (GlcNAc) disaccharide substituted by two phosphate groups. From the anchor extend six fatty acyl chains comprising only tetradecanoic acid (CI4) and its 3-hydroxylated derivative [42]. Some H. influenzae strains contain small amounts (<5% w/w) of C16 fatty acids most likely representing phospholipid. Two antigenically distinct lipid A components have been indicated by mAb analysis [59]. The epitope directing this antigenic shift is apparently a chloroform-soluble moiety present in the majority of nt/// and type b strains tested and specific for H. influenzae. Such variation within species is not uncommon and is likely due to substitutions of the fatty acids. The oligosaccharide portion of the LPS from several H. influenzae strains has been analyzed in fine detail, including one nt/// and two type b strains as well as strain Rd [60-63]. In each case, the LPS has a common inner core consisting of three heptoses linked via a single 2-keto-3-deoxyoctulonosonic acid (Kdo) molecule to lipid A [42, 64, 65]. The degree and pattern of extension of mainly neutral hexose sugars from each heptose varies between strains [63, 66, 67]. An influence on LPS heterogeneity is the specific genetic mechanisms that promote antigenic or phase variation through so-called contingency genes [68] (see §in.H). Further heterogeneity of LPS results from the degree of substitution of the molecule by modifications such as phosphate, phosphoethanolamine, pyrophosphoethanolamine, 0-acetyl, phosphorylcholine (PC) groups, or sialic acid (NANA). A deletion mutation of lid results in lack of incorporation of PC as a substituent to the hexose sugars of //. influenzae LPS [69]. The addition of sialic acid (A^-acetylneuraminic acid, NANA) to terminal sugar residues is often regarded as a modification of LPS. Neuraminidase (sialidase) treatment enhances reactivity of an antibody, mAb 3F11, which recognizes an LPS structure that is proposed as an acceptor for sialic acid addition, in up to 50% of strains [70]. For one type b strain, this observation was confirmed as loss of NANA by HPLC analysis of the
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
LPS. In a subsequent survey, a change in the LPS profiles on neuraminidase treatment was observed for 7 out of 24 (29%) type b H. influenzae and 22 of 31 (71%) xiiHi strains [70a], with some correlation between serotype and the sialylated LPS phenotype. None of the type b, e, or f strains showed any change in LPS profile after neuraminidase treatment. However, a proportion of type a, c, and d strains showed evidence of LPS sialylation, although the degree varied. No sialyltransferase activity has been demonstrated for Haemophilus, but the gene encoding the CMP-NANA synthetase (siaB), which catalyzes formation of the nucleotide sugar donor used by sialyltransferases, has been studied. In strains containing sialic acid, the highest-molecular-weight LPS glycoforms are absent in siaB mutants. Structural analysis of the LPS from an niHi strain, 375, has shown that when present NANA is linked to a p-galactose of a (hex)3 glycoform. Structural analysis of the H. influenzae type b strain A2 LPS has also confirmed the presence, but not detailed the linkage, of NANA to the rest of the molecule [64].
C. Pili and Fimbriae Haemophilus, like other bacteria, has evolved adherence mechanisms that facilitate its close association with host cells, particularly at its normal site of colonization in the respiratory mucosa. H. influenzae can adhere to a range of cell types and is known to agglutinate human erythrocytes. Electron microscopy has shown that hemagglutinating Haemophilus strains project long filamentous appendages that extend from the bacterial cell surface and that are not present on nonhemagglutinating bacteria (reviewed in [71]). Such organelles have variably been termed fimbriae or pili, and we will refer to them as pili given their structural and functional homology to the Pap pili of Escherichia coli. The primary role for pili is believed to be initiation of contact between the bacterium and the human cell. Long, thick hemagglutinating pili (LKP) mediate binding of//, influenzae to buccal epithelial cells [72, 73], bronchial epithelial cells [74] but not to human fibroblasts, HeLa cells (human cervical carcinoma origin), A549 cells (human alveolar epithelium carcinoma origin), human nasal epithelial cells, and human tracheal fibroblasts or Chang cells (human conjunctival origin) [74]. Elimination of piliation results in decreased levels of colonization of monkeys [75]. Piliated organisms bind randomly over the surface of buccal epithelial cells. Binding increases over time to a maximum at between 24 and 36 hours incubation [76]. The expression of pili is a phase-variable phenomenon with rates of transition between 10~^ and 10^ per bacterium per generation [77]. The majority of type b and niHi clinical isolates do not express pili after several laboratory subcultures. It is suggested that incubation in the presence of epithelial cells "selects" for piliated variants. In other bacteria, pili bind to glycoconjugated receptors present on eukaryotic cell-surface glycoproteins and glycolipids. Consistent with this.
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689
Studies have shown that neuraminic acid-containing lactosylceramides—the gangHosides GMl, GM2, GM3, and GDI a—can inhibit pilus-mediated binding of H. influenzae to the Anton antigen (An-Wj) common to buccal epitheUal cells and erythrocytes [78, 79]. Neither erythrocytes nor buccal epithelial cells from An-Wj-negative hosts bind piliated strains. EM investigation of pili show them to be 4.7 to 18 nm in diameter and 209 to 453 nm in length, with a hollow core [72]. They appear as relatively thick, flexible rods with a short, thinner fibrillum at the top similar to E. coli Pap pili. H. influenzae pili have regularly spaced horizontal striations with crossover repeats suggesting that they are double-stranded helices. The majority of cells (95%) in strains capable of expressing them do so, and they are distributed in a peritrichous manner [80]. Estimates of the number of pili per bacterium range from 16 upward [72]. The pilin subunit proteins from different strains can show strain-to-strain variability in migration rates on SDS-PAGE analysis. DNA and amino-acid sequence analysis indicates that the pilin of the type b strain Eagan comprises 196 amino acids with a molecular mass of 21 kDa. The chromosomal region encoding pilus determinants (hifA-hifE) has been studied in detail. The major pilus subunit gene (hifA) has been cloned and sequenced from several independent type b and niHi strains (reviewed in [81]). The amino-acid sequence for each of 10 hifA gene products is significandy homologous (63-81% amino-acid identity) and contains a 17- to 20-aa leader sequence preceding the mature peptide of between 191 and 196 amino acids. The sequences have homology to pilins from other organisms, including E. coli, Klebsiella pneumoniae, Serratia marsescens, and Bordetella pertussis. H. influenzae pilins contain two cysteines, a tyrosine, and a glycine residue in a pattern characteristic of E, coli pilus structural proteins. A single copy of the pilin gene is present in most Haemophilus strains studied, the notable exceptions being its absence from the completely sequenced genome of Haemophilus strain Rd [11] and Haemophilus biogroup aegyptius strains [82], which possess two copies of the gene [83]. Transcribed in the opposite direction and upstream of hifA are four further genes—hifB, hifC, hifD, and hifE—comprising with the hifA gene the Hi pilus gene cluster. This gene cluster when cloned and expressed in E. coli facilitated both pilin and pili synthesis and cells demonstrated adherence to buccal epithelial cells [84]. The hifB gene has significant nucleotide-sequence identity to pilus chaperone proteins of other organisms and encodes a 214-aa protein. HifB forms complexes with HifA and HifD diat protect the structural protein from proteolytic digestion when crossing the cell wall [85]. hifB mutants of H. influenzae do not express piH [85] but still transcribe hifA, suggesting that absence of the chaperone results in degradation of HifA in the periplasm. HifC has strong homology to the family of pilus assembly platform proteins (ushers) like PapC of E. coli, and is a large 837-aa protein [86]. Mutation of hifC causes a pilus- and hemagglutinationdeficient phenotype, although the pilin subunit is still synthesized [87].
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
HifD has amino-acid sequence homology to a number of bacterial gene products, including HifA above, particularly in the C-terminal regions [87, 88]. HifD is a 216-aa lipoprotein-like protein incorporated at the pilus tip. Deletion mutations in hifD produce mutants that express the pilin subunit but do not assemble pili and do not hemagglutinate [88]. Although the function of HifD remains to be confirmed, it may be involved in the polymerization of pilins into pih [81]. The hifE gene encodes a protein with significant homology to HifA and HifD, particularly at the C-terminal region [87, 88], and which is located at the tip of the pilus. Mutation of hifE results in strains with intact pilin production but lessened pilus expression [88] and negligible buccal cell adherence. The similarity of HifA, HifD, and HifE in the C-terminal region may reflect a conserved structure to maintain the correct subunit-subunit interactions between the structural pilus components and the chaperone proteins that carry them across the cell wall. As with HifA, two cysteines and the tyrosine and glycine residues at positions 2 and 14, respectively, are conserved suggesting that the proteins are assembled in a similar manner. HifE shows complete identity between two type b strains tested, but only around 50% homology to the protein from niHi strains. HifE would appear to be associated with the binding site for epithelial cells as anti-HifE antibody completely abrogate pilus-mediated hemagglutination [89]. Pilus genes are present in the same relative location in piliated type b and niHi strains but are also found in nonpiliated variants of piliated strains [90]. The transcription of all pili genes is determined by reversible changes in the number of dinucleotide (TA) repeats located within the bidirectional promoter region between hifA and hifB [91]. Changes in the number of repeats between the -10 and -35 regions alter binding of RNA polymerase by repositioning the promoter binding components, and are responsible for the phase-variable phenotype of pilus expression. In the type b strain AM20, 10 copies of AT allowed maximum pilus expression, 11 copies allowed reduced expression, and 9 copies resulted in no expression. Also contained in the hif gene cluster are multiple inverted direct repeats that may form hairpins, stabilizing the mRNA. In niHi strains, the finding that the /z//locus is flanked by inverted repeats has led to speculation that the fimbriae gene cluster was originally contained on a mobile genetic element [92] and may form part of a pathogenicity island. The chromosomal insertion site has been compared between 20 H. influenzae strains, and it is proposed that an extended cluster of genes was obtained by horizontal transfer to a progenitor strain that gave rise to the various forms of the locus seen in strains today [93]. A role for pili in adherence of H. influenzae to isolated respiratory epithelial cells is clear, but the evidence for pili being important in establishing or maintaining nasopharyngeal colonization in the host is less convincing. Piliated variants have been shown to bind more efficiently following intranasal inoculation of infant rats [94] and rhesus monkeys [75]. However, the organisms recovered
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691
from the nasopharynx of the monkeys were predominantly nonpihated, indicating perhaps the transient need for pilus expression in the infection process. This is similar to the transient, often phase-variable expression of other cell surface determinants observed during colonization and infection, emphasizing the complexity of the processes involving multiple bacterial and host factors. A role for pili in invasive disease is less clear. It has been shown that pihated Haemophilus type b strains produce decreased levels of bacteremia after intranasal, intraperitoneal, or intravenous inoculation compared to nonpiliated variants. Bactericidal antibodies directed against nonpilus structures bind to piliated bacteria more effectively than nonpiliated strains [95]. Furthermore, piliated variants of strains show enhanced opsonophagocytosis by neutrophils [96], although the molecular basis for this is not fully understood. Hemagglutinating pili were initially identified in type b strains, but similar structures are present on nt/// strains. Assembly of the pilin protein into a pilus may be different in nt/// strains, accounting for the relatively weak correlation between their presence and binding to host cells [73]. Nonpiliated isolates adhere more efficiently to Hep-2 cells (human laryngeal carcinoma origin), confirming that nonpilus adhesins are also present. As well as hemagglutinating pili, other types of filamentous appendages have been described in //. influenzae. Biotype IV strains express peritrichous pili that do not hemagglutinate but that mediate adherence to HeLa cells [97]. Surface fimbriae have been described on otitis media isolates that mediate adherence to chinchilla tracheal cells but do not bind to human erythrocytes [98]. By EM these structures are thinner than pili, and analysis has indicated that the major component has strong homology with outer-membrane protein A of £". coli [98]. Thin surface fibrils termed Hsfs have also been described for type b Haemophilus strains, that consist of a single protein which mediate attachment to human epithehal cells [99].
D. Outer-Membrane Proteins The finding that nonpiliated isolates of //. influenzae adhere to human epithelial cells in vitro indicates that the bacterium possesses adhesins other than pilin that can mediate host cell interactions [100]. Two high-molecular-weight proteins (HMWl and HMW2) were identified [101] that are expressed at the bacterial cell surface and are responsible for attachment of nt/// to human epithelial cells [102]. The two proteins are similar to each other and related to the filamentous hemagglutinin, a well-characterized adhesin from Bordetella pertussis [103]. Both HMWl and HMW2 confer adhesive properties when introduced into nonadhesive E. coli, but exhibit different binding specificities. HMWl mediates binding to human conjunctival cells through a glycoprotein receptor, while the target of HMW2 has not as yet been defined.
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
HMWl and HMW2 function as monomeric proteins (rather than assembled subunits), and are processed by cleavage of a 441-aa N terminus to produce the mature protein. The expression of both HMWl and HMW2 has been shown to be dependent on repetitive DNA sequence in the promoters of the corresponding genes; expansion of a 7-bp tandem repeat located between two transcriptional start sites represses expression of the proteins [104]. This mechanism is often seen in the control of eukaryotic gene expression. The adhesive properties of HMWl and HMW2 are also dependent on the accessory proteins HMWIB/IC or HMW2B/2C that are responsible for correct secretion and translocation of the adhesins [105]. The two sets of accessory proteins are interchangeable. HMWIB and HMW2B are predicted to have 20 membrane-spanning domains forming a pore in the outer membrane through which HMWl or HMW2 may be translocated. In strains lacking HMWIB, HMWl is degraded in the periplasmic space, and fails to reach the outer membrane. HMWl and HMW2 have been assessed as vaccine candidates in the chinchilla model of otitis media. Purified HMWl and HMW2 were administered to animals on three occasions prior to challenge by intrabulbar inoculation with 300 cfu of ntHi. Some degree of protection was observed in vaccinated animals, and, interestingly, all bacteria recovered from them were downregulated in expression of the HMWs, probably resulting from selection due to the immune response [106]. Although HMWl and HMW2 contribute to the binding of some ntHi, a significant proportion of nonpiliated isolates do not express either of these adhesins. Further outer-membrane proteins have been implicated in the adherence of these isolates. Hia (Haemophilus influenzae adhesin) was isolated by transforming E. coli with a genomic library from an ntHi isolate, and identifying clones that conferred adhesion to Chang conjunctival cells [107]. Southern analysis revealed an hia homolog in 13 of 15 HMWl/HMW2-deficient ntHi isolates, and hia was absent in all 23 strains that expressed HMWl/HMW2-like proteins. Hia is a 115-kDa protein that promotes adherence to many cell lines in vitro and is related to the adhesin Hsf, characterized in type b isolates [99]. Although HMW proteins and Hia are largely responsible for adherence of niHi to human cells, the observed entry of bacteria into cells appears to be mediated by other factors. Hap has been shown to promote uptake of Haemophilus into epithelial cells [108], and is related at the amino-acid level to the serine IgAl proteases found in Haemophilus, although it lacks proteolytic activity. Hap is processed in a similar way to the IgAl proteases [109], with the C terminus forming a P-barrel structure in the outer membrane that is required for correct translocation of the N terminus. There is now evidence that this residual C-terminal portion of Hap mediates attachment to epithelial cells and aggregation of bacteria [110]. Therefore, ntHi possess a variety of adhesins that are responsible for interactions between the bacterium and human cells. Isolates of ntHi can present varied
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profiles of adhesins that mediate different stages of bacterial attachment to and entry into human cells. Many other surface-expressed proteins of H. influenzae have been identified and characterized. Most of these have received attention as potential immunogens in the search for a vaccine to prevent infections caused by both typeable and ntHi. Many, though not all, of these proteins are highly polymorphic as a result of accumulation of point mutations in surface-exposed loops. The exceptions appear to be P4 [111, 112], P6[l 13], D15 [114], and lipoprotein D [115, 116]. In addition, some proteins are subject to antigenic switching. This variability is characteristic of surface molecules that are subject to selection by host immunity and is evidence of phenotypic adaptation of Haemophilus to increase its fitness within hosts. Although some antigens have been shown to elicit bactericidal antibodies [112, 116, 117] or protection in animal models [114], structural heterogeneity poses a problem since escape mutants [118] are likely to render these vaccines ineffective.
E. IgA Proteases Several organisms elaborate proteases that cleave immunoglobulin Al (IgAl) at different peptide bonds within the hinge region [119]. This leaves intact Fab^ fragments without the Fc^ or (FCot)2.SC portions that are required for the activity of secretory IgA. These proteases are specific in their activity for human or primate immunoglobulin Al. The Haemophilus IgAl proteases are serine type enzymes that are synthesized as 169-kDa preproteins [120]. On cleavage, the 50-kDa C-terminal domain of the protein remains in the bacterial outer membrane while the proteolytically active N terminus is secreted. Secretion of IgAl proteases is also dependent on a sequence 3' to the coding region of the enzyme since deletion of this downstream portion results in accumulation of the IgAl protease in the periplasm. For H. influenzae, at least two classes of IgA 1 proteases have been described based on cleavage at either a prolyl-sery 1 (designated type 1) or four amino acids away at a prolyl-threoryl bond (type 2) [121-123]. In addition to differences in cleavage specificity, these proteins display considerable polymorphisms and antigenic variation so that more than 30 types have been described based on serological responses in humans [124, 125]. In at least one instance, a change in both cleavage specificity and antigenicity of IgA 1 protease apparently occurred in a strain harbored in the respiratory tract of an individual with chronic obstructive respiratory tract disease [124], suggesting that these polymorphisms are relevant to adaptation or evasion of host defenses. Comparison of the DNA sequences of the polymorphic variants has indicated that there is considerable interstrain recombination. These proteases are elaborated in vitro into the culture media and show variable activity [126].
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
Unrelated bacteria have acquired IgAl proteases by convergent evolution; streptococci secrete metalloproteases, and Prevotella spp. produce a cysteine protease with similar specificity to the Haemophilus enzymes. This indicates the considerable biological importance of IgAl proteases. Despite the interest in their role in pathogenesis, any demonstrable function in enhancing colonization or infection remains speculative since the relevant experiments are constrained by lack of appropriate models. One set of experiments using organ cultures of human respiratory tract tissue failed to show any differences between an igar mutant when compared to its isogenic iga"" parent [127]. Several roles have been postulated for IgAl proteases based on indirect evidence or on the basis of analogous functions in other pathogens. These include: 1. Functional host deficiency of local (respiratory tract) IgA through cleavage. It has been proposed that the binding of the cleaved Faba fragment to H. influenzae would not only lack its usual host defense function but might competitively block the functional binding of other host proteins [128], 2. A signaling molecule capable of transducing a response in host cells through nuclear binding of a post-translational fragment of IgA. 3. Facilitation of intracellular survival through interactions with the lysosomal-membrane protein LAMP-1 and perturbation of lysosomal fusion [129]. The last two mechanisms have not been investigated specifically in Haemophilus but have been proposed as mechanisms of action for the IgAl proteases of A^. gonorrhoeae. It has also been suggested that IgAl proteases may contribute to the development of atopic disease [130]. A comparison of infants colonized with Haemophilus showed that those who went on to have evidence of atopic eczema had strains with greater activity of IgAl protease when compared to infants that remained healthy. This association has not been confirmed in other studies and does not address the issue of whether the relationship is causal.
F. Iron Acquisition The survival of//, influenzae in the human host depends on the bacterium's ability to acquire and utilize nutrients from its habitat. Iron is an important cofactor in cellular processes in prokaryotes, but it is of limited availability within human hosts. Levels of free iron are very low at mucosal surfaces, and most iron is bound to specific host molecules involved in the storage or transport of iron in the body. Therefore, Haemophilus must procure iron from limited resources, and then transport the charged iron molecule across the hydrophobic outer and inner membranes.
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PATHOGENESIS OF HAEMOPHILUS INFLUENZAE INFECTIONS
The strategy employed by a range of bacteria is to secrete low-molecularweight iron-binding molecules called siderophores. Siderophores laden with iron are then taken up from the extracellular milieu to the cytosol in an energy-dependent process. The manner by which H. influenzae acquires iron, however, is entirely distinct. Instead of using siderophores, Haemophilus and other members of the Pasteurellaceae and Neisseriaceae express proteins at their cell surface that specifically bind host iron-carrying molecules (such as transferrin and lactoferrin), and scavenge iron from these host molecules. The process is highly specific and adapted for life in mammalian hosts. The current view of how //. influenzae acquires iron has been derived from studies on Haemophilus itself, and well as inferred from findings in other related bacteria. In plasma, iron is bound to transferrin (TF), a glycoprotein with a molecular weight of 79 kDa. The N and C termini of TF both have a globular sialoprotein moiety that can independently bind a single molecule of iron. Lactoferrin (LF) is structurally related to TF but is found predominantly in polymorphoneutrophil (PMN) granulocytes at mucosal surfaces and in breast milk. Type b H. influenzae isolates recovered from patients with invasive disease can grow on TF as the sole iron source, and the uptake of iron from TF is mediated by two TF-binding proteins, TbpA and TbpB, that appear to be ubiquitous in invasive disease isolates [131, 132]. Tbps are predicted to have both membrane-spanning and extracellular domains. Most of the sequence variation in Tbps and the related LF-binding proteins (Lbps) occurs in the extracellular domains of the molecules. The extracellular portion of TbpA binds the C domain of TF, from which it removes the single iron molecule. The functional importance of the transmembrane domains is shown by their high degree of conservation of Tbps and Lbps across a range of bacteria [133], with one of the putative membrane-spanning domains being virtually invariant. The multiple short membrane-spanning domains of TbpA has been proposed to form 26 amphipathic p-sheets, making a channel through the bacterial outer membrane; iron is believed to be transported through the center of this barrel into the periplasmic space. A putative Lbp was identified in the whole genome sequence of H. influenzae strain Rd, but it is not known if it is functional, as it lacks the proposed consensus sequence for an iron channel. However, a potential Lbp has been partially characterized in nt/// [134] by demonstrating that iron-saturated lactoferrin can bind to and enhance the growth of nt/// isolates. Heme is a potential source of both iron and porphyrin, and is required for aerobic growth of Haemophilus (see §I.B). Reidl and Mekalanos [135] showed that the hel gene from Haemophilus is able to complement an E. coli hemA mutant that cannot synthesize its own porphyrin, hel encodes the protein moiety of an outer-membrane lipoprotein, eP4, that is essential for growth under aerobic conditions, and is thought to permit the E. coli hemA mutant to utilize exogenous heme as a source of porphyrin. A domain in the N terminus of eP4 is related to the heme-binding domain of HbpA, a 120-kDa hemoglobin-binding protein
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
isolated from H. influenzae by affinity purification [136]. An Hbp has been more fully characterized in H. ducreyi in which the protein is required for full virulence in an animal model of infection [137]. The utilization of heme-hemopexin as a source of iron is dependent on the 100-kDa HxuA protein found both on the bacterial cell surface and in culture supematants [138, 139]. Isolates of type b H. influenzae express a further receptor with a molecular weight of 57 kDa that also binds heme-hemopexin [140]. In a survey of strains, the latter protein was present in all 21 H. influenzae and six nXHi isolates tested, but not in any of six H. parainfluenzae isolates [141]. HxuA was absent in all the H. parainfluenzae and one H. influenzae strain. Genetic analysis has identified three systems that are responsible for transport of iron across the periplasmic space once it has been removed from host molecules. Uptake requires a functional TonB/ExbB system that provides the energy required to dissociate iron from the Tbps [142, 143]. Once in the periplasmic space, the ferric-binding protein Fbp directs iron to an inner-membrane permease. Isogenic mutants lacking Fbp are unable to use TF as a source of iron and exhibit reduced growth rates on media with iron citrate or protoporphyrin as iron sources [144]. Therefore, Fbp is part of a common pathway m the transport of iron from a number of sources. The Haemophilus iron transport {hit) operon was characterized initially in niHi and contains three genes—hitA, hitB, and hitC—that encode a periplasmic binding protein, a cytoplasmic permease, and a nucleotide-binding protein, respectively [145, 146]. Similar to Fbp, proteins encoded by the hit operon mediate the uptake of iron from a number of sources including TF. HbpA is a 60-kDa lipoprotein involved in binding heme in the periplasmic space and its import across the inner membrane [147]. The suggested structure of this protein has been modeled [148], and shows a high degree of similarity with the periplasmic dipeptide-binding protein of E. coli. A number of loci involved in iron acquisition may be controlled by a Fur homolog. The expression of Tbps, the Hbp, and the heme-hemopexin-binding proteins are all repressed by exogenous elemental iron, heme, and protoporphyrin [149], and potential Fur binding motifs have been identified upstream of genes encoding the Tbps and TonB/ExbB. The relative contribution of each iron uptake system during pathogenesis is unknown. Levels of TF are elevated in the sputum of patients with chronic bronchitis who are predisposed to //. influenzae pneumonia [150], and tbpA, tbpB, hgpA, and huxA are transcribed during otitis media [151]. This indicates that microenvironmental levels of iron are low during otitis media. It is clear that Haemophilus possesses a wide repertoire of mechanisms for iron acquisition that allows it to exploit a diverse yet highly specific range of potential sources. This reflects the importance for the bacterium to scavenge iron effectively from limited environmental resources and that this must exert strong selective pressures on the success of H. influenzae and its adaptation to human hosts.
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Peptidoglycan
Although LPS is an important mediator of host tissue damage during Haemophilus infection (§III.B), it is not the only bacterial component with cytotoxic and proinflammatory activity. Peptidoglycan is a structurally important component of the bacterial cell wall that has immunomodulatory properties. The composition of//, influenzae peptidoglycan has been analyzed [152], and low concentrations (down to 0.42 nM) can induce meningeal inflammation, and cerebral edema when administered to the cerebrospinal fluid (CSF) [153]. It has been suggested that during bacterial meningitis LPS is responsible for CSF leukocytosis, while peptidoglycan mediates cerebral edema and disruption of the blood-brain barrier [154]. Additionally, peptidoglycan may play an important role during otitis media. Injection of purified peptidoglycan from nt/// into the middle ear of chinchillas induces histopathologic changes similar to those in otitis media, although doses in excess of 3 |ig are required [155].
H. Phase Variation and IdentiHcation of Genes Involved in Pathogenesis A notable feature of the //. influenzae genome is the presence of multiple loci containing tandem repetitions of tetranucleotides. These sequences of repetitive DNA are located within the translated reading frames [58]. The significance of these DNA repeats is their relative instability, which results in increases or decreases in the number of repeats and thereby altered gene expression. The resultant switching on or off of translation results in phenotypic (phase) variation (Fig. 4). This switching is reversible and occurs at high frequencies, typically 10"^ per bacterium per generation. Many of the proteins encoded by these phase-variable genes are cell-surface proteins, or encode biosynthetic enzymes such as glycosyl transferases of the biosynthesis of LPS (see §III.B). It is proposed that this mechanism of phenotypic variation has evolved so that bacterial pathogens can adapt to the changing microenvironment within and between hosts. Many infections involve clonal expansion of the microbial population and phase variation provides a combinatorial mechanism whereby pathogenic bacteria can vary, reversibly and independently, several host-interactive surface molecules at a high rate. Contingency genes make up a very small fraction of a bacterium's DNA. In the case of //. influenzae strain Rd, there are 12 contingency genes out of a total of about 1800 genes. Nonetheless, these contingency genes can provide considerable flexibility in function. For microbes to persist in a host population, the average number of new infections caused by a single infected host (RQ) must exceed unity [14]. To survive in a new, genetically different host, microbes must be able to adapt rapidly
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
A Transcriptional -35
-10
Dinucleotide repeats Alteration in repeat number Change in promoter structure e.g. hifA, hifB
B Translational -35
-10
: ^ Tetranucleotide repeats Alteration in repeat number Frameshift mutation e.g. lidA, lex2A Fig. 4 Representation of reiterative DNA sequences mediating phase variation at a transcriptional (A) or a translational (B) level. The transcriptional start site is indicated by an arrow.
to a new host microenvironment in which human genetic polymorphisms and receptors as well as differences in specific and nonspecific host defenses may pose a precipitous challenge. Here again, mechanisms producing diversity at host-interactive loci within a clonal population could improve the probability of survival. The activities of contingency loci may make an important contribution to the value of RQ. Although contingency genes may offer fitness advantages by increasing the probability that descendants of individual cells can escape some of the unpredictable challenges that threaten their extinction, these mechanisms must also increase the probability of the emergence of pathogenic clones that cause disease. Causing disease may be one of the prices that bacteria pay for their ability to produce so many variants, since occasional ones may stray beyond their usual ecological niche and result in disseminated infection in the body such as meningitis. Provided that such events are rare—and meningitis is a relatively rare consequence of infection—the benefits of contingency genes for the survival of the bacterial species should outweigh the disadvantages of killing a minority of hosts. Although a considerable amount is known about the pathogenesis and biology oi Haemophilus, our understanding is far from complete. This is evidenced by the fact that nothing is known about the function of over 30% of the genes in H. influenzae strain Rd [11]. Given the intimate relationship between the bacterium and humans, it is likely that many of these genes are involved in host-microbe
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interactions and pathogenesis. Therefore, much remains to be learned about the biology of H. influenzae. Previous work on bacterial pathogenesis has largely centered on studying the behavior of microbes with immortalized cell lines in tissue culture. These screens only identify a subset of the virulence genes required for pathogenesis because they do not reflect the diverse environments that bacteria encounter in a host. More complex assays are required for a fuller appreciation of bacterial virulence, and the application to H. influenzae of novel genetic methods that allow pools of bacterial strains to be screened in animal models [156, 157] should be extremely informative.
IV. Pathogenesis Many of the steps in the pathogenesis of invasive Haemophilus infections are well understood (Fig. 5A,B). Our knowledge has come through a combination of molecular genetics, and animal and in vitro models of infection. Indeed, the study of H. influenzae has been a paradigm for understanding the pathogenesis of bacterial meningitis. Several distinct stages have been recognized during Haemophilus pathogenesis.
A.
Colonization
H. influenzae efficiendy colonizes the upper respiratory tract. The nasopharynx represents the sole reservoir of Haemophilus and is the site from which infection is disseminated to new, susceptible individuals. Therefore, colonization is crucial both to the maintenance and propagation of the bacterium, and is a prerequisite for both local and invasive disease. The primary interaction between Haemophilus and humans is the binding of the bacterium to mucin, which has been observed both in overlay assays [158, 159] and in human organ cultures. Binding appears to be mediated by the bacterial outer-membrane proteins P2 and P5, and the sialic acid-containing oligosaccharides of mucin. This may be a transitory event in colonization, as the mucociliary layer exhibits a high rate of turnover with foreign material effectively cleared from the upper airways. The bacterium elaborates cell wall components (including peptidoglycan) that impair ciliary function and cause epithelial cell damage. LPS has been studied in organ cultures of rat and guinea pig trachea and shown to cause loss of ciliary activity and disruption of ciUated epithelial cells [160-162]. This was consistent with observations of injury to respiratory epithelia observed in human respiratory mucosa [160]. LPS is a major factor compromising mucociliary clearance
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CHRISTOPH TANG, DEREK HOOD, AND E. RICHARD MOXON
A Mucus layer
B Mucosal epithelium
C Endovascular compartment
H. influenzae
B Location in the host
Mucus layer
Mucosal epithelium i) initial damage
ii) attachment
iii) entry
Bacterial factor involved
Not known
LPS Peptidoglycan Pili High molecular weight proteins Fimbriae Haemophilus surface fibrils Haemophilus influenzae adhesin Hap
Endovascular compartment i) tissue damage ii) avoidance of clearance
iii) nutrient acquisition
Lipid A portion of LPS Capsule LPS sialylation Phosphoryl choline addition to LPS Iron uptake system
Fig. 5 Stages in H. influenzae pathogenesis (A) and the bacterial factors known to be involved during progression of disease (B).
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mechanisms, probably promoting prolonged attachment to mucus and epithelial cells to facilitate replication and contact with areas of damaged epithelium. This damage may promote subsequent stages in colonization, and the association of bacteria with areas of damaged upper respiratory epithelium. The initial interactions between Haemophilus and human cells appear to be mediated by a number of high-molecular-weight outer-membrane proteins and pili but inhibited by the presence of the bacterial capsule [161]. It was initially shown that attachment of H. influenzae to human oropharyngeal cells occurs through pili [163]. Subsequendy, many strains isolated from the nasopharynx were found to be nonpiliated, and to enter cells in more complex models such as nasopharyngeal organ culture [160] or in primates [75]. The uptake of nonpiliated nt/// isolates is mediated by several bacterial surface proteins such as HMWl, HMW2, and Hap (see §III.D for details). The precise site where Haemophilus resides during long-term persistent carriage in individuals has not been determined conclusively. It has been assumed that capsulate bacteria such as Haemophilus exist extracellularly. However, several independent lines of evidence suggest that the bacterium establishes itself in an intracellular niche. First, it is known that colonization with Haemophilus is difficult to eradicate with antimicrobials [164] unless agents are used that are active within human cells, such as rifampicin and the quinolones. Also, in vitro studies have shown that H. influenzae can be taken up by, and survive within, human epithelial cells [165] and macrophages [166, 167]. Finally, careful, albeit limited, in situ hybridization studies using a fluorescein-labeled probe for H. influenzae 16S rRNA have been performed on sections of human tissue obtained from 10 children undergoing adenoidectomy [168]. H. influenzae was present in the adenoids of all patients and was found in macrophage-Hke cells located in the subepithelial layer, some of which contained up to 200 intracellular bacteria. This supports the view that long-term colonization results from the intracellular survival of Haemophilus, However, the results should be interpreted with caution, given the limited sample size and that all the sections were obtained from children with abnormally hypertrophied adenoids.
B. Local Disease nt/// cause a number of diseases resulting from the local, mucosal spread of the bacterium. These include bronchitis, sinusitis, conjunctivitis, and otitis media. Following the successful control of type b invasive infections, interest has now switched to prevention of local disease caused by nt/// that accounts for significant morbidity. The prevailing view of the pathogenesis of otitis media is that it follows nasopharyngeal colonization with nt///, then contiguous spread to the middle ear via the eustachian tube. Evidence in support of this includes the comparison of
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paired ntHi isolates from the middle ear and the nasopharynx from patients with otitis media. By simple phenotypic and genotypic analyses, most paired isolates appear identical. More detailed examination of the profile of adhesins in paired isolates has confirmed these observations [169]. It was also found that all of 17 ntHi isolates were nonpiliated and expressed at least one of HMWl, HMW2, or Hia. Indeed, nonpiliated strains are overrepresented among middle ear isolates [81]. This suggests that, while pili contribute to nasopharyngeal colonization, they are not required for otitis media. The chinchilla has been used to study the pathogenesis of ntHi infections and to evaluate vaccination protocols. The chinchilla has been used because the anatomy of its middle ear is similar to that of humans. Most models, however, have relied on direct inoculation of bacteria into the middle ear, or nasopharyngeal inoculation followed by applying negative pressure in the middle ear to aspirate bacteria up the eustachian tube. More recently, a model has been described in which animals are pretreated with antibiotics before nasopharyngeal inoculation with antibiotic-resistant bacteria that results in reproducible otitis media. In this model, it was found that, while colonization was independent of the age of animals, the incidence of otitis was much higher in 3-month-old compared to 1-year-old animals [170]. This model should prove informative in future studies of otitis media.
C.
Bacteremia
Much of what is understood about Haemophilus bacteremia comes from study of the behavior of the bacterium in the infant rat model. This system has been extensively characterized [171] and has the advantages that invasive infection develops following inoculation of the normal portal of infection, is reproducible, and the pathologic changes observed in the meninges are virtually indistinguishable from those seen in affected children. Bacteria reach the bloodstream by translocating through the vascular endothelial cells, rather than via the lymphatic system. H. influenzae has the capacity to be taken up by and translocate through human endothelial cells in culture. Although the molecular details of this event have not been entirely elucidated, it is known that endocytosis into, and translocation through, endothelial cells are inhibited by expression of a polysaccharide capsule on the surface of the bacterium. Following intranasal inoculation, distinct stages of bacteremia have been documented. Immediately after challenge, there is a transient low-level bacteremia that precedes a "lag" phase during which Haemophilus is undetectable in the circulation. A few hours later, bacteria appear in the bloodstream in increasing numbers with the incidence of bacteremia being dependent on the level of inoculum and the age of the animal [172, 173]. The level of bacteremia then
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continues to rise, and reaches a plateau often in excess of lOVml at around 48 hours. Additionally, it was demonstrated that bacteremia can arise from the proliferation of a very few bacteria within the bloodstream. For instance, after animals are given 10^ bacteria intranasally, in most instances bacteremia results from the replication of only a single organism [28]. This observation provides two important insights into Haemophilus pathogenesis. First, it shows that there is a bottleneck to infection between the mucosal surface and the bloodstream, with only a few bacteria from the inoculum "successfully" going to cause disease. The nature and site of this bottleneck have not been defined, and it is also not known whether "successful" bacteria have attributes that distinguish them from the unsuccessful ones. Second, it demonstrates that the replication rate of Haemophilus must be extremely fast (with a mean generation time of around 50 minutes), as bacteremia rises rapidly to high levels within 12 hours. This replication occurs within the circulation itself, as, despite an extensive search, no extravascular source of bacteria could be identified to account for the rise in bacteremia. Once in the systemic circulation, Haemophilus avoids clearance through mechanisms that are at least in part due to the presence of a polysaccharide capsule. Therefore, the polysaccharide capsule is beneficial to the bacterium at certain stages during pathogenesis, but deleterious in other situations. The ability of Haemophilus to switch on and off expression of capsular polysaccharide is likely to be crucial in its capacity to cause disease (see §III.A). The clearance of encapsulated H, influenzae from blood requires deposition of C3 on the bacterium, and this is independent of the later complement components, C5 to C9. The bacteria are then removed from the circulation following phagocytosis by tissue macrophages. The type b capsule inhibits the initial binding of C3, thereby reducing uptake by phagocytic cells [174]. It is not known whether the type b capsule is more efficient than other capsular types at preventing bacterial clearance, and whether this accounts for the preponderance of type b strains among invasive disease isolates. Both the oligosaccharide and lipid A moieties of LPS have important roles during bacteremic disease. The lipid A of Haemophilus LPS is very similar to that of most other Gram-negative bacteria in exhibiting heat-stable endotoxic properties. Rabbits injected with Haemophilus LPS show the typical endotoxic Shwartzman reaction and a biphasic febrile response on exposure. A comprehensive study of the contribution of each component of the LPS oligosaccharide to pathogenesis has been possible since the postgenomic sequence elucidation of the biosynthetic pathway of Haemophilus LPS. In general, the loss of two or three hexose sugars from H. influenzae strain Eagan LPS results in about a 10-fold drop in the level of bacteremia following intraperitoneal inoculation of an infant rat [56]. A core oligosaccharide containing one or two hexose sugars attached to the heptose backbone and any further truncation results in complete attenuation of the mutant strains. Thus, a minimum of five sugars attached to the lipid A are required for efficient intravascular survival in the infant rat.
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An impairment of virulence has also been noted for a liclllicl double mutant strain, which constitutively lacks the digalactoside epitope and phosphorylcholine (PC) substituent, when compared to the wild-type strain. In this case, the mutant was comparable with the wild type in its ability to survive in the bloodstream after intraperitoneal inoculation and to colonize the nasopharynx. However, the mutant showed a reduced incidence and magnitude of bacteremia after intranasal colonization [175]. The combined results from these experiments would indicate that the digalactoside structure and/or PC substitution of H. influenzae LPS has a role in bacterial virulence. It has been reported that //c7-dependent incorporation of PC into H. influenzae LPS alters the bacterium's susceptibility to the bactericidal activity of human serum [176, 177]. Expression of PC on the LPS of encapsulated organisms renders the organisms more sensitive to normal human serum, presumably due to natural antibodies against PC and the interaction of C-reactive protein (CRP) [176]. Also, during nasopharyngeal carriage in infant rats there was a gradual selection for PC"^ variant cells. These results would indicate that phase variation of PC plays a role in invasive disease, whereby a PC"^ phenotype is optimal for organisms to persist on the mucosal surface, but that a PC~ phenotype may contribute to increased invasion and serum resistance. For an nXHi strain it has been demonstrated that PC in LPS is an epitope directing serum killing involving CRP and complement. The terminal digalactoside, on the other hand, blocked host-directed antibody-mediated serum bactericidal activity. Thus, it would appear that distinct LPS epitopes are responsible for interactions with the acquired and innate humoral immunity systems [177].
D.
Localization
Meningitis is the most devastating consequence of invasive Haemophilus infection, and the infant rat model has proved to be extremely informative for studying the pathogenesis of the disease. It has been demonstrated that meningitis occurs as a consequence of bacteremia, and not via spread from the nasopharynx through the cribiform plate at the base of the skull [178]. Furthermore, meningitis invariably occurs in association with, and is preceded by, bacteremia by several hours. The incidence of meningitis correlates with the magnitude of bacteremia, meningitis being extremely rare in instances when bacteremia was <10^ organisms/ml, while at above lOVml animals consistently develop meningitis [173]. Analysis of the cerebrospinal fluid from affected animals shows that, as the number of bacteria increased in the CSF, so did the number of inflammatory cells. The mediators of the inflammatory changes have been determined in part by direct inoculation of purified components of the bacterium. Instillation of purified H. influenzae type b LPS intracistemally in rabbits rapidly induces an inflammatory reaction in the CSF, evidenced by inflammatory cells, increased serum proteins, and a meningeal exudate [179]. There is also evidence of a role for characterized
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peripheral phase-variable LPS epitopes in host-parasite interactions of H. influenzae. Organisms contained in the CSF of newly diagnosed cases of meningitis showed a majority (>99%) of organisms binding mAbs 4C4 and 12D9 compared to only <0.1% when organisms were cultured in vitro [180]. This would imply that the mAb 4C4 and 12D9 reactive LPS epitopes are selected in vivo\ the occasional bacteria that did not bind the mAbs were consistent with the occurrence of phase variation in clinical situations.
V. Conclusions H. influenzae is still a major cause of death and morbidity despite the availability of a vaccine that is highly effective in preventing the overriding majority of invasive infections. However, the cost of the current conjugate vaccine restricts its use to certain parts of the world, and it fails to protect against infections caused by niHi. Therefore, new interventions to prevent H. influenzae infections are still required. Molecular epidemiology and genetic analysis of pathogenesis emphasized the role of the type b capsule in invasive infections, and lead to the development of vaccines directed at this target. It is hoped that further understanding the relationship between the host and the microbe, a process accelerated by the advent of whole-genome sequencing, will lead to the development of strategies to protect individuals from niHi disease and provide affordable vaccines against type b infections on a worldwide basis.
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154. Burroughs, M., Prasad, S., Cabellos, C , Mendelman, P M., and Tuomanen, E. (1993). The biologic activities of peptidoglycan in experimental Haemophilus influenzae meningitis. J. Infect. Dis. Ul, 464-468. 155. Leake, E. R., Holmes, K., Lim, D. J., and DeMaria, T. F. (1994). Peptidoglycan isolated from nontypeable Haemophilus influenzae induces experimental otitis media in the chinchilla. J. Infect. Dis. 170, 1532-1538. 156. Mahan, M. J., Slauch, J. M., and Mekalanos, J. J. (1993). Selection of bacterial virulence genes that are specifically induced in host tissues [see comments]. Science 259, 686-688. 157. Hensel, M., Shea, J. E., Gleeson, C , Jones, M. D., Dalton, E., and Holden, D. W. (1995). Simultaneous identification of bacterial virulence genes by negative selection. Science 269, 400-403. 158. Reddy, M. S., Bernstein, J. M., Murphy, T. F., and Faden, H. S. (1996). Binding between outer membrane proteins of nontypeable Haemophilus influenzae and human nasopharyngeal mucin. Infect. Immun. 64, 1477-1479. 159. Davies, J., Carlstedt, I., Nilsson, A. K., Hakansson, A., Sabharwal, H., van Alphen, L., van Ham, M., and Svanborg, C. (1995). Binding of Haemophilus influenzae to purified mucins from the human respiratory tract. Infect. Immun. 63, 2485-2492. 160. Read, R. C , Wilson, R., Rutman, A., Lund, V., Todd, H. C , Brain, A. P, Jeffery, P K., and Cole, P. J. (1991). Interaction of nontypable Haemophilus influenzae with human respiratory mucosa in vitro. J. Infect. Dis. 163, 549-558. 161. Read, R. C, Rutman, A. A., Jeffery, P K., Lund, V., Brain, A. P, Moxon, E. R., Cole, P J., and Wilson, R. (1992). Interaction of capsulate Haemophilus influenzae with human airway mucosa in vitro. Infect. Immun. 60, 3244-3252. 162. Johnson, A. P., and Inzana, T. J. (1986). Loss of ciliary activity in organ cultures of rat trachea treated with lipo-oligosaccharide from Haemophilus influenzae. J. Med. Microbiol. 22, 265-268. 163. Guerina, N. G., Langermann, S., Clegg, H. W., Kessler, T. W., Goldman, D. A., and Gilsdorf, J. R. (1982). Adherence of piliated Haemophilus influenzae type b to human oropharyngeal cells. J. Infect. Dis. 146, 564. 164. Sundberg, L., Cederberg, A., Eden, T, and Ernstson, S. (1984). The effect of erythromycin on the nasopharyngeal pathogens in children with secretory otitis media. Acta Otolaryngol. (Stockholm) 97, 379-383. 165. St. Geme III, J. W, and Falkow, S. (1990). Haemophilus influenzae adheres to and enters cultured human epithelial cells. Infect. Immun. 58, 4036-4044. 166. Williams, A. E., Maskell, D. J., and Moxon, E. R. (1991). Relationship between intracellular survival in macrophages and virulence of Haemophilus influenzae type b. J. Infect. Dis. 163, 1366-1369. 167. Noel, G. J., Barenkamp, S. J., St. Geme III, J. W., Haining, W. N., and Mosser, D. M. (1994). High-molecular-weight surface-exposed proteins of Haemophilus influenzae mediate binding to macrophages. J. Infect. Dis. 169, 425-429. 168. Forsgren, J., Samuelson, A., Ahlin, A., Jonasson, J., Rynnel-Dagoo, B., and Lindberg, A. (1994). Haemophilus influenzae resides and multiplies intracellularly in human adenoid tissue as demonstrated by in situ hybridization and bacterial viability assay. Infect. Immun. 62, 673-679. 169. Krasan, G. P, Cutter, D., Block, S. L., and St. Geme III, J. W. (1999). Adhesin expression in matched nasopharyngeal and middle ear isolates of nontypeable Haemophilus influenzae from children with acute otitis media. Infect. Immun. 67, 449-454. 170. Yang, Y. P, Loosmore, S. M., Underdown, B. J., and Klein, M. H. (1998). Nasopharyngeal colonization with nontypeable Haemophilus influenzae in chinchillas. Infect. Immun. 66, 1973-1980.
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171. Moxon, E. R., Glode, M. R, Sutton, A., and Robbins, J. B. (1977). The infant rat as a model of bacterial meningitis. J. Infect. Dis. 136 (Suppl.), S186-S190. 172. Moxon, E. R., Smith, A. L., Averill, D. R., and Smith, D. H. (1974). Haemophilus influenzae meningitis in infant rats after intranasal inoculation. J. Infect. Dis. 129, 154-162. 173. Moxon, E. R., and Ostrow, P. T. (1977). Haemophilus influenzae meningitis in infant rats: Role of bacteremia in pathogenesis of age-dependent inflammatory responses in cerebrospinal fluid. /. Infect. Dis. 135, 303-307. 174. Noel, G. J., Mosser, D. M., and Edelson, R J. (1990). Role of complement in mouse macrophage binding of Haemophilus influenzae type b. J. Clin. Invest. 85, 208-218. 175. Weiser, J. N., Williams, A., and Moxon, E. R. (1990). Phase-variable lipopolysaccharide structures enhance the invasive capacity of Haemophilus influenzae. Infect. Immun. 58, 3455-3457. 176. Weiser, J. N., Shchepetov, M., and Chong, S. T. (1997). Decoration of lipopolysaccharide with phosphorylcholine: A phase-variable characteristic of Haemophilus influenzae. Infect. Immun. 65, 943-950. 177. Weiser, J. N., Pan, N., McGowan, K. L., Musher, D., Martin, A., and Richards, J. (1998). Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein. J. Exp. Med. 187,631-640. 178. Ostrow, P. T, Moxon, E. R., Vernon, N., and Kapko, R. (1979). Pathogenesis of bacterial meningitis. Studies on the route of meningeal invasion following Hemophilus influenzae inoculation of infant rats. Lab. Invest. 40, 678-685. 179. Syrogiannopoulos, G. A., Hansen, E. J., Erwin, A. L., Munford, R. S., Rutledge, J., Reisch, J. S., and McCracken Jr., G. H. (1988). Haemophilus influenzae type b lipooligosaccharide induces meningeal inflammation. J. Infect. Dis. 157, 237-244. 180. Weiser, J. N., Love, J. M., and Moxon, E. R. (1989). The molecular mechanism of phase variation of H. influenzae lipopolysaccharide. Cell 59, 657-665.
CHAPTER 15
Pathogenic Mechanisms in Streptococcal Diseases MICHAEL CAPARON
I. Introduction II. Three Basic Mechanisms of Pathogenesis: Example of S. pyogenes III. Steps Common to All Three Pathogenic Mechanisms A. Adherence B. Multiplication C Host Cell Signahng IV. First Mechanism: Invasion and Multiplication in Tissue A. Avoid Recognition by Phagocytic Cells B. Avoidance of the Immune Response C Damage and Spread in Tissue V. Second Mechanism: Toxin-Mediated Disease A. Hallmarks of the Diseases B. Superantigens VI. Third Mechanism: Immunopathological-Based Diseases A. Hallmarks of the Diseases B. Antigenic Mimicry VII. Concluding Remarks References
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/. Introduction The streptococci are a heterogeneous group of bacteria and are responsible for a wide variety of diseases that range from caries in human teeth to meningitis in Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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fish. To fully appreciate the streptococci it is necessary to appreciate their diversity. However, despite their heterogeneity, the streptococci have numerous characteristics in common and share many similarities in pathogenic processes. Common characteristics include a Gram-positive cellular morphology and a tendency for cells to divide only in a single plane and remain attached to each other such that they form long chains. They share a fermentative metabolism in which lactic acid is the major end-product, and they lack the ability to either synthesize heme or incorporate heme into enzymes. Despite these characteristics, most species of streptococci are tolerant of oxygen and will readily grow in ambient air. Since their metabolisms also allow them to grow equally well in the complete absence of oxygen, they are traditionally referred to as facultative microorganisms. While standard biochemical and molecular criteria are used to classify streptococci into species, several additional classification schemes are also commonly utilized. The most common of these are based on the appearance of colonies grown on blood agar plates and a serotyping scheme developed by Lancefield [52] that recognizes differences and similarities in a cell-wall carbohydrate. Since these schemes are overlapping and are nonexclusive, the result can be confusing, can place several unrelated species in the same classification, and can result in one species being referred to by several different names. For example. Streptococcus pyogenes is also commonly known as a P-hemolytic streptococcus and a group A streptococcus. The Lancefield classification scheme provides a useful framework for considering the pathogenic range of the streptococci. The group A streptococci are responsible for a large number of different diseases of humans (described in greater detail below), the group B streptococci are the most frequent cause of sepsis and meningitis in newborn infants. The group C and G streptococci resemble the group A streptococci but cause a number of different diseases in animals like horses, cows, cats, dogs, and pigs. For example, group C S. dysgalactiae causes mastitis in cows and group G 5*. equi causes an often-fatal infection of the throat (known as "strangles") in horses. Certain group G streptococci can infect humans and cause diseases of the throat and skin. Several species of group D streptococci formerly classified in the genus Streptococcus have recendy been reclassified within the genus Enterococcus and commonly infect both the urinary tract and the valves of the heart (endocarditis). Among species not classified by the Lancefield scheme are a large collection of species known as the viridans streptococci, which are commonly found as residents of the oral cavity. While usually harmless commensuals, the viridans group includes the organisms responsible for dental caries {S. mutans and several other closely related species), and most viridans species can be associated with endocarditis. Also of significance are S. pneumoniae, an important agent of pneumonia and otitis media in children, and S. iniae, a pathogen of fish and a rare cause of cutaneous infections in humans.
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//. Three Basic Mechanisms of Pathogenesis: Exampie of S. pyogenes Despite their heterogeneity in the types of hosts they infect and the types of diseases they cause, the streptococci essentially use one of three basic mechanisms to cause disease (Fig. 1). For specific examples of how these three mechanisms cause disease, we will consider the example of 5. pyogenes (also frequently referred to as group A streptococcus), perhaps one of the most versatile of any of the pathogenic microorganisms that can infect humans in terms of the different diseases it causes and the different tissues it infects. The three mechanisms include the following: 1. Attachment and/or invasion into tissue followed by multiplication in extracellular spaces. Damage is caused by a combination of the products from multiplying streptococci and the host's own inflammatory response to the infection. The location of the streptococci within the host determines the characteristics of the resulting disease. For S. pyogenes, infections typically begin
tliniugii
2 Production of Toxins oke an Autoimmune RBSfMinse
Fig. 1 Pathogenesis of streptococcal infections. Streptococci produce disease through three basic mechanisms. (1) Invasion and spread through tissue produces disease by damaging host tissue at the location of multiplying streptococci through the combined actions of bacterial growth and the host's inflammatory response. (2) Toxins produced by streptococci multiplying at localized sites diffuse to other areas in the host and damage susceptible cells. (3) Streptococcal antigens may be molecular mimics of host structures, and infection triggers an autoimmune response that leads to tissue damage.
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either in the throat or the skin, and examples of this class of disease include those that range from the relatively mild pharyngitis commonly known as "strep throat" to those that involve increasingly deeper layers of tissue that can frequendy be fatal. This range of tissue involvement is most striking in the different diseases caused in the skin. Disease can result from localized infection of the superficial epidermis (impetigo), or the entire epidermis (ecthyma), to infections that spread laterally through the upper dermis (erysipelas) or through the lower dermis (cellulitis). The infected tissues will contain numerous streptococci, and the lesions are characterized by a vigorous host inflammatory response. As a general rule, elimination of the streptococci terminates the disease process and the streptococci produce no permanent damage to the infected tissue. This is not the case for infection of deeper layers of tissue. For example, infection of the fascia or underlying muscle results in disease (necrotizing fasciitis and myositis, respectively) and includes numerous streptococci in the tissue, evidence of inflammation, and the spread of organisms laterally through the dssue. These infections are life-threatening and characterized by severe irreversible necrotic damage. Treatment must include removal of the damaged tissue. 2. Toxigenic mechanisms. In this case, the streptococcus secretes a soluble toxin that diffuses into the surrounding tissue or even systemically through the vasculature. The toxin binds to a receptor on suscepdble host cells and triggers an inappropriate response by that cell, which results in pathology. The damaged tissues are often at sites distant from the location of the streptococci, and a common characteristic of these toxin-mediated diseases is that no trace of the streptococci is observed in the damaged tissues. Examples of this class include scarlet fever and toxic shock syndrome. In the latter example, the focus of infecUon is at a localized site in the dssue, and can range from either a mild nondestructive form of infection to a severe necrodc infecdon. However, what disdnguishes toxic shock syndrome is the development of flu-like symptoms followed by pronounced systemic hypotension, frequendy accompanied by failure of multiple organ systems, disseminated soft tissue necrosis, and desquamation of skin, often on the palms or soles of the feet. In many cases, the streptococci remain localized to a specific focus of infection throughout the entire course of the disease. 3. Immunopathological phenomena. In this instance, pathology is not a direct consequence of infection by the streptococci. Rather, disease is the result of an inappropriate host response that is triggered by the host's response to the streptococcal infection. The inappropriate host response targets and damages the host's own tissues, resulting in disease. A hallmark of this class of diseases is that symptoms often begin days to weeks after the host has cleared the original infecdon and the streptococci are typically no longer present in the host during an attack of the disease. A large number of diseases occur as immunopathological phenomena triggered by prior infection with S. pyogenes. These include rheumatic fever and rheumatic heart disease, acute glomerulonephritis, and even
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certain forms of psoriasis. There is also evidence that links certain forms of obsessive-compulsive disorder syndrome to prior infection by S. pyogenes [89]. In general, these disorders are referred to as "nonsuppurative sequelae" to streptococcal infection. Rheumatic fever is a classic example of a nonsuppurative sequelae. The primary symptoms of acute rheumatic fever include a generalized arthritis-like inflammation of joints and can also include the heart. In severe cases, repeated attacks of rheumatic fever lead to scarring of and eventual failure of the heart valves. An attack of rheumatic fever is only triggered by an infection of the pharynx; however, during the acute phase of the disease it is generally not possible to culture S. pyogenes from the pharynx. Instead, the patient will have rising titers to several streptococcal antigens, suggestive of a very recent infection. Finally, rheumatic fever can be completely prevented by treating streptococcal pharyngitis in susceptible individuals with penicillin, even though the symptoms themselves do not respond to penicillin once they appear.
///. Steps Common to All Thiree Pattiogenlc Mectianisms
A.
Adherence
Regardless of the ultimate class of disease, all streptococcal infections share certain steps in common. For example, infection is initiated by transfer of the streptococcus from person to person (humans are the only significant reservoir of S. pyogenes), probably via aerosol transmission of infected droplets or through direct contact with an infected individual or with environmental surfaces that have been contaminated by an infected individual [100]. In the skin, it seems that S. pyogenes has only a very limited ability to penetrate across an intact epidermis, and infection of this tissue usually is associated with entry of the streptococcus mediated by some type of external trauma, either through a preexisting wound, or by a traumatic event to skin previously colonized by the streptococcus that implants the organism into the underlying tissue [100]. An ominous feature of these infections is that disease can be established following entry of only a few organisms. Once in contact with a new host, a critical step in pathogenesis is for the entering streptococcus to come into contact with an epithelial cell of the skin or pharynx and attach to that cell. This adherence event is thought to be mediated by stereospecific interactions between a microbial adhesin and its cognate host
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receptor molecule [44]. In Gram-negative bacteria, this attachment event generally involves interaction of a pilus adhesin with a carbohydrate epitope on a lipid or protein-based glycoconjugate. In contrast, while some species of viridans streptococci do make pili, pili have not been described for other streptococci, including S. pyogenes. The best-characterized adherence mechanisms of streptococci involve recognition and binding of host molecules that are, in turn, associated with the surface of a host epithelial cell or tissue. The host proteins that participate in this type of interaction are often those that are components of the extracellular matrix [74]. Numerous extracellular matrix proteins—including laminin, various types of collagen, fibrinogen, vitronectin, and fibronectin—are known to bind to S. pyogenes. Perhaps the best-characterized streptococcal adhesin that binds to an extracellular matrix component is a fibronectin-binding protein known as protein F or Sfb [35, 90]. Protein F is a cell-wall-associated protein that binds soluble fibronectin essentially irreversibly and at high affinity (apparent ATj ~ 1.0 nM) and immobilized tissue forms of fibronectin with even greater affinity [69, 70]. Genetic and biochemical analyses have provided much insight into the structure of protein F (Fig. 2). As is typical for many proteins associated with the cell walls of Gram-positive cocci, protein F has a conventional signal sequence at its N terminus and a cell-wall-attachment domain at its C terminus. The latter domain is distinguished by the characteristic pentapeptide "LPXTG" motif. Studies in Staphylococcus aureus [85] have shown that this sequence is recognized by a sorting pathway following secretion of the molecule across the single cellular membrane of a Gram-positive bacterium, with the result that the protein is cleaved following the threonine residue of the LPXTG motif. A pentaglycine bridge then covalently couples the threonine to a lysine residue of the cell wall peptidoglycan. The end-product of this sorting pathway is a cell-surface protein that is covalendy tethered to the cell wall at its processed carboxy terminus and has its amino terminus exposed to the environment. What is very interesting about protein F is that its exposed domains contain two distinct but overlapping segments that can independently bind to different regions of the fibronectin polypeptide [70]. The first of these fibronectin-binding domains is known as RD2 (repetitive domain-2), and it consists of a 37 amino-acid motif that is repeated in tandem. Considerable variation in the number of repeats exists between different alleles of protein F and can range anywhere from 1 to 6 copies. Interestingly, the functional binding unit does not consist of a single repeat unit. Rather, the minimal fibronectin-binding domain is 44 amino acids in length and consists of the 25 carboxy-terminal amino acids of one repeat and the 19 amino-acid residues from the amino terminus of an adjacent repeat. This domain begins and ends with a direct repeat of the sequence motif MGGQSES. The RD2 domains bind to the very amino-terminal 29-kDa fibrin and heparin-binding domains of fibronectin. The second fibronectin-binding
15.
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PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
NH2
HH2
NH2
4,
fiegkiri
Variibie
Repeats
i
Variable < fBgton
Repeat domam i
l^streain FrNblnd^ ftomafn (UR) IrrHnimai Fn-binciiig ypit Repeat domahfi II (RD2)
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X
M pnoteiii
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Fig. 2 Comparison of two surface proteins of S. pyogenes. The M protein and protein F have in common that both are adhesins, that they are composed of blocks of repeating sequence, that they contain conserved and variable regions, and that they are anchored to the cell-wall peptidoglycan at their carboxy termini by the LPXTG sorting pathway. Both bind host proteins: factor H and albumin to the C repeat of M protein, and fibronectin to two distinct domains of protein F (UR and RD2). Some M proteins also bind fibrinogen and the Fc regions of immunoglobulin (IgG or monomeric IgA) at sites located amino-terminal to the C repeat domain. M protein is a coiled-coil dimer, while the structure of protein F is not known.
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MICHAEL CAPARON
domain in protein F binds to a different region of the fibronectin molecule: the N-terminal 70-kDa domain of fibronectin that includes the fibrin- and heparinbinding domains along with an adjacent collagen-binding domain. This second domain of protein F, UR ("upstream region"), encompasses 49 amino-acid residues. Of these, 43 are located immediately N-terminal to the first repeat of the repetitive domain, and the rest are derived from the first six residues of the repetitive domain. Despite the fact that UR and RD2 overlap, considerable evidence suggests that they can independently and simultaneously bind their target regions of fibronectin [70]. The UR domain is responsible for the very high binding affinity of the whole protein F molecule and may be the more important of the two domains for interaction with immobilized tissue forms of fibronectin [69, 70]. Mechanistically, the ability to bind fibronectin and/or other components of the host extracellular matrix confers on streptococci an enormous potential for adherence. Fibronectin is widely distributed throughout the host and is found in the vasculature, in tissue, in the extracellular matrix, and in many secretions. In addition, fibronectin itself is recognized by a wide variety of host cellular receptors [46]. Thus, by having the capacity to bind to a single host protein, S. pyogenes gains the ability to interact with a large number of different host cells. Its preference for binding to immobilized over-soluble fibronectin may act to prevent competition for binding to a solid substrate under circumstances in which the organism becomes exposed to both forms of fibronectin. Finally, because the most notable characteristic of the fibronectin molecule itself is its ability to bind to other host structures, including other components of the extracellular matrix, by binding to soluble fibronectin, S. pyogenes gains the adhesive capacities that are possessed by fibronectin itself. For example, by itself, S. pyogenes has only a poor capacity to adhere to the acellular regions of the dermis. However, its capacity to bind dermis increases dramatically if the streptococcus is first allowed to bind soluble fibronectin on its surface [68]. This enhancement is apparently due to the capacity of the streptococcal-bound fibronectin to bind to collagen in the dermis. The importance of the ability to bind fibronectin for adherence is further suggested by the observation that some isolates of S. pyogenes contain two additional surface proteins with a fibronectin-binding domain highly homologous to RD2 (protein FII and opacity factor) [48, 78]. In fact, many other species of streptococci have protein F-like molecules, including some belonging to groups C and G, which are pathogenic for animals, and group G species, which are pathogenic for humans [74]. Similar surface proteins are also found in strains of Staphylococcus aureus that are pathogenic for humans or animals [74]. Many of these other adhesins in the protein F family also have a two-domain mechanism for interacting with fibronectin, although it appears that neither the primary sequence nor the location of the second binding domain is conserved [48]. In
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PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
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addition, a number of other streptococcal adhesins have been described that interact with fibronectin (ZOP, LTA, GAPDH, FBP54, M protein) [36, 54]. However, it should be noted that there are strains of S. pyogenes described that lack a fibronectin-binding protein of the protein F family [63]. Other species of streptococci pathogenic for humans also bind exclusively to immobilized fibronectin (most notably the group B streptococci [92] and S. pneumoniae [95], even though they do not possess an adhesin related to protein F). As if its extracellular matrix-binding capacity was not adequate, S. pyogenes possesses a number of additional adhesins. These include lipoteichoic acid (LTA), the M protein, and a polysaccharide capsule (the M protein and the capsule are described in more detail below). A number of cellular receptors may be recognized by the M protein, including fucosylated glycoconjugates [99] and CD46, a host surface protein that regulates activation of the complement system [67]. The polysaccharide capsule, a homopolymer of hyaluronic acid, can be recognized by the host hyaluronate receptor (CD44) [86]. Cellular receptors for LTA include fibronectin [36] and the macrophage scavenger receptor [26]. In summary, entry and adherence are the first steps in the pathogenesis of any streptococcal disease. The variety of the different types of diseases that can be caused by S. pyogenes is matched by the variety of its adhesive capabilities. Despite the heterogeneity of the streptococci as a group, several common themes exist for adherence. Due to its capacity to bind components of the host extracellular matrix and many other host receptors, S. pyogenes has the potential to adhere to virtually any cell or structure within the host. However, its actual pattern of adherence in vivo is likely dictated by signal transduction pathways that regulate expression of specific adhesins (see below).
B.
Multiplication
Following attachment to host tissues, the next step common to all streptococcal diseases is the requirement for multiplication of the organisms in a tissue environment. This is a largely unexplored topic of streptococcal pathogenesis, but one that may be of considerable interest given the rather limited metabolic capacity that is conferred by the lactic acid fermentative metabolism of the streptococci. For example, most streptococci are highly aerotolerant even though there is no clear metabolic rationale for this property. Furthermore, since they lack heme-containing enzymes like catalase, which are thought to be important for survival in an aerobic environment, it is not clear how they have the capacity to be aerotolerant. Interactions with the atmosphere play an important role in regulating the virulence properties of S. pyogenes (Fig. 3). Oxygen stimulates expression of
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MICHAEL CAPARON
M locus
^r^^^
y^
'»8
emm
emm
scpA
Protein F
t growth ceases
Cysteine Protease (SpeB)
I "N ^^^ ^ V ropB
A SpeB ^— I nutrients
Fig. 3 Regulation of virulence. Interaction with the environment regulates expression of adhesins. For the M locus, the concentration of carbon dioxide stimulates transcription of mga, which upregulates its own transcription. Each gene in the M locus has its own promoter and Mga binds to specific sites located upstream of each gene and activates transcription. Other signals stimulate expression of emm genes, including osmolarity, temperature, and iron limitation. For protein F, oxidative stress activates transcription of prtF by an unknown mechanism. In some strains, anaerobiosis activates transcription of the adjacent gene that encodes RofA, which then binds to specific sites in the promoters for both wfA and prtF to activate transcription. Transcription of the gene encoding the cysteine protease {speB) is activated at the end of logarithmic growth by a pathway dependent on the adjacent gene ropB. Presence of certain nutrients (e.g., glucose) leads to a reduction in expression of SpeB.
several fibronectin-binding adhesins, including protein F and ZOP [54, 96]. Transcription of the gene that encodes protein F is enhanced in an aerobic atmosphere by a mechanism that may involve a signal transduction pathway that senses the concentration of superoxide [30]. The ZOP fibronectin-binding adhesin is formed by an oxygen-dependent posttranslational modification of an as-yet-unidentified surface component [54]. In contrast, expression of a second streptococcal adhesin, the M protein, is stimulated in environments that are typically low in oxygen [13]. Regulation of the gene that encodes the M protein {emm) is at the level of transcription, involves the transcriptional regulator Mga, and responds to the environmental concentration of carbon dioxide [66]. In addition, some strains will also express protein F under low-oxygen conditions through a mechanism that involves activation of the RofA transcriptional regulator under anaerobic conditions [28]. These observations suggest that sensing the gas composition of the atmosphere is a general strategy used by the streptococcus to ascertain its
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location in tissue and modulate its adhesive capacity by utilizing different adhesins in different tissue compartments. Additional evidence that interaction and sensing of the atmosphere play a role in regulation of virulence comes from the observations that the activities of several secreted proteins are modulated posttranslationally by oxygen, including the cysteine protease and the oxygensensitive hemolysin streptolysin O.
C. Host Cell Signaling Once attached and multiplying on the surface of a host cell, the next step common to all streptococcal infections is the communication between the signaling pathways of both the bacterium and the host cell. Adherence has been implicated as a mechanism by which the streptococcus modulates the proinflammatory responses of epithelial cells since adherent and nonadherent streptococci induce different patterns of cytokine and prostaglandin responses in several different types of epithelial cells. In particular, adherent S. pyogenes do, but nonadherent S. pyogenes do not, provoke a vigorous IL-6 response in host epithelial cells [20, 97]. However, while adherence is necessary to stimulate IL-6 expression, it is not sufficient to provoke this high-level cytokine response [97]. Since one particular characteristic of the streptococci and of S. pyogenes is that they have the capacity to secrete a very large number of different enzymes and toxins into their surroundings (described in greater detail below), it is likely that many of these molecules are involved in modulation of host cell signaling responses. For example, it has been shown that the pore-forming hemolysin streptolysin O acts synergistically with adherence promoted by the M protein to modulate signaling interactions in the epithelium, including the IL-6 response of keratinocytes [97]. A second streptococcal surface protein, the surface-associated glyceraldehyde 3-phosphate dehydrogenase, has been implicated in activation of epithelial cell protein tyrosine kinase activity [72]. S. pyogenes, as well as several other streptococcal species including group B streptococci and S. pneumoniae, has the ability to enter into cultured epithelial cells [22, 53, 81]. For some strains ofS. pyogenes, the fibronectin-binding adhesin protein F acts as an invasin to stimulate uptake and its invasin-like properties appear to function independently of its role in promoting adherence [60]. The precise role that invasion into cells contributes to pathogenesis has yet to be established, but the fact that it can occur and is dependent on specific streptococcal proteins makes a strong case for a highly structured communication between S. pyogenes and epithelial cells. While this area of investigation is just beginning to be explored, the fact that different clones of S. pyogenes are so heterogeneous in terms of the different types of surface proteins and toxins that each has the potential to produce and in terms of the patterns by which these products are regulated may suggest that the synergistic interactions of different subsets of these
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products on epithelial cell signaling responses plays an important role in determining the subsequent course of infection and the characteristics of the disease that ultimately develops.
IV. First Mechanism: Invasion and Multiplication in Tissue A.
Avoid Recognition by Phagocytic Cells
The first major class of diseases caused by S. pyogenes are those that result from its ability to invade and multiply extracellularly in host tissues. As a general rule, multiplication of streptococci in tissue provokes an intense inflammatory response by the host. A key component of this response is recruitment of polymorphonuclear leukocytes (PMNs) to the site of streptococcal multiplication. Also, as a general rule, the streptococci have almost no ability to survive the microbicidal defenses of the PMN, such that their principle virulence mechanisms involve avoidance of recognition, ingestion, and killing by these host defense cells.
1.
M PROTEIN
One mechanism that S. pyogenes uses to avoid recognition by PMNs involves the surface M protein (Fig. 2) (for a detailed review, see [27]). The M proteins represent a related family of proteins that share a common structure. In general, they are fibrous rodlike molecules that are highly a-helical homodimers arranged in a coiled-coil conformation. This structure basically consists of two a-helices that coil around one another. In this regard, their structure resembles other classic coiled-coil proteins like mammalian tropomyosin. This includes the signature periodicity of a coiled-coil protein that consists of a repeating heptad motif (a-b-c-d-e-f-g),j in which the residues at positions a and d are hydrophobic and oriented to pair with the hydrophobic residues of the other polypeptide in the dimer to stabilize the coiling of the two helices around each other. Residues at the other positions are generally those that promote formation of helices. This periodicity is reflected in the overall sequence of the polypeptide, which is itself highly repetitive. The basic structure of an M protein (the M6 protein, for example) consists of three distinct blocks of tandem repetitive sequences referred to as the A, B, and C repeat domains. A short nonhelical region is found at the very N terminus of the proteins, and the C terminus contains sequences characteristic of the sorting pathway (i.e., the LPXTG pathway) that involves
15.
PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
729
processing and covalent linkage to the cell wall peptidoglycan. While this basic structure is conserved between different M proteins, they can differ quite dramatically in their primary sequences. In particular, the degree of variability generally decreases from the N terminus to the C terminus. The N terminus and the A and B repeat domains vary extensively between different M proteins, while the C repeats and the cell-wall-attachment domains are highly conserved. The number of individual repeat units that make up each of the three repeat domains can vary extensively as well, even within highly related M molecules in closely related strains. Additional heterogeneous features of the protein and its genetic structure will be described in more detail below. The classic work of Rebecca Lancefield identified the M protein as having a role in avoidance of phagocytosis [52]. She demonstrated that streptococci rich in surface M protein had the capacity to survive and multiply in the presence of PMNs in human blood, while those isolates deficient in M protein were killed. Furthermore, addition of an anti-M protein antibody effectively opsonized the M-rich streptococci and promoted their ingestion and killing by the PMNs. Work subsequent to this has focused on determining the molecular basis of the ability of the M protein to block recognition by PMNs, and present evidence suggests several possible mechanisms. Most studies have suggested that PMNs recognize S. pyogenes via complement deposited on the streptococcal cell wall, and that the presence of M protein, while not preventing deposition of complement, somehow alters an essential characteristic of the deposited complement, either in the pattern of deposition or in the quantity of complement deposited [27]. Most, if not all, M proteins implicated in avoidance of phagocytosis can bind the host protein fibrinogen [27]. While the molecular nature of this binding has not been established, it is interesting that fibrinogen seems to bind toward the N terminus of M protein at a region that is highly variable between different M proteins [82], suggesting that some structural characteristic not apparent in the primary sequence mediates this binding phenomenon. One school of thought is that the ability of M protein to block recognition is dependent on this bound fibrinogen, which may serve to somehow mask the cell wall and impede deposition of complement. A second school of thought suggests that the ability of conserved domains in the C repeat region to bind host proteins, like factor H, which can downregulate activation of the complement cascade, can alter the pattern of deposition of complement on the streptococcal cell surface in a manner that influences the ability of PMNs to ingest the streptococcal cells [42]. However, more recent studies have shown that the entire C repeat region can be deleted from the M6 protein without an effect on its ability to block recognition by PMNs [75]. The ability of streptococci to bind host proteins, which in turn bind to other host proteins, confers on the organisms an immense functional potential. In this regard, it has been shown that, after having been bound to M protein, fibrinogen can recruit and bind factor H, suggesting multiple pathways for interaction with factor
730
MICHAEL CAPARON
H [43]. It is also interesting that M proteins are not restricted to S. pyogenes, but are also found in group G strains that are pathogenic for humans [19] and group C strains that are pathogenic for horses [93].
2.
CAPSULE
Genetic studies that have manipulated M protein expression in several different strains using several different M proteins have confirmed the seminal observations of Lancefield on the role of the M protein in the avoidance of recognition by PMNs. However, it appears that not all M proteins are equally efficient at this task [23, 45]. In these strains, the polysaccharide capsule makes a major contribution to this property [23]. Polysaccharide capsules play a key role in preventing uptake of several other streptococcal species, most notably S. pneumoniae and the group B streptococci. The capsule of S. pyogenes is a glycosaminoglycan and is a relatively simple homopolymer of hyaluronic acid consisting of repeating subunits of glucuronic acid and N-acetylglucosamine. Three genes for production of the capsule have been described and are designated hasA, hasB, and hasC, encoding hyaluronan synthase, UDP-glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively [21]. The three genes are organized into a single operon (the has operon), and introduction of the has operon into unrelated streptococcal species is sufficient to allow them to produce a hyaluronic acid capsule [25]. A role for the capsule in virulence has been established in studies where mutation of the has operon renders some strains of S. pyogenes susceptible to killing by PMNs, even though the mutants continue to express high levels of M protein [101]. While the mechanism by which the capsule of S. pyogenes interferes with recognition by PMNs is not yet established, mechanisms involving steric interference of phagocyte recognition of complement deposited on the cell surface, and the recruitment of complement inhibitory factors to the capsule surface to block the deposition of complement, have been described for different capsular types of S. pneumoniae [11]. M proteins differ not only in their efficiency in providing an avoidance of recognition by PMNs, but different strains recovered from human infections will also differ quite dramatically in the amount of capsule they produce in vitro and range from strains that produce very little detectable hyaluronic acid to strains that produce large capsules that form colonies that have a very characteristic mucoid morphology [103]. In at least one strain, it appears that the relative importance of M protein vs. the capsule is dependent on the concentration of complement the organisms are exposed to, with the capsule being relatively more important at lower concentrations of complement and the M protein of more importance at higher concentrations [62]. This may have implications for survival in different host compartments that differ in their concentration of complement components (e.g., cutaneous tissue vs. the bloodstream). These observations
15.
PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
731
emphasize how important it is for the streptococci to have the ability to avoid recognition by phagocytic cells and illustrate how adaptable the streptococci are in maintaining this property. 3.
OTHER FACTORS
S. pyogenes possesses a number of additional products that act to protect it from interacting with phagocytic cells, including at least two proteases and a hemolysin. The first of these is a serine protease known as the streptococcal C5a peptidase (SCPA), whose mature form becomes associated with the streptococcal cell wall [102]. The only known substrate of SCPA is the C5a cleavage product of the fifth component of complement. This product of complement activation is a powerful chemoattractant for PMNs. SCPA makes a single cleavage in C5a between His67 and Lys68 that are located in a region of the molecule that is recognized by the PMN C5a receptor. As a result, the action of SCPA neutralizes the C5a molecule's ability to serve as a chemoattractant. Thus, SCPA is likely to contribute to the early stages of streptococcal pathogenesis by inhibiting the recruitment of PMNs to an initial site of streptococcal multiplication in tissue. The second protease that protects streptococci from interacting with phagocytic cells is the streptococcal cysteine protease (also referred to as SCP and SpeB; see below), which differs from SCPA in that it is freely secreted from the streptococcal cell and has a very broad substrate specificity. For example, SCP can cleave and activate several host proteins including pro-IL-ip [50a], tissue metalloprotease [12], and kininogen [38]. It can degrade host extracellular matrix proteins [50] and can also cleave and release active forms of streptococcal surface proteins from the cell wall, including SCPA [5]. In addition, while these two proteases are regulated at the level of transcription, they are under control of distinct pathways. The C5a peptidase is under the control of a positive regulator known as Mga [88], and its regulation is sensitive to carbon dioxide and alterations in atmosphere (see below), while the cysteine protease is subject to catabolite repression, is expressed preferentially postexponentially [15], and requires the positive-acting transcriptional regulator RopB [59]. Inactivation of the gene that encodes the cysteine protease (speB) has a wide range of effects on virulence that depend on the specific strain under analysis. These range from no effect at all [2] to a substantial attenuation of virulence [58]. Similarly, while the gene that encodes the cysteine protease (speB) is virtually universally present in all S. pyogenes isolates [50, 91], there is a dramatic variation in the amount of protease different strains will secrete in vitro [91]. For those strains that require protease activity for virulence, inactivation of the gene that encodes the protease results in both a higher degree of recognition and killing of the streptococci by PMNs in vivo and a decreased ability of the streptococcal mutants to kill the phagocytic cells themselves [57]. This latter property is a hallmark of many streptococcal diseases. In fact.
732
MICHAEL CAPARON
streptococci are often referred to as "pyogenic cocci" based on their ability to kill phagocytic cells at the site of infection. Killing the phagocytic cells themselves provides an obvious advantage in a bacterium's attempts to evade recognition, uptake, and killing by these host defense cells. Aside from the cysteine protease, S. pyogenes makes and secretes a number of additional gene products that can affect the viability and function of PMNs. Most notable among these is streptolysin S (SLS), so named because, unlike the thiol-dependent pore-forming hemolysin streptolysin O, SLS is active in the presence of oxygen and is primarily responsible for the zone of p-hemolysis produced by colonies of S. pyogenes on the surface of blood agar (for a detailed review, see [32]). SLS is a nonimmunogenic toxin of relatively low molecular weight whose molecular composition is far from understood. It does appear that SLS is composed of a short peptide of streptococcal origin that requires a host "carrier" molecule in order to be released from the streptococcal cell surface. Carrier molecules can include albumin, RNA, and certain detergents. The resulting toxin is one of the most potent cytolytic agents known; however, its mechanism of membrane damage, while only poorly understood, does not appear to involve the formation of large pores. On the other hand, it is known that SLS is a potent cytotoxin for PMNs. Mutational analyses have identified several loci that are required for expression of SLS activity [8, 55], and, while it has not yet been established if any of these loci are direcdy involved in the production of the toxin, it is clear that the SLS-defective mutants are highly attenuated in murine models of infection of cutaneous tissue [8]. B. Avoidance of the Immune Response A powerful strategy used by the host to defend itself against pathogens that escape recognition by PMNs is the production of antibodies that recognize and neutralize the specific molecules the bacterium uses to escape the recognition mechanisms of the host. Thus, successful pathogens often have evolved powerful strategies to avoid this host immune response. 5. pyogenes is a master of this strategy and has evolved several highly effective mechanisms to evade the host immune response. These include strategies for both antigenic mimicry and antigenic variation. In the case of antigenic mimicry, both SLS and the hyaluronic acid capsule are highly nonimmunogenic. For SLS, the basis of this is not known but may involve the fact that the active toxin includes components derived from the host. The glycosaminoglycan polymer that comprises the capsule is commonly found in host tissue structures rich in hyaluronic acid, and as a direct molecular mimic of a host molecule is likely immunologically privileged. An additional aspect of mimicry can be found in the ability of the streptococcal cells themselves to bind many host proteins to their surfaces, including fibrinogen, plasmin, fibronectin, laminin, collagen, vitronectin, factor H, IgG, IgA, and albumin (Fig. 4) (for a
15.
PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
733
Fibronectin Laminin
^
-\
Vitronectin
k^
IgG igA C4b-binding y^ V ^ ^ - ^ ^ _ , . ^ ^ protein /^ ^ \^ CD46 H-Kinlnoqen -Kininogen
1
Plasmin(ogen)
Collagen
Factor H Fig. 4 Host proteins bound by S. pyogenes. A partial list of the many host proteins that are known to bind to the cell surface of S. pyogenes. In many cases, streptococcal receptors for the host proteins have been identified. Streptococcal binding structures may recognize a single or multiple host proteins, and a single host protein may be bound by multiple streptococcal surface products.
review, see [9]). In this case, the organism has not produced a structure that mimics a host molecule, but has produced a receptor that captures a host molecule in order to mimic a host surface. The classic work of Lancefield described above demonstrated that antibodies directed against M protein can effectively direct the recognition, uptake, and killing of S. pyogenes by PMNs in human blood [52]. S. pyogenes has responded to this pressure by using a strategy of variation of the antigenic structure of M protein. In fact, the antigenic variability of M protein serves as the basis of a serotyping scheme developed by Lancefield as an epidemiological tool where isolates of S. pyogenes are classified into serogroups based on their reactivity to antisera developed against different M proteins. The current typing scheme recognizes upwards of 80 distinct antigenic types, and this likely represents a significant underestimate of the serologic diversity of these molecules in streptococcal population. As described above, while M proteins share a similar structure, there is virtually no conservation of primary sequence in their N-terminal variable domains. The molecular epidemiology of M proteins is very interesting and very complex. This is apparent in the structure of the chromosomal locus (emm) that encodes M protein (Fig. 5), which itself is highly heterogeneous between different clones of S. pyogenes. The study of a large number of different emm loci has revealed an underlying pattern to the organization of this chromosomal region. The emm locus can contain one, two, or up to three genes in tandem encoding for an M protein.
734
MICHAEL CAPARON
Pattern A mg«L
SF-1
B
MjfA
> ^ SF-l
SF-I
:^>— D
1
SF-l
SF.3
SF.4
SF-l
SF.5
SF.4
SF-2
SF.3
X>*^ X><
F/g. 5 Structure of the M locus. The genes that encode the M proteins (emm) are located between a transcriptional activator (mga) and scpA, which encodes a cell-surface protease. Different strains can have one, two, or three genes encoding an M protein. Based on the scheme devised by Hollingshead and colleagues [39], sequences in the conserved regions and cell-wall-attachment domains (see Fig. 2) are used to assign genes into one of four subfamilies (SF-l through SF-4). By number and arrangement as defined by subfamily designation, most loci are organized into one of five chromosomal patterns (A through E). Some chromosomal patterns correlate with specific disease states. For example, pattern E strains are most frequently isolated from impetigo and are infrequently associated with rheumatic fever. The SF-1 and SF-2 genes demonstrate the most variability and are usually the M protein recognized by specific typing sera. The convention is to designate the gene encoding the type-specific M protein by its associated serotype (e.g., emm6 encodes a serotype 6 M protein) and to further identify specific alleles within a serotype by a number (e.g., emm6.1 is the prototype emm6). Examination of the serotype-specific emm genes reveals a mosaic structure that has likely arisen through horizontal transfer.
The genes are oriented so that they are all transcribed in the same direction, but each gene is transcribed from its own promoter. At the 5' end of the cluster is the gene mga, which encodes a positive acting transcriptional regulator (formerly known as Mry and VirR, this regulatory protein was recendy renamed Mga [87]) that binds to a characteristic site located in each emm gene's promoter. The distal end of the locus consists of the gene {scpA) that encodes the C5a peptidase, which is also under the regulatory control of Mga [88]. Based on nucleotide polymorphisms in defined regions of the emm genes that encode the highly conserved carboxy-terminal region of the M proteins, it is possible to classify different emm genes into one of four subfamilies. Using the scheme developed by Bessen and Hollingshead [6, 7, 39, 40], which is based on both the number of emm genes in the locus and their arrangement and composition as defined by subfamily designation, it is possible to assign greater than 99% of
15.
PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
735
the different emm loci examined to one of five different chromosomal patterns [39]. When examined in this context, there is a correlation between the chromosomal pattern and the general pattern of diseases a particular strain will produce. One group of patterns (A, B, and C) is preferentially associated with pharyngitis (and, thus, rheumatic fever, which only occurs following a pharyngeal infection), one pattern (D) is most often associated with impetigo of the skin, and one pattern (E) demonstrates no clear preference [7]. In correlating the structure of the emm locus to serological classification, as a general rule, some of the emm subfamily genes are more variable in their N-terminal sequences, and all chromosomal patterns typically contain at least one of these highly variable emm subfamily genes, which usually represents the serospecific M protein that defines the strain serologically [6]. In general, when not considered in the context of chromosomal pattern, no clear correlation exists between serological M type and specific streptococcal diseases. The exception to this rule is acute glomerulonephritis, which is a nonsuppurative sequela that has been associated with only a few serological types (for review, see [41]). Some emm genes encode M proteins that bind to the Fc region of immunoglobulin, and these can be further subdivided into those proteins that bind IgA or those that bind to IgG. The IgG-binding types can be further subdivided into groups based on affinities for the different subtypes of human IgG (for review, see [9, 16]). Comparisons of large numbers of different emm genes and loci strongly suggest that the polymorphism has arisen through horizontal transfer and that the individual genes and loci are mosaics that have been generated by homologous recombination [6, 51]. In this regard, it is important to note that streptococci are rich in different bacteriophages, many of which are proficient at transduction. An additional mechanism that may contribute to the divergence of defined lineages may involve intramolecular recombination between nearly-identical repeats in the same emm gene that themselves have diverged through antigenic drift [40]. The implication of this variation for pathogenesis is that, because of the very large number of different genes, it is very difficult for any one individual to develop immunity that will protect against all the potential M proteins in the streptococcal population. Thus, variation does not occur at high frequency during infection of a single individual; rather, over time, any one population tends to become infected with strains that express different M types. In this regard, it is interesting to note that the cases of severe invasive disease that have occurred over the last few years have, more often than not, been caused by one of only a few different M types. This has fueled speculation that these strains represent the emergence of novel hypervirulent clones. However, more recent data have indicated that in some communities that underwent a dramatic increase in the number of severe invasive infections there was an extraordinarily high rate of mild or asymptotic carriage of the same clone in the community, particularly in pediatric populations [17]. Thus, rather than being hypervirulent, the increased incidence of severe invasive disease may be more a reflection of the success of a
736
MICHAEL CAPARON
particular clone at penetration into a community. The increased incidence in severe disease may then arise as a consequence of the large number of individuals who have become exposed to the clone, rather than to an increased capacity of the clone to cause severe disease. Similar questions concerning variation and epidemiology have been raised over the ability of other streptococcal species to antigenically vary the structures they use to avoid recognition by phagocytic cells. The most notable among these is S. pneumoniae, whose different strains have the capacity to express upwards of 80 different types of capsular polysaccharide [34]. C. Damage and Spread in Tissue Once the streptococci have entered host tissue, attached and interacted with an epithelial cell, they begin to multiply and will induce an intense inflammatory response by the host and then actively kill and evade recognition by the host phagocytic cells that have entered this initial site of infection. At this juncture, the streptococci will begin to spread in the tissue and the tissue will become damaged. The degree of spread and the nature of the damage are characteristic of specific diseases and can range from a very localized mildly inflammatory lesion to complete necrotic destruction of tissue. The extensive heterogeneity that exists even between strains isolated from similar disease syndromes [14] is apparent at the level of the complement of different possible known virulence genes the strain may possesses: most strains do not contain all known virulence genes, but, rather, they typically have only some subset of these genes. The heterogeneity is also apparent at the level of the different possible alleles of any given gene the strain possesses and in the different patterns of regulation of these genes by various strains. This heterogeneity has also complicated analysis of the different mechanisms the streptococcus uses to invade and damage tissue. However, it is likely that these processes involve damage generated both by the direct action of some streptococcal product(s) on host tissues and by the host's own inflammatory response to those products. 1.
TOXINS AND ENZYMES
A well-appreciated general feature of S. pyogenes is its prodigious ability to secrete different products into its environment (Fig. 6) (for a detailed review, see [1, 31]). These include numerous high-molecular-weight proteins with known enzymatic and/or toxic activities. Many of these secreted enzymes have degradative activities that may also assist in multiplication of the streptococci by helping the cells to acquire nutrients, or may directly participate in damage to tissues. These include hyaluronidase and the up to four different types of DNases. This group of enzymes may also assist the streptococcus in spreading through tissue through their abilities to degrade high-molecular-weight polymers, like DNA, that become viscous when released by damaged cells. Other secreted enzymes include an NADase, glucuronidase, and esterase and various peptidases, neuraminidases, and phosphatases. The cysteine protease may directly damage tissue or may
15.
PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
GAPDH
LTA
N^
737
glucuronidase
f
NADase
SSA
^ Amylase
Streptokinase ^f--^ / DNase A, B, C, D ^
V
\
- > phosphatase
/
"ya'^'-o^'dase
^-.__-^<^ bacteriocins y ^ ^ / \ SpeA,B,C,F Streptolysin O Streptolysin S
^ ^. ^ Cysteine protease
Fig. 6 Extracellular products of S. pyogenes. A partial list of the many products secreted from S. pyogenes. Abbreviations: GAPDH = glyceraldehyde phosphate dehydrogenase; NADase = nicotinamide adenine dinucleotide glycohydrolase; SpeA, B, C, F = streptococcal pyrogenic exotoxin serotype A, B, C, and F; SSA =streptococcal superantigen; LTA = lipoteichoic acid.
activate several host-derived degradative pathways in an unregulated fashion, such that the unchecked degradative pathway becomes destructive. For example, the cysteine protease can activate host tissue metalloproteases that are normally involved in degradation and remodeling of tissue (see above). 2.
INTERACTION WITH THE PROCOAGULATIVE AND FIBRINOLYTIC CASCADES
The cysteine protease can also release active kinin through cleavage of its inactive precursor H-kininogen [38]. Active kinins, including the nonapeptide bradykinin, are small peptide hormones that are the principal effector molecules produced by the host contact-phase system. This system involves a number of different proteins that assemble on a host cell surface, such as an activated endothelial cell, which leads to activation of prokallikrein, which in turn cleaves H-kininogen to liberate kinins. The activated kinin peptides are potent effectors and are part of the coagulation cascade, where they act to increase vascular permeability through stimulation of production of prostaglandins and nitric oxide. Production and release of kinins at the site of infection and the resulting increase in vascular permeability is thought to facilitate penetration and spreading of the infecting streptococci into the surrounding tissues. In addition, S. pyogenes can capture and bind H-kininogen to its surface through an interaction with M protein [4], and active bradykinin can be released from H-kininogen that is bound to the streptococcal cell surface [3]. Thus, S. pyogenes can mimic and pirate multiple aspects of the host contact-phase system. Capture of host-derived proteolytic activity may also contribute to streptococcal spread through a tissue and invasion across tissue barriers. A key component
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MICHAEL CAPARON
of this activity is the streptococcal-derived plasminogen activator streptokinase (for a review, see [10]). Almost all streptococcal strains isolated from human infections secrete streptokinase. Unique among plasminogen activators, streptokinase itself has no enzymatic activity. Rather, it binds and forms an equimolar complex with plasminogen that induces a conformational change in plasminogen such that the resulting complex becomes an potent plasminogen activator. Activated plasmin is a serine protease whose physiological role is primarily in fibrinolysis, but it is actually a very potent protease of broad specificity. For example, it can degrade proteins of the extracellular matrix, including fibronectin and laminin, and, like the streptococcal cysteine protease, can activate other host proteases. This fact may explain why its activation is tighdy regulated under normal circumstances, and why the host also produces several highly active inhibitors of the activated protease. In contrast, the streptokinase-plasminogen complex is not subject to inhibition by host plasmin inhibitors. Thus, S. pyogenes can activate several host degradative pathways in an unregulated fashion. Another interesting feature of streptococcal interaction with plasminogen is that streptokinase is produced by many different streptococcal species aside from S. pyogenes. For example, the streptokinase developed by biotechnology to attack clots during myocardial infarction is derived from a group C species that causes human infection. Also of interest is that streptokinases from strains that preferentially infect humans, horses, or pigs are very species-selective in plasminogen activation and will only efficiently activate plasminogen derived from their natural hosts. A more insidious aspect of the streptococci's ability to activate plasminogen is that they also have the capacity to capture the active protease on their cell surface. This can occur through multiple pathways, suggesting that it is an important virulence property. In the first pathway, plasmin binds directly to a streptococcal plasmin receptor located on the surface of the cell wall. Two such plasmin receptors have been described to date, and they are both of unexpected origin since both are enzymes essential for glycolysis that were not expected to be displayed on the cell surface. One of these is glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an enzyme essential for glycolysis that catalyzes conversion of glyceraldehyde 3-phosphate to 3-phosphoglyceroyl phosphate [56, 73]. Furthermore, there appears to be only a single gene that encodes this essential enzyme in the streptococcal genome, the enzyme is found simultaneously exposed on the cell surface and in the cytoplasmic compartment, and the surface-exposed enzyme retains enzymatic activity. A second cell-wall component that was purified on the basis of its ability to bind plasmin with a higher affinity than GAPDH was revealed to be a-enolase [71]. Like GAPDH, a-enolase is an essential glycolytic enzyme that catalyzes dehydration of 2-phosphoglycerate to phosphoenolpyruvate. Also, like GAPDH, a-enolase is also located in the cytoplasmic compartment, and the surface-exposed enzyme retains its enzymatic activity. A surface location has been established by both cell-fractionation and crosslinking studies, and in the case of GAPDH iron starvation causes release of the enzyme into the surrounding medium. When plasmin is bound to purified
15.
PATHOGENIC MECHANISMS IN STREPTOCOCCAL DISEASES
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a-enolase, it retains its proteolytic activity and becomes resistant to inactivation by host plasmin inhibitors. Furthermore, almost all streptococcal species examined have a cell-surface-exposed structure with antigenic similarity to the S. pyogenes a-enolase [71], which suggests a high level of conservation of this pathway for interaction with plasmin. A second mechanism for capturing host plasmin involves formation of a complex among streptokinase, plasminogen, and fibrinogen [98]. The complex becomes tethered to the streptococcal surface through recognition of fibrinogen by a streptococcal fibrinogen-binding protein, of which there are several (see above). A key common feature of both pathways of plasmin capture is that the streptococcal cell becomes coated with proteolytically active plasmin that is not subjected to inactivation by the normal pathways that regulate activated plasmin in the host. It is then thought that the streptococcal cells gain a strong degradative capacity, which causes destruction of tissue, spreading through tissue, and the ability to degrade and penetrate across tissue barriers like a basement membrane. This latter property is expected to play a major role in the ability of the streptococci to pass between different tissue compartments, or from tissue into the bloodstream. As of yet, there is no direct evidence to support this mechanism, and it should be considered that, while plasmin-binding is a highly conserved trait, there is no correlation between how invasive a particular strain can be and how efficient it is at capturing plasmin [10]. However, this mechanism is not without precedent in that metastatic tumor cells often have the ability to activate and capture plasmin, and it is thought that this property contributes to their ability to cross tissue barriers to enter and invade tissue [24]. In this regard, it is of interest to note that a-enolase is expressed on the surface of some types of cancer cells and that it can serve as a plasmin receptor [76]. The different mechanisms used by S. pyogenes to interact with the fibrinolytic cascade are summarized in Figure 7.
V. Second Mechanism: Toxin'Mediated Disease A. Hallmarks of the Diseases The defining characteristic of a streptococcal disease that is mediated by a toxin is that the most serious damage to the host occurs at sites distinct from the primary site of infection. This classification is not mutually exclusive of other types of severe disease and can often be manifested as a more terminal complication of a serious invasive disease in tissue, and even bacteremia. However, these diseases can also be associated with a very local and mild or even nonapparent streptococcal infection. Children in particular are more vulnerable to a toxin-mediated disease in the absence of severe local disease. For group A streptococci, the classic examples of toxin-mediated diseases are scarlet fever and the streptococcal toxic shock syndrome (for a more detailed review, see [83]). In the pathogenesis of toxic shock syndrome, infection of a local site in tissue is followed by a profound
740
MICHAEL CAPARON
Streptokinase < + Plasmin(ogen)
^ ^
L< [protein
- cysteine protease ^
M pruit;iii r/
Plasmin(ogen)
y GAPDHV a-enolase
Fig. 7 Capture and exploitation of host molecule function. Several pathways used by 5". pyogenes to capture and exploit the function of several host molecules are shown. Streptokinase secreted by S. pyogenes binds and activates plasminogen, and the complex is bound by the streptococcal cell. Alternatively, surface dehydrogenase enzymes (glyceraldehyde phosphate dehydrogenase, GAPDH, or a-enolase) directly capture plasmin(ogen). In both cases, active plasmin is sequestered on the streptococcal cell surface and protected from normal mechanisms of inactivation and the streptococcal cell acquires a strong surface-associated proteolytic activity. Similarly, H-kininogen is captured by the streptococcal cell surface in a conformation that allows the action of the secreted streptococcal cysteine protease to release active kinins, potent effectors of the coagulation cascade. Streptococcal protein F can capture fibronectin, allowing the streptococcal cell to acquire the multiple binding activities of fibronectin, including the ability to bind to collagen-containing substrates.
life-threatening systemic hypotension. In addition, the onset of hypotension can be accompanied by some combination of additional serious complications, including renal dysfunction, an erythematous rash, coagulopathy, damage to the liver, adult respiratory distress syndrome, and soft-tissue necrosis. In many cases, the patients are healthy with no predisposing conditions and present initially with complaints of severe pain, often in an extremity, that is out of proportion to physical findings. In some cases, the onset of pain is preceded by gastrointestinal symptoms characteristic of the flu. Even with aggressive treatment the mortality rate is very high. B.
Superantigens
The pathogenesis of the toxin-mediated diseases is not understood. However, there is considerable speculation that they involve superantigens. Superantigens are proteins that have the ability to bind to an invariant region of the class II major histocompatibility complex (MHC) on an antigen-presenting cell and to crosslink this receptor to a T cell through binding to the variable region of the p-chain of the T cell antigen receptor. In both cases, binding of the superantigen occurs
15.
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outside of the site of antigenic-peptide specific binding region of the receptors. S. pyogenes is a prodigious producer of protein toxins with the properties of a superantigen (for review, see [77, 83]). These toxins are freely secreted by the streptococci into their surroundings and have a number of biological properties in common. When the purified toxins are injected into an animal, they typically induce a transient fever and can dramatically sensitize the animal to lipopolysaccharide- (LPS-) mediated lethal endotoxic shock. Superantigens trigger many effects in both the antigen-presenting cell and the T cell, and these effects occur in the absence of specific antigen. One consequence is prolonged and unregulated production of cytokines by these host cells, including interleukin 1 (IL-1) and tumor necrosis factor alpha (TNF-a) by the antigen-presenting cells and gamma interferon (y-IFN) by the T cells. These cytokines may then be responsible for triggering the systemic effects, including the profound hypotension. As mentioned, the list of superantigens that 5. pyogenes can produce is extensive. Many of these are related, both in sequence and structure. The first class was historically known as the streptococcal erythrogenic toxins based an their association with the red rash produced during scarlet fever but are now referred to as the streptococcal pyrogenic exotoxins (SPEs) to reflect their identity as superantigens. Originally classified serologically, the SPEs consist of three distinct antigenic types. Of these, the genes for SPE-A and SPE-C are encoded in the genomes of lysogenic bacteriophage [33,49]. The structure of SPE-C has been determined, and this has revealed that it has an overall structure similar to the superantigens of Staphylococcus aureus that is characterized by an amino-terminal P-barrel domain and a carboxy-terminal domain that includes a P-grasp motif [80]. However, it lacks the binding site used by the staphylococcal superantigens for binding to the MHC molecule and instead binds as a dimer to the MHC using a zinc-dependent mechanism. The gene for the third type (SPE-B) appears to lie on the streptococcal chromosome and has not been reported to be associated with a bacteriophage. Sequence analysis of genes encoding both SPE-B and the streptococcal cysteine protease revealed that they were identical, and this is why the gene for the protease is designated speB [37]. An additional superantigen called the streptococcal superantigen (SSA) has been described that has some sequence similarity to SPE-A [61]. Another secreted protein, mitogenic factor (MF or SPE-F), was identified on the basis that it is mitogenic for T cells and was subsequently found to have superantigenic properties [65]. MF (SPE-F) is one of the several DNases secreted by S. pyogenes [47]. The genes for SPE-A, SPE-C, and SSA are present in a subset of all streptococcal strains, and any one strain may have the genes for one or several of these factors, or may have none at all [18, 91]. In contrast, the gene for SPE-B and SPE-F (encoding a protease, and a DNase, respectively) are found in virtually all isolates of S. pyogenes [50, 91, 104]. This pattern of distribution of the genes for the various superantigens has made it difficult to establish a correlation with the presence of a specific superantigen and any one particular type of disease. However, there is a strong correlation between production of certain specific superantigens and toxic shock syndrome caused by Staphylococcus aureus. Superantigen activity has also been
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described for group B, C, F, and G streptococcal species isolated from cases of necrotic and toxic shock-like illnesses [84].
VL Third Mechanism: Immunopathologicol'Based Diseases A.
Hallmarks of the Diseases
The immunopathological-based diseases include several seemingly unrelated diseases that have in common the fact that they occur several days to weeks after an acute streptococcal infection has been cleared by the host. These types of diseases are almost exclusively associated with infection by S. pyogenes and apparently only occur in humans, even to the extent that has been difficult to impossible to reproduce their pathology in experimental animals. As a consequence, the pathogenesis of these diseases are among the most poorly understood of the different streptococcal infections. These diseases are commonly known as the streptococcal nonsuppurative sequelae and include rheumatic fever and rheumatic heart disease, acute glomerulonephritis, certain forms of psoriasis, and possibly, even some forms of obsessive-compulsive disorder [89]. B.
Antigenic Mimicry
While the development of the nonsuppurative sequelae may involve participation of superantigens [94], and the pathogenesis of acute glomerulonephritis may involve trapping of certain streptococcal proteins in the basement membrane of kidney, followed by activation of complement [64], current thinking has focused on the potential role of antigenic mimicry in these diseases (for review, see [29]). According to this mechanism, the streptococcus expresses epitopes that are molecular mimics of host structures. As a consequence of the host's response against the streptococcal infection, there is a breakdown of the normal prohibition against responding to self-antigens that results in an autoimmune pathology. While there is no direct proof for this hypothesis, compelling circumstantial evidence comes from the historical observation that prompt treatment of streptococcal pharyngitis with penicillin completely blocks subsequent onset of rheumatic fever in susceptible individuals. Furthermore, once an individual has contracted a case of rheumatic fever, they remain extremely susceptible to recurrences following subsequent cases of streptococcal pharyngitis and that recurrences are associated with a substantial immune response against many different streptococcal antigens. These observations suggest that by removing the organisms prior to the development of an immune response development of the disease can be blocked. However, once it has responded, this pathological immune response retains a memory that can easily be triggered on reintroduction of antigen.
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The principal molecular evidence in support of the antigenic mimicry hypothesis is that individuals with acute rheumatic fever often have circulating antibodies that react with numerous human antigens present in heart tissue, including myosin. These same populations of heart-reactive antibodies will also recognize streptococcal surface proteins, most notably the M protein, a structural mimic of myosin. It is generally not thought that these antibodies directly contribute to the pathology observed in these diseases, rather that they are symptomatic of the expression of molecular mimics by the pathogen that is accompanied by a breakdown of T-cell anergy. What triggers this breakdown is not understood [79].
Vll. Concluding Remarks As stated in the introduction, to understand the streptococci it is important to embrace their diversity. The heterogeneity of the streptococci as a group are reflected by the heterogeneity of the different lineages that make up the species S. pyogenes. However, despite this heterogeneity, the different species, and even the different lineages that make up a single species, most of the diseases caused by these organisms share underlying common mechanisms. What distinguishes the different species is the specific gene products used to accomplish similar steps in pathogenesis. In the case ofS. pyogenes, an emerging theme is that the different lineages have become specialists, and have adapted different virulence factors or even combinations of different virulence factors to accomplish similar tasks. Thus, it will become increasingly important to analyze virulence and the contribution of specific genes to pathogenesis in the context of the repertory of different virulence factors any one particular strain has the capacity to produce.
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94. Valdimarsson, H., Sigmundsdottir, H., and Jonsdottir, I. (1997). Is psoriasis induced by streptococcal superantigens and maintained by M-protein-specific T cells that cross-react with keratin? Clin. Exp. Immunol. 107, 21S-24S. 95. van der Flier, M., Chhum, N., Wizemann, T. M., Min, J., McCarthy, J. B., and Tuomanen, E. I. (1995). Adherence oi Streptococcus pneumoniae to immobilized fibronectin. Infect. Immun. 63, 4317-4322. 96. Van Heyningen, T., Fogg, G., Yates, D., Hanski, E., and Caparon, M. (1993). Adherence and fibronectin-binding are environmentally regulated in the group A streptococcus. Mol. Microbiol. 9, 1213-1222. 97. Wang, B., Ruiz, N., and Caparon, M. G. (1997). Keratinocyte proinflammatory responses to adherent and nonadherent group A streptococci. Infect. Immun. 65, 2119-2126 98. Wang, H., Lottenberg, R., and Boyle, M. D. R (1995). A role for fibrinogen in the streptokinase-dependent acquisition of plasmin(ogen) by group A streptococci. J. Infect. Dis. Ill, 85-92. 99. Wang, J. R., and Stinson, M. W. (1994). Streptococcal M6 protein binds to fucose-containing glycoproteins on cultured human epithelial cells. Infect. Immun. 62, 1268-1274. 100. Wannamaker, L. W. (1970). Differences between streptococcal infections of the throat and skin. New Engl. J. Med. 1^1, 23-30. 101. Wessels, M. R., Moses, A. E., Goldberg, J. B., and DiCesare, T. J. (1991). Hyaluronic acid capsule is a virulence factor for mucoid group A streptococci. Proc. Natl. Acad. Sci. U.S.A. 88, 8317-8321. 102. Wexler, D. E., Chenoweth, E. E., and Cleary, P. P. (1985). Mechanism of action of the group A streptococcal C5a inactivator. Proc. Natl. Acad. Sci. U.S.A. 82, 8144-8148. 103. Wilson, A. T. (1959). The relative importance of the capsule to the M-antigen in determining colony form of group A streptococci. J. Exp. Med. 109, 257-270. 104. Yutsudo, T, Okumura, K., Iwasaki, M., Hara, A., Kamitani, S., Minamide, W., Igarashi, H., and Hinuma, Y. (1994). The gene encoding a new mitogenic factor in a Streptococcus pyogenes strain is distributed only in group A streptococci. Infect. Immun. 62, 4000-4004.
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CHAPTER 16
Listeria
monocytogenes
HAFIDA FSIHI PIERRE STEFFEN PASCALE COSSART
I. General Overview of Listeria monocytogenes and Listeriosis A. History B. Other Listeria Species C. Ecology and Specific Properties of L. monocytogenes D. Human Listeriosis: Epidemiology and Clinical Features E. Successive Steps of Infection by L. monocytogenes in Animal Models IL Genetic Tools and Cell Biology Techniques to Study L monocytogenes Infection III. Molecular Mechanisms for Entry and Spread of L. monocytogenes in Nonphagocytic Cells A. Entry of L monocytogenes into Mammalian Cells B. Escape from the Primary Vacuole C. Intra- and Intercellular Spreading D. Other Virulence Determinants of L. monocytogenes IV Regulation of L. monocytogenes Virulence Gene Expression A. PrfA, a Pleiotropic Activator of Virulence Gene Expression B. PrfA, a Protein of the CAP/FNR Family C. PrfA Boxes D. PrfA as a Repressor E. Control of prfA Expression and PrfA Production F Requirement for a PrfA Cofactor G. Global Regulation of Virulence Gene Expression V Conclusion References
Principles of Bacterial Pathogenesis Copyright © 2001 by Academic Press All rights of reproduction in any form reserved. ISBN 0-12-304220-8
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/. General Overview of Listeria monocytogenes and Listeriosis A.
History
Listeria monocytogenes was presumably first isolated at the beginning of the twentieth century as a Gram-positive rod in tissue specimens of infected patients. In 1919, Hulphers called Bacillus hepatis the bacterium he isolated from necrotic foci in a rabbit liver. In 1926, the species name monocytogenes was given by Murray and colleagues to describe a new bacillus with potent monocytosis-producing activity in rabbits and guinea pigs [1]. In 1927, in honor of Lord Joseph Lister, Pirie named Listerella hepatolytica the bacillus responsible for an epizooty among rodents in the Tiger river region of South Africa. In 1929, the first unambiguous isolations of these bacteria from humans were reported by Nyfeldt [2]. Another human case was described when "diphtheroids" were isolated from spinal fluid cultures of a patient with meningitis [3]. In 1940, the organism was given its definitive name Listeria monocytogenes [4, 5]. B. Other Listeria Species The genus Listeria consists of six different species: L. monocytogenes, L. seeligeri, L. welshimeri, L innocua, L. ivanovii, and L. grayi. The various Listeria species can be distinguished from each other by their hemolytic phenotypes and their abilities to reduce nitrate and ferment specific sugars [6, 7]. Whereas L. monocytogenes infects both humans and animals causing meningitis, sepsis, and abortion [8, 9], L. ivanovii is restricted to sheep and cattle, in which it causes septicemic disease, neonatal sepsis, and abortion, but no brain infection [10]. The other species are generally considered nonpathogenic, although L. seeligeri and L. welshimeri have each been reported as the causative agent of human infections [11, 12] and L. innocua has been implicated in a case of ovine meningoencephalitis [13]. C. Ecology and Specific Properties of L. monocytogenes L. monocytogenes is widespread in nature and can be commonly found in soil, decaying vegetation, sewage, and wastewater [14]. This microorganism has also been isolated from over 50 different animal species, including birds, fish, and crustaceans [7]. It is estimated that 1 to 5% of humans are asymptomatic intestinal carriers of L. monocytogenes [9]. Because of the multiple ecological niches in which L. monocytogenes can be isolated, the origins of the human infection have long been uncertain, but epidemiologic studies provided evidence for a foodborne origin for both outbreaks and sporadic episodes of listeriosis [8, 15-18]. Listeria contamination has been traced to milk, cheese, raw vegetables, fish, poultry, and
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meat. Association of listeriosis with this wide variety of both raw and processed food products is mainly due to the ability of L. monocytogenes to survive and multiply in extreme conditions, such as refrigeration temperatures (4-10°C), low pH (3.6-5.0), and high NaCl concentrations (5-10%) [19, 20]. Other characteristics of L. monocytogenes include its catalase-positive and oxidase-negative phenotypes, and its ability to be motile at 28°C by means of peritrichous flagella, but not at 37°C, at which flagellin production is highly reduced [21]. D. Human Listeriosis: Epidemiology and Clinical Features At least 16 serovars of L. monocytogenes have been recognized. Both flagellar and somatic (carbohydrate-containing) surface antigens contribute to the serotypic designation of L. monocytogenes [22]. Three serotypes (4b, l/2a, and l/2b) are responsible for about 90% of human disease [14, 23]. However, no direct correlation has been found with any particular genotype that could account for the particular virulence characteristics of these serotypes. Although listeriosis can affect healthy persons [15], L. monocytogenes remains an opportunistic pathogen that has a predilection for individuals with impaired immunity such as the fetus, newborn infants, pregnant women, and elderly individuals [9]. Transplantation patients, cancer patients on therapy, or individuals treated with corticosteroids are at particular risk in acquiring a severe listerial infection. The risk of listeriosis is also markedly increased among HIV-infected individuals [24, 25]. As outlined above, L. monocytogenes has a broad host range and can infect both humans and animals, with infections involving mainly the central nervous system and the fetoplacental unit having a high mortality rate (30%). However, if one refers to human listeriosis, the name ''monocytogenes'' is somehow misleading, because monocytosis is a rare feature of human infection. The so-called monocytosis-producing agent of L. monocytogenes is a lipid that produces monocytosis in rabbits and rodents but not in humans [26]. 1.
CENTRAL NERVOUS SYSTEM INFECTIONS
The main clinical forms of central nervous system (CNS) infections by L. monocytogenes are meningitis and diffuse or focal encephalitis (brain abscesses) [9, 27]. Compared to Streptococcus pneumoniae. Neisseria meningitidis, and Haemophilus influenzae, which also cause meningitis, L/5^r^na-associated meningitis has one of the highest mortality rates [28]. Brain stem infection with L. monocytogenes predominantly causes rhomboencephalitis, a complication of human listeriosis similar to the listerial ovine circling disease [29, 30]. 2.
LISTERIOSIS DURING PREGNANCY
During the third trimester of gestation, cell-mediated immunity is thought to be impaired [31], predisposing pregnant women to develop listerial bacteremia.
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HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
Symptoms are not severe and resemble influenza-like illness often associated with myalgias, arthralgias, headache, and backache. For unknown reasons, CNS infection is extremely rare during pregnancy [32]. 3.
NEONATAL INFECTIONS
While usually not severe for the mother, listeriosis during pregnancy can be devastating for the infant. Deficiencies in local immunoregulation at the placental level probably contributes to perinatal infections [33]. L. monocytogenes is one of the few bacteria that can cross the transplacental barrier to infect the fetus, thus resulting in premature labor, fetal death, or severe neonatal sepsis. Neonatal infection acquired in utero (early-onset neonatal listeriosis) may lead to the fatal syndrome of granulomatosis infantisepticum, characterized by disseminated abscesses and granulomas in the liver, spleen, lungs, kidneys, brain, and skin [34]. Infection acquired during delivery can also lead to meningitis in newborns (late-onset neonatal listeriosis).
E. Successive Steps of Infection by L. monocytogenes in Animal Models With the exception of transmission from mother to fetus, and sporadic cases due to direct contact with infected animals [35], human infection with L. monocytogenes begins with consumption of contaminated food [15, 36, 37]. As L. monocytogenes is responsible for animal infections with clinical features similar to those of human listeriosis, various experimental animal models including mice, rats, rabbits, and guinea pigs have been widely used to dissect the successive steps of listerial infections. Bacteria are inoculated to these animal models by oral, intraperitoneal, subcutaneous, or intravenous routes [38]. Virulence of the L. monocytogenes strains can be assessed by determination of the median lethal dose (LD50) values or by monitoring the bacterial growth of the tested strains in target organs such as the liver and spleen. Following ingestion, bacteria can cross the intestinal barrier. Reports vary as to the first phases of infection. In some reports, L. monocytogenes is shown to preferentially target the Peyer's patches, the lymphoid follicles of the gut, as demonstrated by bacteriological and histopathological analyses of orally and intragastrically inoculated mice [39,40]. In a recent study, the authors were unable to locate bacteria within murine M cells following an oral listerial infection [41]. In a study using a rat ligated ileal loop system, L. monocytogenes has been shown to replicate preferentially in the Peyer's patches, but there did not seem to be a preferential site of translocation in the intestine [42]. In another study carried out in murine ligated intestinal loops L. monocytogenes penetrates the intestinal mucosa through M cells [43]. Finally, using oral infections of guinea pigs, Racz
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et al were able to show that Listeria invades enterocytes. These different results may be due to the different animal models used and the different experimental conditions such as the size of the bacterial inoculum. Whatever the mechanism used, crossing the epithelial barrier provides the bacteria with a portal of entry into the lymphatic system and the blood. Intravenous injection bypasses this first phase of the infection. In the murine model, it has been shown that 10 minutes after an intravenous injection of a sublethal dose of L. monocytogenes, approximately 90% of the injected bacteria are recovered from the liver and 10% from the spleen [44]. In the liver, the bacteria are readily phagocytosed by Kupffer cells. After an initial phase of bacterial killing resulting in the disappearance of the majority of the inoculum, bacteria can spread to adjacent hepatocytes, where they may induce apoptosis [45]. Dying cells then trigger recruitment of neutrophils, which eliminate cellular debris as well as most surviving bacteria, while a sterilizing T-cell response rapidly appears. It is thought that in the case of immunodeficient individuals residual bacteria multiply and spread hematogenously to the brain and/or placenta. During a listerial infection several tissues are infected, suggesting that L. monocytogenes is able to invade a wide variety of nonphagocytic eukaryotic cells. Indeed, electron microscopic studies long ago demonstrated the capacity of the bacteria to penetrate in vivo epithelial cells of both the cornea, after instillation of the eye, and the intestine, after oral infection [46, 47]. Tissue culture assays of bacterial invasion reveal that L. monocytogenes is capable of penetrating various cell types, including hepatocytes [48-50], fibroblasts, epithelial [51], and endothehal cells [52, 53].
//. Genetic Tools and Cell Biology Techniques to Study L monocytogenes Infection Following its identification, L monocytogenes first received attention mainly as a model for the study of cell-mediated immunity [54, 55]. The increasing number of small outbreaks of listeriosis and their tracing to contaminated food products has provoked a real concern in the food industry as well as in public health services. Interest in understanding how the organism infects and survives in its host exploded in the 1980s, when tools and efforts to address the molecular aspects of bacterial pathogenesis in general were rapidly developing. Electron microscopy then allowed identification and further dissection of the successive steps of the infectious process in L. monocytogenes-inftcied cells [56] (Fig. 1). L. monocytogenes has now become one of the best-understood intracellular pathogens.
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HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
Fig. 1 Thin sections of the different steps of the L monocytogenes infectious process [168, 281]
Compared to other intracellular pathogens L monocytogenes has at least two important properties. First, it grows well in vitro, with a doubling time of 1 hour, facilitating genetic studies. This is in contrast to other Gram-positive intracellular pathogens that grow very slowly or may even fail to grow in vitro [57]. Second, it has a susceptible and easy manipulatable animal model, the murine model, in which virulence of genetically modified organisms can be easily assessed. This is not the case for some other well-studied invasive bacteria such as Shigella flexneri [58]. Genetic tools have allowed an in-depth analysis of the genes involved in the pathogenesis of L. monocytogenes. The first hemolysin-negative mutants of L. monocytogenes were isolated by conjugative-transposon mutagenesis [59-61]. Nonconjugative transposons such as Tn977, Tn9]7lac that allow random generation of lacZ fusions and other derivatives have also proven to be powerful tools for identification of virulence genes, gene cloning, and expression analysis [62-64]. DNA electroporation of penicillin-treated bacteria allows plasmid transformation [65]. Gene replacements to introduce point mutations or in-frame deletions into the chromosome are performed via allelic exchange [66, 67]. The
16.
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LISTERIA MONOCYTOGENES
Fig. 1 continued
development of a transducing phage may be very useful [282]. The Gram-positive species Bacillus subtilis [68-70] and the related avirulent L. innocua [71, 72] are routinely used for heterologous gene expression and subsequent functional and regulation studies. Pulsed-field gel electrophoresis permitted the establishment of a physical map of the 3.15-Mb circular chromosome of L. monocytogenes [73]. Determination of its complete nucleotide sequence (strain EGD) has been recendy achieved in the framework of a European consortium. Tissue culture from various cell lines are widely used for evaluating invasion of mammalian cells by L. monocytogenes and cell-to-cell spread of the bacteria.
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HAFIDA FSIHI, PIERRE STEEPEN, AND PASCALE COSSART
Uptake by host cells is evaluated by the gentamicin survival assay, which involves incorporation of the bactericidal agent gentamicin into the medium just after infection. As gentamicin does not efficiently penetrate mammalian cells, internalized bacteria survive addition of the antibiotic whereas extracellular bacteria are killed. Lysis of infected cells and direct plating of serial dilutions allow quantification of viable internalized Listeria, Invasion can also be evaluated by double immunofluorescence labeling. In this technique, after infection, cells are labeled before and after permeabilization with two different antibodies. Cell-tocell spread is monitored by the plaque-assay [74]. L. monocytogenes, like S. flexneri, produces plaques in fibroblast monolayers, and the diameter of the plaque may be correlated to virulence [75, 76]. Important advances in understanding the actin-based motility of L. monocytogenes were made by using video microscopy and an in vitro system of cell-free extracts from Xenopus eggs [77], human platelets [78], or bovine brain extracts [79].
///. Molecular Mechanisms for Entry and Spread of L monocytogenes in Nonphagocytic Cells L monocytogenes has the potential to infect in vivo and in vitro a variety of eukaryotic cells, including phagocytic and nonphagocytic cells [80]. The infectious process involves several different steps, and bacterial proteins involved in each of these steps have been identified. The surface proteins intemalin A (InlA) and InlB trigger entry of the bacteria into nonphagocytic cells [80, 81]. Following internalization, bacteria reside within membrane-bound vacuoles until the poreforming toxin listeriolysin O (LLO) [82] and in some epithelial cells phosphatidylcholine-phospholipase C (PC-PLC) [83, 84] allow their release into the cytosol. In this environment, bacteria can grow, multiply, and also move. Intracellular movement is mediated by the ActA protein, which polymerizes the host actin into comet tails that propel the bacteria to the plasma membrane, inducing formation of bacteria-containing protrusions that are then endocytosed by neighboring cells [85, 86]. In the newly infected cell, the bacterium is surrounded by two plasma membranes that are lysed by the cooperative activity of LLO, phosphatidylinositol (PI)-PLC, and PC-PLC [84, 87]. Bacteria are then free to start a new infectious cycle without exposure to the extracellular humoral immune system of the host or other bactericidal components [56] (Fig. 2). Most genes encoding these listerial virulence determinants are clustered on a 10-kb region of the chromosome and form a regulon tightly controlled by the transcriptional factor PrfA (see §IV).
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LISTERIA MONOCYTOGENES
Lysis of the two-membraned vacuole
Entry
Lysis of the primary vacuole
Growth and intracellular movement
Cell-to-cell spread
Fig. 2 Intracellular life cycle of L. monocytogenes. Indicated are the successive steps. The stippled material around the bacteria and in the tails represents F-actin. Adapted from Tilney and Portnoy (1989) [56].
Listeria internalization by macrophages is an important aspect of pathogenesis that has been reviewed elsewhere [88]. In this chapter, we focus mainly on L. monocytogenes invasion of nonphagocytic cells and describe the bacterial determinants as well as their eukaryotic receptors (when identified) involved in the infectious process (Table I). A. Entry of L. monocytogenes into Mammalian Cells 1.
MORPHOLOGICAL ASPECTS OF THE ENTRY PROCESS
Electron microscopy has allowed examination of L. monocytogenes entry into mammalian cells [52, 89, 90]. This bacterium penetrates the mammalian cell without any detectable perturbation of host cell morphology, except at the site of entry, where the plasma membrane tightly enwraps the bacterium. This process, referred to as the "zipper" mechanism, is different from the spectacular membrane ruffles observed on Salmonella or Shigella entry and known as the "trigger" mechanism [91], and is similar to that used by Yersinia, which enters mammaUan cells through the invasin-integrin pathway [92]. 2.
THE INTERNALIN (INLA) AND INLB PROTEINS
a. Structural Features. Transposon-induced noninvasive mutants of L. monocytogenes were isolated using the human intestinal cell line Caco-2 [71].
760
Table I
HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
Identified virulence determinants in L monocytogenes
Protein
Principal characteristics
Function
Listeriolysin
- 58-kDa, SH-activated, cholesterolrequiring pore-forming cytolysin - pH-dependent hemolytic activity
- escape from the phagosome - induces phosphorylation of MAP kinases - induces Ptdlns hydrolysis in HUVEC
Internalin (InIA)
- 84-kDa leucine-rich protein - eukaryotic receptor: E-cadherin - covalently linked to the peptidoglycan via its LPXTG motif
- entry into cultured epithelial cells (Caco-2) - no role identified in vivo
InlB
- 67-kDa leucine-rich protein - surface exposed via its 3 GW modules
- entry into cultured HepG2, HeLa, Vero, Hep-2, HUVEC, and fibroblasts cells - role in multiplication in hepatocytes in vivo - stimulates PI3-kinase activity
ActA
- 67 kDa, polar localization - anchored to the bacterial surface through a hydrophobic C-terminal region
- actin polymerization in infected cells
PI-PLC
- phosphatidylinositol-specific phospholipase C - 33 kDa, basic - active between pH 5.5 and 7.5
- escape from the primary vacuole in murine macrophages - escape from the double-membraned vacuole - amplifies the LLO-related Ptdlns hydrolysis
PC-PLC
- phosphatidylcholine- (PC-) preferring phospholipase C - 29 kDa, active between pH 5.0 and 8.0 - activated on cleavage by the zincdependent metalloprotease Mpl
- escape from the primary vacuole in the human epithelial Henle 407 cells - escape from the double-membraned vacuole and cell-to-cell spread
PrfA
- 27-kDa DNA-binding protein (CAP/FNR family)
- transcriptional activator of most Listeria virulence genes
ClpC
- 27-kDa stress protein (Clp/ATPase family)
- intracellular growth in macrophage
InlC
- 30-kDa leucine-rich protein
- no defined role, but inlC mutants show reduced virulence in mice
Abbreviations used: HUVEC = human umbilical vein endothelial cells; MAP = mitogen activated protein; Ptdlns = phosphoinositides; PI3-kinase = phosphoinositide 3-kinase; LRRs = leucine-rich regions.
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761
Localization of the transposon insertion site in these noninvasive mutants allowed identification of a bicistronic operon composed of two highly homologous genes: inlA and MB [71, 93]. Both gene products—InlA (also called intemalin) and InlB—are required for Listeria uptake by various cell Hues, but each gene seems to play a major, if not exclusive, role for entry in specific cell lines. InlA is required for intemaHzation by the enterocyte-like Caco-2 cells [71] and cells expressing its receptor, E-cadherin. The InlB protein mediates entry into cultured hepatocytes [48, 50], some epithelial or fibroblast cell lines including HeLa, Hep-2, Chinese hamster ovary (CHO), and Vero [51, 94, 95], and the human umbilical vein endothelial cells (HUVECs) [52, 53]. More recent results indicate that the cell specificity conferred by InlB is much wider than that of internalin, in agreement with the significant role in virulence of InlB in the mouse model [48, 49, 94, 96]. When transformed with a plasmid harboring the inlA gene, the noninvasive bacterium L. innocua becomes invasive for Caco-2 cells [71]. InlA- and InlBcoated latex beads are also able to be internalized by Caco-2 and Vero cells, respectively [95,97]. Taken together, these results indicate that both InlA and InlB are sufficient for entry in their respective target cells. Homology searches revealed that InlA and InlB are members of the superfamily of leucine-rich repeats- (LRR-) containing proteins known to be involved in protein-protein interactions [98, 99]. LRRs are sequence motifs (20 to 29 residues) with a defined periodicity of leucine residues that have now been found in over 60 proteins. Members of the LRR family include many eukaryotic proteins, such as the adenylate cyclase of Saccharomyces cerevisiae, the connectin of Drosophila melanogaster, the human LPS cell-surface receptor CD 14, and some bacterial proteins from prokaryotic pathogenic species such as IpaH from Shigella flexneri, the YopM protein from Yersinia pestis, and the filamentous hemagglutinin protein of Bordetella pertussis [100]. InlA possesses 15 LRRs of 22 amino acids and InlB has eight of such repeats (Table I). The LRR region of InlA and part of its following regions and the LRRs of InlB are necessary and sufficient for the entry process [97, 101], suggesting that in both cases these repeats interact with the corresponding InlA or InlB receptors. InlA is a protein of 800 amino acids that contains a C-terminal cell wall-associating motif highly conserved in a number of surface proteins. This motif consists of an LPTTG pentapeptide followed by a stretch of 20 hydrophobic amino acids and a short tail of charged residues [102]. When this region is deleted [102] or absent in natural isolates [103], the InlA protein is completely released and the invasion capacity of the strain severely impaired. The LPXTG motif (X is any amino acid residue) is required for covalent anchoring of these proteins in the bacterial cell wall [104]. It has been proposed that this event occurs by a proteolytic cleavage between the threonine (T) and glycine residues (G) of the LPXTG motif (Fig. 3C) and a subsequent linkage between the carboxyl group of threonine and the free amino group of the pentaglycine cross-bridge within the
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16. LISTERIA MONOCYTOGENES
763
cell wall [105, 106] (Fig. 3D). The putative enzyme (named sortase) involved in this cleavage has been recently identified [283]. The InlB protein is 630 amino acids long, and its 232 C-terminal amino acids are required for surface association [107]. This region contains tandem repeats of 80 amino acids that begin with the dipeptide GW, and therefore are referred to as the GW modules. Similar repeats are found in the bacteriolysin Ami of L. monocytogenes, in lysostaphin of Staphylococcus simulans and in LytA, an amidase of Streptococcus pneumoniae. As is the case for lysostaphin [108], InlB, when exogenously added to an MB mutant is able to associate to the cell wall and to mediate invasion of host cells. Such an association of InlB to the cell wall also occurs with several Gram-positive but not with Gram-negative bacteria [107], suggesting that InlB is associated to a constituent specifically found in the cell wall of Gram-positive bacteria such as lipotechoic acid (Fig. 3E). Increasing the number of GW modules at the C terminus of InlB improves anchoring of the hybrid protein to the cell surface of L. monocytogenes [107], reinforcing the hypothesis that GW modules may constitute a novel motif for associating proteins to the cell wall in Gram-positive bacteria. While intemalin can be easily detected at the surface of L. monocytogenes, InlB is not detected by immunofluorescence [48, 107], indicating that InlB is partially embedded in the cell wall. As InlB is also found in culture supematants, interaction of the protein with the cell wall after release from the bacterial surface is possible. This loose association of InlB with the bacterial surface suggests that this soluble form of the protein could play a role during infection. b. The Intemalin and InlB Receptors. The mammalian receptor engaged by InlA in Caco-2 cells has been identified as E-cadherin [90], a key adhesion molecule in epithelial cells. E-cadherin is a transmembrane glycoprotein whose intracellular domain interacts via catenins with the actin cytoskeleton. It is involved in Ca^'^-dependent homophilic interactions that mediate cell-cell adhesion and maintain tissue integrity in differentiated epithelia. E-cadherin is also involved in heterophilic interactions with a^py integrins present in intraepithelial lymphocytes [109-111]. E-cadherin is present on basolateral surfaces of epithelial cells, in agreement with the observed preferential site of entry of L. monocytogenes from this side in Caco-2 cells [112]. A receptor for InlB has been recently identified [284]. It is gClqR, the receptor for the globular part of CIQ.
3.
OTHER BACTERIAL FACTORS INVOLVED IN ADHESION AND ENTRY
a-D-galactose present on the surface of some virulent strains of L. monocytogenes was reported to mediate attachment of virulent L. monocytogenes to hepatocarcinoma cell lines (HepG2) that have a well-characterized a-D-galactose receptor [113]. Binding was abolished by pretreatment of the cell line with the
764
HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
sugar or with neuraminidase that renders the galactose-receptor functionally inactive [113]. a-D-galactose is lacking in two nonvirulent strains, suggesting involvement of this carbohydrate in the adhesion of L. monocytogenes to hepatocytes. Attachment of L. monocytogenes to both macrophages and epithelial cell lines is inhibited by incubation of bacteria with heparan sulfate, a major glycosaminoglycan component of proteoglycans or by treating mammalian cells with heparinase [114]. These results suggested that binding of L monocytogenes to both professional phagocytes and epithelial cell lines occurs through recognition of the heparan sulfate proteoglycan (HSPG) receptor. Sequence analysis showed that the ActA protein possesses a heparan sulfate-binding domain, suggesting that this bacterial surface protein may play a role in this interaction [114]. Supematants of Listeria cultures contain large amounts of a protein of 60 kDa (p60). Mutants synthesizing reduced amounts of p60 are rough and form long cell chains with double septa between individual bacterial cells. These mutants display both a decreased adherence to and invasiveness into the 3T6 mouse fibroblast but not into Caco-2 cells [115, 116]. The gene coding for this major extracellular protein was named iap for invasion-associated protein. The gene product has a murein hydrolase activity [117]. Pretreatment of an iap mutant with purified p60 restores both colony morphology and Listeria virulence. How p60 is involved in the intracellular uptake of Listeria is not yet clear. The long cell chains of the rough iap mutant could impair its internalization, or p60 could exert an indirect effect on entry by affecting listerial proteins involved in this process. Interestingly, an attenuated recombinant Salmonella typhimurium strain expressing p60 has an increased invasion capacity for hepatocytes and macrophages in vitro, and p60 expression seems to promote transient bacterial growth in the liver and spleen in mice [118].
4.
HOST CELL SIGNALING DURING L. MONOCYTOGENES ENTRY
Evidence that L. monocytogenes exploits mammalian signal transduction pathways to promote its uptake was first provided by the use of inhibitory drugs. For instance, genistein, which competes with the binding of ATP to protein tyrosine kinases and therefore inhibits the activity of these enzymes, and cytochalasin D, which prevents actin polymerization, both inhibit entry but not adherence of Listeria in the epithelial cells Caco-2, HeLa, Henle-407, and Vero [51, 71, 119-121], indicating that tyrosine phosphorylation of host proteins and an intact actin cytoskeleton are needed for Listeria invasion. By using a combination of pathway-specific inhibitors, genetically inactivated cell lines, and bacterial mutants, identification of the mammalian pathways activated during infection as well as the listerial factors triggering these events is progressively emerging. As will be mentioned in a following section (IILB), proteins involved in either entry or later steps of infection, in particular listeriolysin implicated in escape from the
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fisteria monocytogenes
Cytopiasm
Fig. 4 Interaction of invasive L monocytogenes with host signaling pathways. The various host responses triggered on L. monocytogenes infection have been reported in different cell lines and, unless experimentally proven, should not be extrapolated to all cells. For details, see text (§111.A.4).
primary vacuole, trigger host-signaling pathways. Figure 4 depicts signals occurring during Listeria infection. a. Activation of Mammalian PI S-Kinase on Entry. Two inhibitors of the mammalian phosphatidylinositol 3-kinase (PI 3-K, p85/pllO), wortmannin and LY294002, reduce entry of L. monocytogenes in Caco-2, Vero, and HeLa cells [51]. In addition, transfection of CHO cells with a dominant negative form of PI3-K (Ap85a) severely impairs bacterial internalization, indicating that this enzyme is required for Listeria entry [51]. PI 3-K phosphorylates the D3 position of the inositol ring of phosphatidylinositol (PI), PI 4-phosphate (PI4P), and PI 4,5-biphosphate [PI(4,5)P2] to produce PI 3-phosphate (PI3P), PI(3,4)-biphosphate [PI(3,4)P2], and PI(3,4,5)-triphosphate [PI(3,4,5)P3]. These PI 3-K products are not substrates for any known phospholipases [122] and have been shown to be involved in growth factor-dependent mitogenesis, membrane ruffling, vesicle trafficking, and glucose uptake [123]. The cellular amounts of [PI(3,4)P2] and [PI(3,4,5)P3] increase in response to Listeria infection of Vero cells, indicating that Listeria entry stimulates PI 3-K activity [51]. Efficient stimulation of PI 3-K requires the InlB protein since infection of Vero cells with an inlB mutant led to strikingly reduced levels of the lipid kinase products [51]. Purified InlB alone is in fact able to stimulate activation of PI 3-K, and represents the first nonmammalian agonist of PI 3-K [124].
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Host cell tyrosine phosphorylation is required for stimulation of PI 3-K activity by L. monocytogenes since there is no increase in PI(3,4,5)P3 levels after pretreatment of Vero cells with genistein. Activation of PI 3-K in mammalian cells requires binding of its regulatory subunit p85 to tyrosine phosphorylated proteins [123, 125]. In the case of L. monocytogenes, coimmunoprecipitation experiments have led to identification of three adaptor proteins—Gabl, She, and c-Cbl—that are tyrosine phosphorylated on Listeria infection in Vero cells [124]. Whether a single complex of p85 with these three proteins is formed or whether multiple complexes coexist is unknown. It is also still not known which tyrosine kinase(s) is (are) involved in this pathway. How InlB-mediated stimulation of PI 3-K activity affects bacterial invasion is unknown. One of the PI 3-K products, PI(3,4)P2 has been shown to bind with high affinity the actin-binding protein profilin and the actin-severing protein gelsolin [126, 127] and to uncap barbed actin filaments in permeabilized platelets [128]. It is therefore tempting to speculate that PI 3-K controls the cytoskeletal rearrangements required for Listeria entry, but this possibility requires further investigation. Wortmannin and LY294002 inhibit entry of both L. monocytogenes and L. innocua expressing inlA in Caco-2 cells [51], suggesting that PI 3-K activity is also needed for efficient InlA-mediated entry. However, PI 3-K activity is constitutively elevated in these cells and no increase in the amount of its products is detected afitr Listeria infection [51]. Taken together, these data suggest that the InlA/E-cadherin pathway probably requires preactivated PI 3-K activity. PI 3-K mediates a variety of cellular responses by generating PI(3,4)P2 and PI(3,4,5)P3. These lipid products bind and regulate the activity of several proteins, including some isoforms of protein kinase C (PKC) and certain signaling proteins containing Src homology 2 (SH2) or pleckstrin homology (PH) domains [129]. The PH domain of the phospholipase Cy (PLCy) binds PI(3,4,5)P3 [130]. Recent data indicate that PLCy is activated by InlB [285], although not required for entry. b. Activation of MAP-Kinase Pathways. HeLa cell infection with L. monocytogenes results in tyrosine phosphorylation of the four isoforms of the mitogenactivated protein (MAP) kinase: ERK-1, ERK-2, JNK, and p38 [132]. Listeriolysin (LLO) has been identified as a bacterial factor that induces ERK-1 and ERK-2 phosphorylation in these epithelial cells [133, 134]. With nonhemolytic mutants, ERK-2 activation still occurs—although only at later (30 to 90 minutes) postinfection times—suggesting that bacterial factors other than listeriolysin may be involved in ERK-2 activation [132]. Infections with a double inlAB mutant allowed to eliminate the possibility that "late" ERK-2 activation could involve the InlA or InlB proteins [132]. The MAP kinase kinases MEK-1 and MEK-2 are the upstream activators that tyrosine-phosphorylate ERK-1 and ERK-2 in the MAP kinase pathway. PD98059, a highly specific inhibitor of MEK-1 activation, blocks Listeria invasion into HeLa cells [132], and MEK-1 is phosphorylated in
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response to Listeria infection of HeLa cells [134]. Taken together, these results indicate that Listeria invasion requires the MEK1/ERK2 signaling pathway (Fig. 4). MAP kinases are central in many host responses, including cytoskeletal reorganization [135]. Their role in Listeria infections is not clear yet. c. Induction of Phosphatidylinositol Metabolism. Infection of human umbilical vein endothelial cells (HUVECs) with L. monocytogenes triggers both release of lipid inflammatory mediators such as platelet-activating factor (PAF) and prostaglandin 12 (PGI2) and hydrolysis of phosphoinositides (Ptdlns) [136]. This effect is absent with a hemolysin-negative mutant. Purified LLO and an LLO-expressing L. innocua strain restore PAF and PGI2 production in HUVECs to a wild-type level, but only partially reproduce the hydrolysis of Pdtlns. LLO is thus the bacterial inducer for the PAF-PGI2 response, but another released bacterial factor is required for full Ptdlns response. Using both mutant strains and purified proteins, PI-PLC was identified as the key cooperative agent in LLO-induced Ptdin metabolism during signaling in HUVECs [137] (Fig. 4). d. Involvement ofpp60^'^^^ Family Protein Kinases. Among the cellular proteins that are tyrosine phosphorylated on entry of Listeria in Caco-2 cells, pp60^'^^^ substrates have been identified [138]. Moreover, treatment of Caco-2 cells with herbimycin A, a specific inhibitor of the pp60^"'^'^^ proteins, impairs Listeria entry, consistent with the idea that pp60^"'^'^^ is activated during Listeria invasion of epitheUal cells [138]. B. Escape from the Primary Vacuole After its internalization by the host cell, L. monocytogenes becomes trapped in a membrane-bound vacuole (or phagosome) that has an acidic pH (5.6) [139]. For its intracellular survival and growth. Listeria have to rapidly escape from this hostile compartment before its fusion with lysosomes. Lysis of this primary vacuole is largely mediated by listeriolysin O [68, 82, 140]; however, in some cell lines this process also involves phosphatidylinositol- (PI-) phospholipase C (PI-PLC) or broad-substrate phospholipase C (PC-PLC) [67, 83, 84]. Escaping from the phagosome allows L. monocytogenes to grow and replicate in the cytosol with a doubling time of 1 hour, as in broth medium [76, 141].
1.
LISTERIOLYSIN O
(LLO)
The hemolytic phenotype of L. monocytogenes is due to listeriolysin O (LLO), a member of the family of the sulfhydryl- (SH-) activated toxins, which includes streptolysin O of Streptococcus pyogenes, pneumolysin of Streptococcus pneu-
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moniae, and perfringolysin of Clostridium perfringens [142]. The mode of action of these structurally related toxins involves interaction with cholesterol in target cell membranes in which they oligomerize to form transmembrane pores (cholesterol sequestration), leading to cell lysis. In the case of L monocytogenes, cholesterol inhibits both the hemolytic and cytolytic activities of LLO, Thus, it was assumed to be the toxin receptor on the surface of the eukaryotic cell, since membranes lacking cholesterol are insensitive to the action of the cytolysin [143]. However, recent studies have shown that cholesterol interferes with oligomerization of LLO monomers and does not prevent binding of the cytolysin to target membranes [144]. LLO is a 58-kDa protein encoded by the hly gene that is located in the virulence gene cluster of L. monocytogenes (see Fig. 6). LLO and ivanolysin (the homolog of LLO in L ivanovii) are the only members of the SH-activated toxins produced by an intracellular bacteria. LLO activity is optimal at pH 5.5. The acidic pH encountered in the phagosome thus favors the cytolytic activity of LLO, which damages the phagosomal membrane, allowing release of the bacterium into the cytosol of infected cells. At pH 7.0 (pH of the cytosol), LLO is less active. This reduced activity at neutral pH may represent a protective mechanism to prevent deleterious effects on the cytoplasmic membrane of the host cell by LLO. Consistent with this hypothesis is the observation that a B. subtilis strain expressing perfringolysin is able to lyse the vacuolar membrane and also exerts membrane damage, an effect that is not observed with B. subtilis expressing LLO [145]. Hemolysin-negative mutants are avirulent in mice [59-61, 66, 146]. Definitive evidence supporting the role of LLO in Listeria pathogenicity was brought when introduction of a plasmid harboring the hly gene in a nonhemolytic Listeria strain restored both hemolytic capacity and virulence [147]. LLO has a critical role in mediating the escape of L. monocytogenes from the phagosome. LLO-negative mutants are defective in their ability to breach the phagosomal membrane and to multiply in the cytoplasm of the host cell [59, 61]. Expression of LLO in B. subtilis is sufficient to allow the bacillus to reach the cytosol of macrophage-like J774 cells [68], and an attenuated Salmonella dublin strain expressing a functionally active LLO was partially released into the cytoplasm of J774 cells, in contrast to a nonhemolytic strain that remained in the phagosome [148]. In some cell lines, LLO does not act alone in disrupting the phagosomal membrane. In the human epithelial cell line Henle 407, an hly mutant is still able to escape in the cytosol, whereas a mutant deficient in phosphatidylcholine-phospholipase C (PC-PLC) synthesis remains trapped within this primary vacuole [61, 83]. In bone marrow-derived macrophages, a mutant lacking the phosphatidylinositol-phospholipase C (PI-PLC) is defective in escape from the phagosome [67]. LLO, as stated above, activates MAP kinase pathways, stimulates Ptdlns metabolism (see §III.A.4), and also induces apoptosis in cultured dendritic cells [149].
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THE LISTERIAL PHOSPHOLIPASES
L. monocytogenes secretes two phospholipases of the C type (PLC): a phosphatidylinositol- (PI-) specific PLC and a phosphatidylcholine- (PC-) preferring PLC. PLCs are virulence factors in several bacterial pathogens, such as B. cereus, S. aureus, Clostridium perfringens, and Pseudomonas aeruginosa. These enzymes cleave phospholipids between the glycerol moiety and the phosphate group, and generally lead to alterations in cell membrane composition and function [150, 151]. The two distinct phospholipases C of L. monocytogenes, PI-PLC and PC-PLC, have overlapping roles in escape from the primary vacuole and cell-to-cell spread [84, 87] (see §III.C.4). a. The Phosphatidylinositol Phospholipase C (PI-PLC). PI-PLC is a 33kDa extracellular protein encoded by tht plcA gene [152-154] (Fig. 6). This gene is found in the pathogenic Listeria species L. monocytogenes and L. ivanovii and in the nonvirulent species L. seeligeri [155, 156]. PI-PLC is more basic than its structurally related counterparts from B. cereus and B. thuringiensis [157]. The crystal structure of PI-PLC has been determined and is highly homologous to that of the PI-PLC ofB. cereus [158]. A. picA mutant is twofold less virulent in mice than the wild-type strain, and its growth in vivo is impaired in the liver but, curiously, not in the spleen of the infected animals [67]. Electron-microscopic and immunofluorescence analysis showed that in bone marrow murine macrophages the plcA mutant is deficient in escape from the primary phagocytic vacuole [67, 87]. Thus, PI-PLC is required for efficient lysis of the phagosomal membrane in bone marrow-derived macrophages. This deficiency is enhanced in the case of a double mutant that lacks both PI-PLC and PC-PLC, suggesting overlapping roles for the phospholipases in escape from the primary vacuole in macrophages [87]. That PI-PLC specifically cleaves PI and/or glycosyl-phosphatidylinositol (GPI) anchors found in many eukaryotic membrane proteins [152, 157] is in agreement with its role in lysis of host phagosomal membranes. Furthermore, PI-PLC is as LLO functionally active at acidic pH [157], probably consistent with an action in concert with LLO in providing the bacteria access to the cytosol. Interestingly, PI-PLC may also play a role in intracellular growth, since expression of the picA gene in L. innocua renders this noninvasive bacteria capable of surviving and multiplying inside the phagosomes of host macrophage cells [159]. b. The Phosphatidylcholine-Phospholipase C (PC-PLC). The second listerial phospholipase C, PC-PLC, is active on a broad-range of phospholipids including phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyeHn [160, 161]. PC-PLC, however, shows a higher specificity for phosphatidylcholine, also called lecithin. PC-PLC is therefore referred to as lecithinase, and its activity can be easily detected by the formation of zones of
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Opacity on egg yolk agar. Encoded by gene plcB, PC-PLC is secreted as an inactive precursor of 33 kDa that is processed, in broth culture, to an active mature form (29 kDa) by a zinc-dependent metalloprotease (Mpl) [162, 163] (Table I; see §III.D.l). Zinc-dependent phospholipases homologous to L. monocytogenes PC-PLC include phosphatidylcholine-phospholipase C from B. cereus and atoxin from C. perfringens [164]. As outlined above, an hly mutant is capable of escape from the primary vacuole in Listeria-initcitd. human Henle 407 epithelial cells, strongly suggesting that bacterial proteins other than LLO are needed to lyse the primary vacuole. A double mutant lacking both LLO and PC-PLC is no longer capable of escaping from the vacuole, indicating that PC-PLC is able, in the absence of LLO, to mediate escape from the primary vacuole in epithelial cells [83].
C. Intra- and Intercellular Spreading
1.
INTRODUCTION
The role of actin filaments in mediating the intracellular movement of L. monocytogenes and its cell-to-cell spread was described 10 years ago [56, 165, 166] (Figs. 1, 2, and 5A; see color plate), and the actA gene has been identified as the primary (and probably the only) bacterial factor required for actin-based motility [167, 168]. actA mutants are able to enter and grow in the cytosol of eukaryotic cells but do not polymerize host actin and consequently are unable to move or spread from cell to cell. They form microcolonies in the vicinity of the host cell nucleus [75,167-169]. ActA is a major virulence determinant since ActA mutants are three orders of magnitude less virulent than wild-type bacteria in a mouse model of infection [169]. Actin filament assembly used for propulsion is not a unique feature of L. monocytogenes. It is also observed in other pathogenic organisms such as L. ivanovii [170] or the unrelated bacteria S.flexneri [171] (see Chapter 8 herein), Rickettsia spp. [172], and the Vaccinia virus [173].
2.
ACTIN POLYMERIZATION AND MOTILITY
The events underlying intracellular Listeria motility can be compared to the processes occurring at the leading edge of moving cells, where actin polymerization is driving the formation of protrusive structures such as filopodia and lamellipodia. Temporally and spatially organized actin assembly/disassembly plays an important active role in many cellular processes, including morphogenesis, organelle transport, and cell motility. Crawling cell motility, coupled with
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continuous actin polymerization, is found in many cell types, including amoebae and neutrophils [174-177]. Actin is an abundant globular 43-kDa nucleotide-binding (ATP or ADP) protein (G-actin) found in all eukaryotic cells. Intracellular concentrations in nonmuscle cells range from 10 to 200 |LiM. The actin monomer can spontaneously polymerize under certain conditions, forming filaments of F-actin [178, 179]. Polymerization in vitro starts with the formation of thermodynamically unstable dimers or trimers (nucleation), which can then rapidly grow into filaments by monomer addition. Hence, the nucleation step is rate limiting during actin polymerization. Actin filaments have a helical shape and display a structural and functional polarity with a fast growing (barbed or plus) end, at which subunits associate rapidly and a pointed (minus) end that has much slower dynamics. The critical concentration for incorporation of ATP-G-actin at the barbed end (filament growth) is lower than the critical concentration at the pointed end and ATP hydrolysis throughout the filament accounts for this nonequilibrium situation [179-181]. In living cells, actin polymerization is controlled by numerous actin-binding proteins [174, 182, 183]. The function of some of these proteins is depicted in Figure 5B and will be discussed in the light of actin polymerization by L. monocytogenes later in this section (III.C.3.c). 3.
THE LISTERIA ACTIN-BASED MOTILITY
a. Filament Organization in the Tails and Movement. Early electron microscopy studies of thin sections of L/^r^na-infected macrophages showed that free bacteria in the cytosol are surrounded with a coat of filamentous actin. This actin cloud is then rearranged into the actin tail [56, 165]. F-actin is always absent from newly formed poles of dividing bacteria, so that rearrangement of F-actin around the bacteria seems to be linked to bacterial division. Labeling techniques with myosin S1 were used to define the polarity of the actin filaments around the bacteria and indicated that the barbed ends are all oriented toward the bacteria [184-187]. These studies also showed that filaments in the tails are short in length (maximum 0.3 |Lim) and that a higher density of more parallel oriented filaments exists in the shell of the tail, while a lower density of more randomly oriented filaments prevails in its core. At later stages of the infection, bacteria may be found in membrane protrusions that can be up to 50 |im in length. These protrusions can be phagocytosed by a neighboring cell or become reabsorbed by the mother cell. The actin filament organization in these pseudopods-like structures is quite different from what had been observed for cytoplasmic tails [188]. In the L/^r^na-containing protrusions, two populations of actin filaments exist: long axial ones (up to 2 |Lim) and short radial ones.
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It was suggested early on that the actin filaments in comet tails are crosslinked by specific proteins, thus providing an inert platform from which bacterial propulsion could be driven [166]. Crosslinking and bundling proteins such as a-actinin or fimbrin/plastin have been found to localize with the comet tails [166, 189-191]. Interestingly, a-actinin is absent from the comet tails found in protrusions, and it has been speculated that the membrane of the protrusions may confer the necessary rigidity to the tail, whereas this function would essentially be performed by crosslinking filaments with a-actinin in the cytoplasmic tails [188]. Support for such a functional mechanism of a-actinin is provided by the fact that microinjection into infected cells of a proteolytic fragment of a-actinin, which has a dominant-negative effect, leads to arrest of bacterial movement and disappearance of the actin tails [189, 190]. Bacterial movement is dependent on continuous actin polymerization because cytochalasin D inhibits the spread of L. monocytogenes in cell monolayers and stops moving bacteria in infected cells [165, 166]. Actin monomers are incorporated at the interface of the tails and the surface of one pole of the bacterium, resulting in the bacterium moving away from a stationary tail in the cytoplasm [166, 185, 192, 193]. The rate of bacterial movement is equal to the rate of actin polymerization, and, as the depolymerization rate of filaments seems to be constant, tail length is linearly correlated to bacterial speed [192]. Measured speeds depend on the cell type analyzed (up to 1.5 |Lim/s~*), and tails can reach a length of about 30 |im [166]. Bacteria often move along circular tracks, and moving bacteria always have a tail. Tails are never associated with stationary bacteria (Fig. 5A; see color plate). Force generation necessary for bacterial propulsion is thought to be provided by the continuous actin polymerization itself at one bacterial pole. The underlying biophysical model, the "Elastic Brownian Ratchet" model, is now widely accepted [194]. b. The ActA Protein. The actA gene encodes a 639-aa protein that is surface exposed and anchored to the bacterial membrane by a C-terminal hydrophobic region [167] (Figs. 5C and 3B). ActA is distributed in a polar fashion around the bacteria, with higher accumulation at one pole and actin accumulation around the bacteria that follows the same asymmetrical distribution as ActA [195]. Actin tails therefore always emerge from the pole of high ActA concentration; therefore, ActA distribution predetermines the site of tail formation and the direction of movement. Whereas it seems clear that ActA is absent from the newly formed poles of dividing bacteria and that ActA asymmetry can be generated by cell division [85], asymmetrical distribution of ActA in nondividing bacteria is a matter of debate [85, 187, 195, 196]. In any case, the protein is not released into the tails, confirming tight anchoring to the bacterial membrane [195, 196]. The ActA protein alone does not interact with actin or stimulate assembly of purified G-actin [168, 197], and whole bacteria only poorly nucleate actin
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filaments from purified G-actin [186]. However, several lines of evidence suggest that, in the context of the eukaryotic cytoplasm, ActA is sufficient to drive actin assembly. First, when actA is transfected into mammalian cells, ActA is targeted to the mitochondria by its C-terminal region, and actin polymerization is observed around these organelles [198, 199]. Second, when ActA fused through its C terminus to a CAAX box derived from the K-ras protein is expressed in mammalian cells, the hybrid protein localizes to the inner leaflet of the plasma membrane. At this localization, it induces the formation of actin-rich cell-surface projections by triggering localized actin polymerization [200]. Two lines of evidence clearly suggested that, aside from continuous actin assembly, ActA is also sufficient for bacterial movement. First, expression of ActA in L. innocua confers to this nonmotile bacterium the capacity of actin-based motility in Xenopus egg extracts [72]. Second, when a hybrid ActA protein is tethered via a LytA fusion to the cell wall of the unrelated bacterial species S. pneumoniae, these bacteria can polymerize actin, form the typical actin comet tails, and move in Xenopus extracts [201]. However, movement is only achieved when the bacteria have divided and hence ActA localization becomes polarized. These experiments convincingly pointed out the capacities of ActA to efficiently interact with host factors involved in actin assembly and suggested that ActA could possess mammalian counterparts important for actin polymerization. Mutational analysis defined three distinct functional regions of the ActA protein: an N-terminal domain (amino acids 1-234, ActA.N), a central proUnerich repeat region (amino acids 235-395, ActA.P), and a C-terminal region (amino acids 396-610, ActA.C) [86, 199, 202, 203]. The C-terminal region anchors ActA into the bacterial membrane (Fig. 3B), where the protein probably exists in dimeric form [204], and ActA variants lacking the C-terminal domain are secreted in the medium. The two other regions are both involved in promotion of actin assembly (Fig. 5C). The N terminus of ActA is absolutely necessary for the bacterium to induce actin assembly on the surface. As shown with a Listeria mutant expressing an ActA.N-coLacZ fusion protein, it is solely sufficient for comet tail formation and movement although with reduced speeds [199, 203, 205]. The ActA.N region facilitates actin nucleation in concert with the host cell Arp2/3 complex [197] (see below). Further analysis of the N-terminal domain defined two different subregions: region T (amino acids 117-121, ^'^KKRRK^^O. also termed the nucleation region, and region C (amino acids 21-97). Region T was shown to be responsible for either overall actin nucleation [199] or actin comet tail formation [205], indicating that this region plays a crucial role in actin assembly. Deletion of region C leads to an interesting phenotype where bacteria are moving with periodic oscillatory changes in rate, leaving discontinuous actin tails behind [205]. The exact molecular mechanisms underlying this phenomenon have yet to be elucidated. Although purified ActA seems not to interact with actin, synthetic peptides
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with sequences derived from the N terminus (P33_74 and P52-74) do interfere with actin polymerization and bind to F-actin [205], raising the interesting possibiUty that ActA might actually possess cryptic actin-binding sites. The central proline-rich domain of ActA is not sufficient to trigger actin polymerization but is necessary to stimulate the rate of movement [199, 202,203]. The ActA.P region is composed of four proline-rich repeats (PRR; consensus sequence D/EFPPPPPTDEEL) interspaced by three so-called long repeats. Deletions in the ActA.P region slow the rate of bacterial motility, and each proline repeat stretch contributes with about 1-2 |im/min to the movement [202]. The long repeats have been inferred to stimulate transition from the initially polymerized actin cloud to the tail structures [202]. It has been suggested that the intracellularly observed phosphorylation of the ActA.P region could be important for polarization of ActA in vivo [85,169]. Consistent with the idea that the ActA.P region is not absolutely required for actin polymerization but stimulates movement rates is the finding that deletion mutants of this region display a weakly reduced virulence in mice [202, 206]. The proline-rich region is the binding site for the two ActA host cell ligands thus far identified; VASP, the vasodilator-stimulated phosphoprotein, and Mena, mammalian-enabled homolog, which belong to the EnaA^ASP family of proteins [207, 208] (see §3.c). In vitro, VASP interacts with purified ActA and peptides corresponding to any of the four PRRs, suggesting that ActA might have four functional binding sites for this tetrameric protein [206, 207]. In infected cells, VASPs (and Mena) colocalize to the interface of the bacterial surface and the actin tails, but the number of VASP molecules that actually bind to ActA in vivo is unknown. ActA displays little overall sequence homologies to eukaryotic proteins. However, discrete regions of ActA share sequence homologies with cellular proteins involved in cytoskeleton organization. Sequence alignments revealed significant homology between the proline-rich repeats and a proline-rich region in human vinculin. In addition, both ActA.P and ActA.C regions display an overall sequence identity of 22% with the proline-rich N-terminal domain of human zyxin [86, 167, 168]. Moreover, the proline-rich repeats of ActA and those of zyxin and vinculin have been classified as specific binding partners for the so-called EVHl (Ena/VASP homology) domain of EnaA^ASP family members [206]. The proUnerich regions of ActA and zyxin can exert similar functions in eukaryotic cells, but so far it has not been shown that the proline-rich domain of zyxin can efficiently replace that of ActA for bacterial movement [209]. Another interesting sequence homology is the overall 25% identity found between the N-terminal domain of ActA and the C-terminal region of vinculin (amino acids 879-1066) [86]. Interestingly, this region of vinculin contains a cryptic actin-binding site, unmasked by the interaction of the protein with PIP2 [210]. Vinculin and zyxin are proteins found in actin-enriched structures such as adhesion plaques and the
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leading edge, and both proteins are able to bind proteins of the EnaA^ASP family [211-215]. It will be of interest to establish how far functional relationships between ActA and presumed eukaryotic counterparts like zyxin, vinculin, or yet undiscovered factors really exist [209, 216]. c. Cellular Factors Important for Actin-Based Motility. ActA induces actin rearrangements in cells but does not directly interact with actin, strongly suggesting that host components associate with ActA to control polymerization of actin. Many cytoskeletal proteins have been reported to be associated either with the bacteria, the comet tails, or both in infected cells. In most cases, these proteins have been identified by means of immunofluorescence colocalization studies using antibodies to known cytoskeletal components. It has to be stressed that some proteins that get recruited to the comet tails of Listeria will do so because of simple mass action due to high local F-actin concentrations, and thus they might not play an active role in the process. Cellular proteins found associated to moving bacteria include proteins with many diverse functions. They are listed in Table II, and only proteins for which function has been analyzed are discussed below. The role of a-actinin in crosslinking actin filaments in the tail has already been mentioned. Other crosslinking or bundling proteins will probably have similar functions in providing Listeria tails with the required rigidity and inertia for propulsion. Evidence for functional implication of ADF/cofilin in the movement of L. monocytogenes has in addition permitted light to be shed on the general mechanisms of action of this group of small (15-22-kDa) actin-binding proteins [217, 218]. ADF/cofilin proteins display preferred binding affinities for F- and G-ADP-actin, and increase turnover rates of actin filaments by mainly enhancing depolymerization rates at the pointed ends. As a consequence, when purified ADF/cofilin is added to diluted platelet extracts, enhanced speeds for Listeria movement and reduced tail length are observed [179]. When ADF/cofilin is depleted from Xenopus egg extracts. Listeria actin tails tend to become much longer than what is usually observed. However, rates of bacterial movement are not reduced, probably due to incomplete depletion of extracts [218]. These data suggested that ADF/cofilin is implicated in depolymerization of filaments in the Listeria comet tails, thus adding to the dynamics of bacterial movement. The presence of capping proteins associated with Listeria tails has been demonstrated [79, 219]. Most capping proteins bind to the barbed ends of actin filaments and thus prevent further elongation of the growing filament [179, 183]. The barbed end capping protein capZ (also called capping protein) is detected throughout the tails [79]. Thermodynamic studies of Listeria propulsion in Xenopus egg extracts suggested that the movement could rely on maintenance of uncapped barbed ends at the bacterial surface [220]. Therefore, the discontinuous comets of the ActA A21-97 mutant have been associated with a defect in
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Table II
HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
Cytoskeletal components associated with actin polymerizing intracellular Listeria Localization
Function within cell
a-actinin
cytoplasmic tails
F-actin crosslinking
[1-5]
TVopomyosin
tail
F-actin pointed end binding
[1]
Fimbrin
tail
F-actin bundling
[6]
Vinculin
tail
component of focal adhesions
[2]
Talin
tail
actin nucleation
[2]
Villin
tail
barbed end capping
[7]
Ezrin
tails in protrusions
cortical actin organization
Profilin
bacterial pole
G-actin binding, nucleotide exchange, actin polymerization
[8]
VASP
bacterial pole
component of focal adhesions
[9]
Mena
bacterial pole
component of focal adhesions
[10]
Arp2/3 complex
tail
actin nucleation
Cofilin
tail
F-actin depolymerization
Gelsolin
bacterial pole
barbed end capping, F-actin severing
[16]
Capping protein
tails
barbed end capping
[14]
Rac
7
small GTPase
[14]
Coronin
tails
actin organization during phagocytosis
[14]
Protein
Interaction with ActA
References
[5,7]
likely
[11,12] [13-15]
efficiently protecting barbed ends at the bacterial surface from capping [205]. However, no depletion experiments of capZ have yet addressed its role in Listeria movement, and further experiments are needed to establish whether ActA is able to regulate the activities of capping proteins or to actively maintain barbed ends uncapped. The function of the capping and severing protein gelsolin in Listeria movement remains elusive. Gelsolin is located at the interface of the tails and the bacterial surface [219]. In cells that overexpress gelsolin, Listeria movement has
16.
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higher rates than in cells expressing normal levels of this protein. On the other hand, in cells derived from gelsolin knockout mice and consequently devoid of this protein, Listeria are able to move at the same speeds than in control cells. Similarly, Listeria movement is also unimpaired in Xenopus egg extracts that had been depleted in gelsolin by immunoprecipitation [218]. Therefore, the effects exerted by gelsolin may be subtle. Increased speeds observed at higher concentrations of this F-actin-severing protein could, for example, simply reflect less viscous environments that would facilitate movement of the bacterium. The Arp2/3 complex has been isolated by fractionation of human platelet extracts as a host factor that induces actin assembly on the surface of Listeria in an ActA-dependent manner [78]. The Arp2/3 complex contains seven proteins, two of which are actin-related proteins Arp2 and Arp3, and the other subunits have been termed Arcs (Arp complex). The complex is highly conserved from Acanthamoeba to humans [221-224] and was suspected early on of playing a central role in actin polymerization in vivo, as suggested by its preferred localization to the leading edge of motile cells [221-223]. In L. monocytogenesinfected cells, Arp3 as well as several Arcs localize to the surface of stationary bacteria and to the tails of moving bacteria again [78, 221]. The presence of the two actin-related proteins in the Arp2/3 complex sustained the idea that the complex could stimulate actin polymerization by acting as a nucleus or by providing free barbed ends [223]. However, the purified Arp2/3 complex only weakly nucleates actin filaments in vitro [197, 225], raising the possibility that efficient activity of this complex could be regulated by additional cofactors. The L. monocytogenes ActA protein was shown to be such a cofactor, as Act A amplifies dramatically the Arp2/3-dependent actin nucleation activity in vitro [197]. The N-terminal part of the Act A protein, which, as discussed above, is sufficient for actin polymerization at the surface of the bacteria, is also sufficient for this stimulating effect [197, 205]. A model for the nucleation activity of the Arp2/3 complex suggests that activation occurs through a switch from a mechanism dependent on actin dimers to one dependent on the binding of actin monomers [226]. The Arp2/3 complex binds with high affinity to the pointed ends of F-actin filaments and thus leaves the created barbed end free for elongation [225]. This can help explain why the Arp2/3 complex is found throughout the actin comet tail of moving Listeria and not only at the pole where active actin assembly is expressed. Short actin filaments nucleated by the Arp2/3 complex in the vicinity of ActA could be released with an Arp2/3 cap on their pointed end, allowing the Arp2/3 complex to persist in the older parts of the tail. Meanwhile, ActA-dependent actin polymerization progresses near the bacterial surface, with the barbed ends ideally positioned toward the membrane. Aside from its nucleation and pointed-end capping properties, the Arp2/3 complex has also been proposed to play a role in actin network organization [225, 227, 228]. Whether the Arp2/3
778
HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
complex also influences actin filament organization in the Listeria comet tails remains an open question. In cells, EnaA^ASP family proteins are often associated with focal adhesions, microfilaments, and membrane regions of high dynamic activity, and Mena is able to trigger actin assembly when expressed in mammalian cells [208, 211]. VASP and Mena localize to the bacterial surface/actin tail interface in moving Listeria [207, 208]. It is therefore reasonable to hypothesize that EnaAASP proteins play an important role in actin assembly induced by Listeria. However, experiments that direcdy show the implication of these proteins for optimal actin polymerization by Listeria (e.g., in cells or extracts lacking EnaAASP proteins) have not yet been reported. The current theory of how EnaA^ASP proteins contribute to enhance actin polymerization at the bacterial surface is basically derived from their capacity to bind to the actin monomer-binding protein profilin [211, 229, 230]. Immunofluorescence colocalization studies showed that in infected cells profilin localizes like VASP and Mena to the bacterial pole, where active actin assembly is harnessed into the tail structure [77, 202, 207, 208]. However, profilin does not bind direcdy to ActA or bacteria that express ActA [77]. It has therefore been suggested that the role of VASP and Mena is to recruit actin-profilin complexes at the bacterial surface that serve to subsequently deliver actin to the growing ends of the actin filaments [77,207,229,231]. Microinjection in Listeria-mftcitd cells with peptides corresponding to the consensus proline repeat of ActA or to the profilin-binding site of VASP causes a rapid arrest of the bacterial movement and leads to depolymerization of tails [229, 232]. These experiments sustain the current VASP-profilin-actin-binding model, but this model does not explain the dramatic effects of the injected peptides seen on the whole comet tails. The role of profilin has also been assessed by depletion experiments in Xenopus tgg extracts [77, 220]. The results of both studies are surprisingly different: in one case, extracts depleted in profilin no longer support bacterial motility, whereas in the other case bacteria move at normal speeds. The differences observed can be partially explained by different experimental setups, but these results suggest that profilin may not be absolutely required for Listeria movement. Interestingly, the Acanthamoeba Arp2/3 complex that had first been isolated by the use of profilin-affinity columns [224] has been shown to bind to profilin [233]. Thus, profilin binding to VASP or Mena at the bacterial surface could in a way interfere with the nucleation activity of the Arp2/3 complex [216]. d. Modelfor Actin-Based Motility. In wild-type bacteria, the apparendy continuous movement could possibly rely at the molecular level on the following three steps: 1. Step 1: generation of free barbed ends at the bacterial surface by either nucleation or severing/uncapping of preexisting fila-
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779
ments. There is evidence [197] that makes it reasonable to hypothesize that control (increase) of Arp2/3 nucleation activity by the ActA.N-terminal domain is a key player in this step. To what extent severing and/or uncapping contributes to generation of free barbed ends is currendy not known. 2. Step 2: monomer addition and movement. This step may be controlled by VASP or Mena binding to the proline-rich repeats. These proteins could in turn bind profilin-actin complexes and supply high actin concentrations at one bacterial pole. 3. Step 3: continuous Hlament release/capping/crosslinking (filament loss into the tails). The factors that more specifically rule and/or counterbalance filament release from the polymerization apparatus are not well understood. Studies of actin filament length and arrangement in Listeria tails have suggested that geometric elements could influence filament loss [188]. The authors defined a half-circled polymerization zone at the rear of the bacterium in which actin filaments are generated and elongate continuously. Filaments that due to bacterial movement become at one moment tangential to this polymerization zone are released and become capped at their barbed ends. Hence, filament length will depend on both the location and orientation at which they were generated at the rear surface of the bacterium. A model summarizing the above-mentioned steps is depicted in Figure 5D. An exact balance between generation of free barbed ends (step 1) and filament loss (step 3) is most likely necessary for continuity of the movement [205] and should allow for elongation of a critical number of actin filaments. Any imbalance between barbed-end generation and filament loss would result in a moving phenotype that is different from the wild-type one, with respect to both velocity and tail shape. The discontinuous moving phenotype of the ActA A21-97 mutant could reflect such an imbalance.
4.
LYSIS OF THE DOUBLE-MEMBRANED VACUOLE
Polarized actin tails propel L. monocytogenes toward the cytoplasmic membrane, where filopodium-like structures envelope the bacteria before being engulfed by neighboring cells (Figs. 1 and 2). This process results in the formation of double-membraned vacuoles from which the bacteria rapidly free themselves by the cooperative action of the two listerial phospholipases PI-PLC and PC-PLC encoded by the plcA and plcB genes, respectively (described in §III.B.2; see Table I). Compared to a wild-type strain, a plcB mutant is 20-fold less virulent in mice and forms smaller plaques on fibroblast cells, suggesting a partial defect in
780
HAFIDA FSIHI, PIERRE STEEPEN, AND PASCALE COSSART
cell-to-cell spread and/or intracellular growth [87, 234]. Transmission electron microscopic analysis of thin sections of infected cells revealed that the plcB mutant accumulates in the double-membraned vacuoles, therefore indicating that PC-PLC is required for gaining access to the cytosol of newly infected cells [164]. As the defect in cell-to-cell spread was partial, a search for additional bacterial factors that contribute to this process has been performed and led to the finding that a double mutant lacking both phospholipases (plcAB mutant) exhibits a greater reduction in plaque size, indicating an additive effect of PI-PLC on cell-to-cell spread [87]. Thus, both PI-PLC and PC-PLC promote cell-to-cell spread in Listeria intracellular infection. PlcB-mediated cell-to-cell spread has been shown to be of critical importance in establishment of L. monocytogenes meningoencephalitis in mice. Mice infected intracerebrally with a plcB mutant survive longer, and have a reduced number of plcB bacteria in their brain compared to mice infected with an inlAB double mutant or a wild-type strain. Histopathological analysis has shown delayed intracerebral spread of the plcB mutant, emphasizing the role of PC-PLC in L. monocytogenes virulence [235].
D. Other Virulence Determinants of L, monocytogenes
1.
MPL
Mpl is a 57-kDa zinc-containing metalloprotease. It is encoded by mpl, the first gene of the polycistronic lecithinase operon (Fig. 6). This enzyme exhibits sequence homologies to other metalloproteases of the "thermolysin family" from Gram-positive bacteria and to related proteins from Gram-negative organisms such as the major secreted protein (MSP) of Legionella pneumophila, the elastase of Pseudomonas aeruginosa and a protease from Serratia spp. [236-238]. Transposon insertions in mpl cause a decrease in virulence of L. monocytogenes but the contribution of Mpl to virulence is probably indirect, mpl mutants not only fail to express the metalloprotease but also fail to produce a mature PC-PLC. Processing of PC-PLC seems to be a major function of Mpl [162, 163], which is also able to process ActA [239].
2.
CLPC
The L. monocytogenes clpC gene is the last gene in an operon of four genes [240]. It encodes an ATPase (ClpC) that belongs to the Hsp-lOO/Clp family, a group of highly conserved proteins implicated in the stress tolerance of many
16.
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781
prokaryotic and eukaryotic organisms [241, 242]. Growth of clpC mutants is highly susceptible to several stresses, including oxidative stress, high osmolarity, iron deprivation, and elevated temperature. The virulence of a ClpC-deficient mutant is severely impaired in the murine model. However, this attenuation in virulence is observed at late times during infection (72 hr) when growth of the mutant becomes strongly affected in the spleen and the liver. During the first 2 4 ^ 8 hours, growth in these organs is similar to that of a wild-type strain, indicating that the initial step of intracellular invasion and replication is not affected in this mutant [240]. In bone-marrow-derived macrophages, the clpC mutant remains confined to membrane-bound phagosomes, and only few bacteria can gain access to the cytoplasm after 4-hour incubation, suggesting a role for the ClpC ATPase in promoting early escape from the phagosome of macrophages [243]. This is consistent with the attenuated virulence observed in vivo, since the clpC mutant might be more rapidly killed in the phagosomal compartment of macrophages.
3.
OTHER MEMBERS OF THE INTERNALIN FAMILY
In addition to InlA and InlB, other members of the intemalin family were identified either by hybridization experiments using part of the inlA gene as a probe (InlC2, InlD, InlE, and InlF) [94] or during a search for new Prf A-regulated genes (InlC also called IrpA for intemalin-related protein A) [244, 245]. With the exception of InlC, the role in infection of these proteins is at present unclear. InlC-deficient mutants are still able to invade several cell lines in vitro but exhibit a 50-fold reduced virulence in mice, suggesting that InlC may play a role in dissemination of infection rather than uptake of L. monocytogenes by nonprofessional phagocytes [245]. The genes for InlC2, InlD, and InlE are clustered on the L. monocytogenes chromosome [94]. Analysis of the organization of this cluster in a different isolate of L. monocytogenes has led to identification of intemalin genes that highlight genetic rearrangements in this locus [246]. Indeed, the new cluster contains three genes—m/G, inlH, and inlE—that are flanked by the same housekeeping genes as the inlC2-D-E cluster. Whereas the inlE gene is identical in both isolates, sequence comparison indicates that the inlG gene is a completely new intemalin gene and that inlH is a hybrid gene between the 5'-part of the inlC2 and the 3'-part of inlD genes. Surprisingly, whereas deletion of the inlCl-D-F cluster in a bacterial mutant does not affect vimlence in mice when injected intravenously [94], in-frame deletion of the entire inlG-H-E locus leads to decreased vimlence in orally infected mice [246].
782
HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
IV. Regulation of L monocytogenes Virulence Gene Expression Expression of virulence genes in L monocytogenes is controlled by different cues, including growth phase, temperature, and medium composition. Many of these effects are mediated by a pleiotropic regulatory protein called PrfA. A. PrfA, a Pleiotropic Activator of Virulence Gene Expression The existence of a regulatory protein controlling virulence gene expression in L monocytogenes was suspected after two main observations. First, analysis of the promoter regions of hly and of the open reading frames (ORFs) located upstream and downstream from hly revealed common structural features suggesting common regulation [247]. Second, several nonhemolytic strains of L. monocytogenes, including the type strain, were lacking at least another putative virulence factor, the lecithinase, or were affected in invasion [248, 249]. These strains harbored a deletion in a region located upstream from hly, and hemolytic activity could be successfully restored when transformed with a plasmid carrying the corresponding wild-type region. These data, together with sequencing information, indicated that this region contained an ORF encoding a protein able to activate transcription of not only hly but also that of the operons located upstream and downstream from hly [250-252]. This protein was called Prf A (for pleiotropic regulatory factor). Strains carrying mutations in PrfA were strongly impaired for virulence in the murine model. Identification of prfA occurred somewhat concomitandy with the completion of determination of the nucleotide sequence of the now so-called virulence locus [152, 155, 164, 236]. This 10-kb locus contains prfA and genes specifically involved in virulence [156] (Fig. 6). As previously mentioned, it comprises the bicistronic plcA-prfA operon, the hly gene, and the polycistronic mpl-actA-plcB operon. These three transcriptional units of the virulence gene cluster are tightly controlled by PrfA at the transcriptional level [250, 251]. In addition to genes located in the virulence gene cluster, PrfA controls expression of the invasion locus inlAB [49, 93]. However, the PrfA dependence of inlAB expression is less stringent than for genes in the virulence locus (see §IV.C). In addition, PrfA controls expression of inlC [244, 245]. Finally, PrfA seems to partially repress expression of both clpC [253] and that of motAB, an operon involved in flagellar motility [254]. B. PrfA, a Protein of the CAP/FNR Family PrfA is a protein of 237 amino acids that displays homologies with proteins of the CAP/FNR family of transcriptional activators of E. coli and other Gram-negative bacteria [255, 256] (Table I). CAP (for catabolite gene activator protein) is the pleiotropic activator of catabolite genes in E. coli. CAP is inactive in the
16.
LISTERIA MONOCYTOGENES
pIcA
783
My
mpl
PI-PLC^^UsteriolysinO
_,. Metalloprotease
actA ^
ActA
pIcB Lecithinase
PrfA /fiM Internalin (InIA)
inlB
1 kb
InIB
Fig. 6 Schematic representation of the chromosomal organization of most L monocytogenes virulence genes and their transcriptional activator PrfA.
absence of cAMP and becomes active after binding this cyclic nucleotide, which induces an allosteric change. FNR is a transcriptional regulator very similar to CAP and activates genes such as those encoding fumarate and nitrate reductases during growth in anaerobiosis. Its cofactor is not cAMP but iron. The overall similarity between CAP of E. coli and PrfA is low (-20%), explaining why the homologies were not immediately detected when PrfA was discovered [250, 251, 255, 256]. Structural and genetic studies have demonstrated that the helix-tumhelix (HTH) motif of CAP directly interacts with its DNA targets. Point mutations affecting the putative HTH motif of PrfA strongly affected the capacity of PrfA to activate virulence genes and led to an attenuated phenotype. These data supported the prediction that the C-terminal part of PrfA might display an HTH motif and that PrfA may be functionally homologous to CAP/CRP [256]. Natural isolates of L. monocytogenes expressing elevated levels of virulence factors have been shown to carry a mutation in PrfA changing Glyl45 to Ser [257]. Amino-acid exchanges in the corresponding region of CAP result in constitutively active CAP proteins, that is, CAP proteins that are active in the absence of cAMP, reinforcing the idea that functional similarities between CAP and PrfA exist. Like CAP, PrfA is also a dimeric protein and binds to its DNA target sequences in dimeric form [258] (A. Renzoni, S. Dramsi, A. M. Gilles, and P. Cossart, unpubHshed results).
C. PrfA Boxes PrfA DNA-binding sites or "PrfA boxes" were first identified upstream from the -35 region of the hly promoter and upstream from the -35 region of the diverging picA promoter, so that hly and picA share the same PrfA box [247]. Similar PrfA
784
HAFIDA FSIHI, PIERRE STEFFEN, AND PASCALE COSSART
boxes are also found upstream from mpl, act A, and prfA itself, and in the MA promoter region. PrfA boxes are dyad-symmetric sites, with the consensus sequence 5'TTAACANNTGTTAA3' centered at position -41.5 with respect to the transcriptional start site in PrfA-activated promoters. Most virulence genes are under the control of PrfA, but activation levels vary considerably among the different genes. Several reports converge to demonstrate a hierarchy of virulence gene activation by PrfA, with most efficient activation at the hly and/7/cA promoters [256, 259]. In the latter cases, the sequence of the PrfA box is identical to the consensus sequence. In contrast, the inlAB promoter is only poorly activated, in agreement with its PrfA box being more distandy related to the consensus sequence.
D. PrfA as a Repressor As reported for several other regulatory proteins, PrfA seems to be able to act as a repressor. The first example of such a function concerns thepr^ promoter itself. It is important to notice that transcription of prfA is complex since this regulatory gene is transcribed both from the picA and its own promoters. The two different transcripts can be detected in various amounts (see below). In addition, two, and maybe three, transcriptional start sites have been identified in the prfA promoter region itself, which appears to negatively control its own transcription [260], because in the absence of a functional PrfA transcription levels at the two prfA promoters increase. However, these data are difficult to reconcile with results obtained with point mutations. Indeed, in the case of either an inactive PrfA variant (Serl84Ala), which is produced in lower quantities than the wild-type protein, or a superactive PrfA (Serl83Ala) variant, which is produced in higher amounts, the amounts of the small /77;;^-specific transcript are unaffected [256]. Several other studies indicate that, aside from its own expression, PrfA may also downregulate gene expression from the clpC [253] and motAB promoters [254].
E.
Control of prfA Expression and PrfA Production
The PrfA protein levels are regulated by cues affecting either prfA transcription or posttranscriptional events. During growth in broth medium at 37°C, expression of prfA is not constitutive. Transcripts from both iht picA and ih^ prfA promoters are generated, albeit with a predominance of the long plcA-prfA transcript at the beginning of the growth curve and of the short prfA specific transcript toward the stationary phase [251]. These observations indicate that growth conditions are able to influence prfA expression in L. monocytogenes.
16.
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785
Temperature also regulates expression of prfA. At low temperature (20°C), the short transcript is the only one expressed [261]. Quantification of the PrfA protein levels showed that PrfA cannot be detected at low temperature, suggesting that regulation of prfA expression may at least in part result from a switch from the plcA promoter to iht prfA promoter, with the small transcript being less efficiently translated than the long transcript [262]. Other explanations can account for these observations. For example, it cannot be excluded that at low temperatures a factor necessary for PrfA synthesis is lacking. Interestingly, virulence genes are not expressed at these temperatures, indicating that the presence of PrfA is absolutely required for their expression. Growth medium composition may also affect transcription of prfA [263]. For example, growth in modified Eagle medium (MEM) leads to increased levels of prfA transcription compared to growth in brain-heart infusion (BHI) [264]. However, this increase is not observed in all L monocytogenes strains (e.g., it is observed for strain EGD but not for strain NCTC 7973). Differences in virulence and in expression levels of both PrfA and PrfA-regulated virulence genes have also been observed in L. monocytogenes strains of serogroup 4 [265]. For clarity, we will not extensively discuss these strain variations and only mention them when necessary. PrfA expression is thus regulated at the transcriptional and possibly translational levels. Other regulatory mechanisms have been considered, including readthrough across the plcA-prfA intergenic region and antitermination activity of PrfA itself, but no data concerning these points are yet available.
F. Requirement for a PrfA Cofactor Low temperature and the presence of cellobiose in the growth medium are two conditions that impair virulence gene expression [261, 266]. At low temperatures, the levels of PrfA protein are reduced when compared to 37°C, explaining why virulence genes are less expressed under these conditions. In contrast, in the presence of cellobiose virulence genes are not expressed, whereas PrfA levels are unaffected, suggesting that a cofactor is required for full transcriptional activation by PrfA. Consistent with a requirement for a cofactor, specific DNA band shifts with PrfA produced from the wild-type strain EGD were only obtained when the protein was previously mixed with PrfA-free Listeria extracts or with B. subtilis extracts [267]. The observed gel retardation appeared to be due to a protein, called Paf for PrfA-activating factor, since extracts could be inactivated by heat treatment but not by RNAse. In addition, when iron was added to the extracts, the activity of the extracts was inhibited, in agreement with the observed increased virulence gene expression in growing bacteria when the culture medium was changed from BHI (12 \\M iron) to MEM (0.05 |LIM iron). Moreover, extracts from
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HAFIDA FSIHI, PIERRE STEEPEN, AND PASCALE COSSART
bacteria grown in MEM were highly active in promoting binding of PrfA to its DNA target site. An attractive possibility to explain these results is that in the absence of iron, or at low iron concentrations, Paf binds to Prf A, and the resulting protein complex activates expression of virulence genes. In the presence of iron (in BHI or MEM+Fe), Paf binds iron and can no longer bind Prf A, which in turn remains inactive. Interestingly, after changing the experimental procedure, gel shift assays have recently been obtained with the purified PrfA protein alone [258] and an hly Prf A box-containing DNA fragment as well as a DNA fragment containing the inlA PrfA box. Two major protein-DNA complexes, named CIII and CI, as well as a minor one were obtained. CI is likely the low-mobility complex formed by PrfA interacting with Paf. Considerably higher amounts of PrfA were necessary to obtain band shifts for the inlA fragment, in agreement with the high sequence deviation displayed by the inlA promoter region compared to the consensus PrfA box. There is in addition some evidence that the inlA promoter could bind Paf in the absence of PrfA, thereby preventing inlA transcription. These experiments undoubtedly require further experimental work, including Paf identification and purification, to clarify the mechanism of action.
G.
Global Regulation of Virulence Gene Expression
In addition to cellobiose, other sugars such as the monosaccharide glucose and the disaccharides maltose and trehalose, which are readily utilized carbohydrates, are effective in repressing virulence gene expression in L. monocytogenes. In contrast, fructose has no repressing effect. This sugar effect is strain dependent: again strain NCTC7973 behaves differently, being the only one where virulence gene expression is exclusively repressed by cellobiose and not by the other sugars. This strain expresses a PrfA with the Gly 145Ser mutation and which renders PrfA constitutively active. The PrfA protein of strain NCTC 7973 also carries a Cys229Tyr substitution in the C terminus of the protein, and there is evidence that this strain carries at least another mutation, explaining its upregulation of a-glucosidase activity, a second glucose-repressed enzyme [268, 269]. It is also interesting to note that a strain carrying the Glyl45Ser mutation in PrfA is able to utilize glucose-1-phosphate (GIP) as a carbon source, in contrast to the wild-type strain, which is unable to do so in conditions where PrfA is inactive, suggesting that GIP utilization is also PrfA dependent [257]. It appears now in the light of these results that carbon source availability is critical for regulation of virulence gene expression in L. monocytogenes. This regulation is mediated by PrfA, a protein similar to CAP. It seems to require a protein cofactor, Paf, whose synthesis or depletion would be regulated by the presence of readily metabolized carbohydrates much like cAMP in E. coli. As mentioned by Youngman and colleagues, this situation has a counterpart in P.
16.
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ISl
aeruginosa where an apparent CAP homolog is partly responsible for regulation of virulence genes [268, 269].
V. Conclusion In the field of bacterial pathogenesis, L monocytogenes has become one of the best-studied intracellular pathogens and is one of the most striking examples of bacteria that exploit mammalian cell functions [92, 270-273]. However, the relatively recent interest in the molecular mechanisms of listerial pathogenesis should not divert the reader from the fact that, since the early work of Mackaness [54], L monocytogenes has been used as a model of choice to study the induction of T-cell-mediated immunity. Indeed, in the murine model immune response to Listeria infection is very rapid, very efficient, and long lasting. Immunological studies with L. monocytogenes are still the object of intense investigations with the aim to develop protective or therapeutic vaccines against many intracellular pathogens that are still important health problems [55, 274]. Following the elegant studies of Pamer et al [275] and their discovery of the first bacterial cytotoxic T-lymphocyte (CTL) epitopes, several groups have successfully used various recombinant L. monocytogenes strains as live vectors to drive CD8-^ T-cell responses [276-279]. In addition, the property of L. monocytogenes to enter a wide variety of cells and reside in the cytosol has been exploited to deliver DNA into mammalian cells [280]. Although this type of transfection experiment uses attenuated self-destructing Listeria, they raise technical problems that hamper the immediate use of such DNA-delivery vectors in gene therapy. In conclusion, many properties of L. monocytogenes have attracted creative research programs. It is hoped that, in the end, they will help to completely eradicate a disease that is still responsible for many sporadic cases of foodbome infection or epidemics with dramatic consequences for patients.
Acknowledgments We apologize to those whose work could not be cited because of space constraints. We are indebted to Keith Ireton for fruitful discussions. Work in P. C.'s laboratory received financial support from the Institut Pasteur, ARC (CTG223), DRET (DGAG7/69), and EC (BMH4CTG60659). P S. was funded by fellowships from the Pasteur-Weizmann Foundation and NATO.
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Index
Aggregative adherence (AA), 415 agrC gene, 113 AgrD peptide, 113 AgrD protein, 112 Agrohacterium tumefaciens, 185, 210 attachment factors, 201 cell-density-dependent gene regulation, 112 plant pathogenesis, 181-183 protein transport from, 55-56 secretion system, 186-189 VirA protein, 89-90 AHL, 110 Ail protein. 232-236, 311 airS gene. 105 Alcaligin. 647 alilA gene. 482 Angular leaf spot. 184 Antibiotic-resistant Salmonella spp.. 312-314 Antigenic mimicry, 742-743 Antimicrobial peptides CAMPs, 278, 295-296 plant pathogens, 204-205 Salmonella and. 278-279. 295-296 Antisense RNA, 115-116 Apoptosis Helicobacter pylori, 532 Neisseria gonorrhoeae, 589-590 araBAD operon, 86 aro gene, 292 Arp2/3 complex, 776-778 ATP-binding cassette transporters, 44, 52-54 ATR. See Acid tolerance response Atrophic gastritis, Helicobacter pylori, 511, 515,540 Attachment factors, plant pathogens, 201-202 Autoexporters, 631-632 Autotransporters. 44. 4 7 ^ 9 ^/r/gene. 190, 199,208 Avr proteins. 196, 198-199
AA. See Aggregative adherence ABC transporters, 44, 52-54 Accessory factor, 53 (iceK gene, 24 acfgene, 462, 482 Achromobactin, 204 Acid tolerance response (ATR). 300-301 cictA gene, 770, 772 ActA protein, 359, 760, 772-775 Actin, 249-250, 356-361, 770-771 a-Actinin, 354, 776 Acute glomerulonephritis. 720, 742 Adaptive mutation, 114 Adenylate cyclase, Bordetella spp., 636-637 ADF/cofilin, 775 Adhesins afimbrial, 421-422 Escherichia coli. 394, 421-422 Haemophilus infhieuz.ae, 692 H. pylori. 528 identifying, 143-144 Neisseria spp., 143, 568-569, 585-586 S-fimbrial, 427 Shigella spp., 366 Streptococcus pyogenes, 143, 725 Yersinia spp., 231-234, 246 Adhesion factors aggregative adherence, 415 enteroaggregative E. coli (EAEC), 416 Helicobacter pylori, 528-529 Salmonella spp., 302 Streptococcus pyogenes, 721-725 Yersinia spp., 231-236 Aerobactin siderophores, 372 Affinity binding, pathogenicity factors, 136, 143-144 Afimbrial adhesins, 421-422 a^c^/gene, 292, 302
805
806
babA2 gene, 523 BabA2 protein, 525 Bacillary dysentery, 106 Bacteremia, Haemophilus influenzae, 702-704 Bacteria compartmentalization, 44 iron uptake mechanism, 203-204 pathogenicity, 133-170 population genetics, 9-28 secretory pathway, 43-61 virulence genes, 2-8 Bacterial soft rot, 183 barA gene, 105 Bartonella henselae, surface proteins, 141 BFP, 400, 402^03 BGP, 581 Biotinylation, 141 "Black holes," 5, 372 Bordetella avium, 14, 620, 635 Bordetella bronchiseptica, 14, 16, 620 cytokines, 640 dermonecrotic toxin, 635 evolutionary relationships, 625, 627 fimA gene, 633-634 host, interaction with, 639-641 infections, 623-624 LPS, 630 pertactin, 631-632 secretory system, 639 siderophore, 647 toxins, 635 Bordetella hinzii, 620 Bordetella holmesii, 620 Bordetella infection, 621-622, 641, 655 Bordetella parapertussis, 14, 16,620 dermonecrotic toxin, 635 evolutionary relationships, 627 fimA gene, 633, 634 infections, 622-623 LPS, 630 pertactin, 631-632 toxin, 635 Bordetella pertussis, 14, 16, 620 bvgAS regu\on, 154 bvgS gene, 101 BvgS protein, 89, 90 cytokines, 640 dermonecrotic toxin, 635 evolutionary relationships, 625, 627 fimbria, 633-634 fim genes, 101, 102
INDEX
infections, 621-622 LPS, 628, 630 pertactin, 631-632 pertussis toxin, 637-638 siderophore, 647 toxin, 55, 635, 637-638 vaccine, 621 virulence factors, 635 Bordetella spp., 620 adenylate cyclase, 636-637 bvgAS regulon, 629, 642-650 dermonecrotic toxin, 634-636 evolutionary relationships, 624-628 FHA, 634-635 fimbriae, 632-634 host, interaction with, 639-642 infections. See Bordetella infections iron uptake, 647 life cycle, 654-658 lipopolysaccharides, 628, 630 motility, 646-647 pertussis toxin, 637-638 phenotypic modulation, 646-650 population genetics, 14-16 secretory system, 639 siderophores, 647 toxins, 635-636, 637-638 virulence factors, 628-639 virulence genes, 650-658 Bordetella trematum, 620 Bordetella vaccine, 641 Borrelia afzelii, 16 Borrelia burgdorferi, 15, 16-17 Borrelia garinii, 16 Borrelia japonica, 16 Borrelia spp., population genetics, 15, 16-17 Brain abscess. Listeria spp., 753 Brazilian purpuric fever (BPF), 681 Brown spot disease, 183 Bundle-forming pilus. See BFP Burkholderia cepacia, plant pathogenesis, 209-210 BvgA protein, 642 bvgAS gene, 650, 651 bvgAS regulon, 154, 629, 642-650 bvgS gene, 101 BvgS protein, 89, 90, 642
CadC protein, 91 Cadherins, 360-361,763
807
INDEX
cagA gene, 521, 522 cAMP, cholera toxin and, 467 cAMP-CRP protein, 94 CAMPs. See Cationic antimicrobial peptides Cancer, Helicobacter pylori, 511,516, 517-518,540 Cancer therapy, based on Salmonella spp., 316 CAP, 782 Capping protein, 775, 776 Capsular polysaccharide, Haemophilus influenzae, 682-684 Capsules E. coli, 426-427 Neisseria meningitidis, 561, 569-570, 597 Streptococcus pyogenes, 730-731 CapZ protein, 775 Carrier state. Salmonella infections, 271-272 Caspase-1, 363,364 Catalase, 525 Cathepsin L, 284 Cationic antimicrobial peptides (CAMPs), 278, 295, 296 CD44, 349, 725 CD46, 575 CDC42, 288, 351,352 CEACAMs, 581-585 Cell-density-dependent gene regulation, 89, 91, 110-113 Cellulitis, 720 CFAs, 394 C/genes, 208 CGM1,581 CGM6,581 Chemical modification screens, 136, 139-141 Cholera host susceptibility, 464 immunity to, 489^90 molecular mechanisms, 465^89, 493 pathophysiology, 463-464 toxin, 138,463 treatment, 464-476 vaccines, 490-493 Cholera toxin, 48, 138, 461-462, 463, 467-468 enzymatic activity, 467-468 extracellular secretion, 471^72 generation of enzymatically active Al, 474-475 periplasmic assembly, 470 prostaglandin induction by, 468^69 regulation, 481-489 structure, 469-470 trafficking model, 477 uptake into host cells, 472-475 vesicular movement of, 476-478
Cholera vaccines, 490-493 Chronic carrier state. Salmonella infections, 271-272 Chrysobactin, 204 Ciprofloxacin, 312, 313 clcC gene, 780 ClpCprotein, 760, 780-781 CNF-1,422-423 Cofilin, 775, 776 Complementation selection strategies, 137, 161-162 Computational screens, pathogenicity factors, 137,167-168 Conjugal transfer systems, 44, 55-57 Conjugate vaccines, Haemophilus spp., 676 Contact-dependent gene regulation, 105 Contact-dependent secretion, 4 4 ^ 5 , 57-61, 186 cooA-D gene, 394 CooA-D protein, 394 Coordinate regulation screens, pathogenicity factors, 136, 154 CorA protein, 297 Coronin, 776 Cortactin, 353 Corynehacterium diphtheria. Fur protein, 82 CRE, 596 Crown gall disease, 182, 183, 186-187 crp gene, 79, 115-116, 301-302,485 CRP protein, 79, 81, 292, 485, 704 CSs, 394 ctxAB gene, 482 ctxA gene, 491 CVD103-HgR,492 CVDll 1,492 CyaA protein, 636-637, 640 cyaA gene, 651 Cya/Crpregulon, 301-302 cvflgene, 79, 301-302, 485 Cya protein, 292 Cystic fibrosis, 202 Cytochalasin D, 233, 595, 772 Cytokines Bordetella spp., 640 Helicobacter pylori and, 531 Neisseria spp. and, 594 Yersinia spp. and, 230-231, 239 Cytotoxic necrotizing factor 1. See CNF-1
daa operons, 104 DAEC. See Diffusely adhering E. coli
808
INDEX
DAF. See Decay-accelerating factor Decay-accelerating factor (DAF), 417 DGI. See Disseminated gonococcal infection Differential display (DD), pathogenicity factors, 136, 147-149 Diffusely adhering E. coli (DAEC) disease, 389, 417 virulence factors, 389, 4 1 7 - H 8 Direct selections, pathogenicity factors, 137, 160-161 Disseminated gonococcal infection (DGI), 564 DNA sequencing, population genetics by, 12 DNA structure, virulence gene regulation and, 94-97 Dnt, 635-636 dps gene, 88 dsDNA tester fragments, 146 J.s/^Egene, 199 Duodenal ulcer, Helicobacter pylori, 511. 540 Dysentery, 106, 336
E EaeB protein, 404 EAEC. See Enteroaggregative E. coli EAST-1,416 E-cadherin, 763 Effector proteins. Salmonella spp., 306-307 EHEC. See Enterohemorrhagic E. coli EIEC. See Enteroinvasive E. coli Electron microscopy, 140 emm gene, 726, 733-735 Ena/VASP proteins, 778 Encephalitis, Listeria spp., 753 Endovascular infections. Salmonella spp., 271 Enteric fever, 268-269 Enteroaggregative E. coli (EAEC) adhesion factors, 416 disease, 389, 414 virulence factors, 389, 415-417 Enterohacter cloacae, adhesin, 143 Enterochelin siderophores, 372 Enterohemorrhagic E. coli (EHEC), 336 disease, 389, 408 hemolysin, 413 toxins, 409 virulence factors, 389, 409-414 virulence gene regulation, 414 Enteroinvasive E. coli (EIEC), 389, 396-398 disease, 386, 389 virulence factors, 389, 396-398 virulence gene regulation, 398
Enteropathogenic E. coli (EPEC), 17, 93 disease, 389-390, 398-400 toxins, 391-393 virulence factors, 389-395, 400-407 virulence gene regulation, 395-396, 407-408 Enterotoxigenic E. coli (ETEC), 336 toxins. 391-393 Enterotoxin, Yersinia spp., 232, 238 EnvZ/OmpR, 82, 84, 86, 89, 292, 301 EnvZ protein. 89 EPEC. See Enteropathogenic E. coli EPEC-secreted proteins. See ESPs Epithelial cell invasion by enteropathogenic E. coli (EPEC), 401 by Neisseria spp., 588-589 by Salmonella spp., 302-307, 336 by Shii^ella spp., 345-355, 366-368 EpsE protein, 471—472 eps gene cluster, 471 EPSs. See Exopolysaccharides Eps system, 471-472, 493-494 ERK, 289 ERM proteins, 354 Erwinia amylovora cIspE gene, 199 exopolysaccharides, 202 harpins, 195 Hrp system, 205 plant pathogenesis, 182, 184, 195, 199 Erwinia carotovora cell-density-dependent gene regulation, 112 pectic enzymes, 200-201, 205-206 plant pathogenesis, 181-183, 210 Erwinia chrysanthemi harpins, 195 pectic enzymes, 200-201, 205 siderophores, 204 Erwinia stewartii, 112 Erysipelas, 720 EsaR protein, 206 Escherichia coli, ?^'i?>-?^9\ adhesins, 394, 421-422 CadC protein, 91 diffusely adhering (DAEC), 388-389, 417-418 enteroaggregative (EAEC), 388-389, 414^17 enterohemorrhagic (EHEC), 336, 388-389, 408-^14 enteroinvasive (EIEC), 388-389, 396-398 enteropathogenic (EPEC), 17, 93, 336, 388 enterotoxigenic (ETEC), 336, 388-389, 390-396
809
INDEX
fimbrial gene expression in, 98-99, 101, 103, 115 harpins, 195 heat-labile toxin, 392, 470 heat-stable toxin, 392-393 hemolysin, 53, 54 HU protein, 93 IHF, 93 Jack-in-the-Box strain, 17 mutator strains, 114 Pappili, 103-105 pathogenicity islands, 3 ^ , 93, 423 as plant pathogen, 184 population genetics, 15, 17-18 porin expression, 82 pullulanase secretion, 51 RssB protein, 91 secretory system, 403, 405 septic (SEC), 388-389, 426-428 SprE protein, 91 toxin, 389, 391-393 uropathogenic (UPEC), 388-389, 418-425 VirP proteins, 81 virulence genes, 8 Escherichia coli infection, 389 diffusely adhering E. coli (DAEC), 389, 417 enterohemorrhagic E. coli (EHEC), 389, 408 enteroinvasive E. coli (EIEC), 389, 396 enteropathogenic E. coli (EPEC), 389, 398^00 enterotoxigenic E. coli (ETEC), 389-390 meningitis, 389, 426 sepsis, 389, 426 uropathogenic E. coli (UPEC), 389, 418 EspA protein, 404-405 EspB protein, 404 EspC protein, 404-405 EspD protein, 404 EspF protein, 404 EspP protein, 413 ESPs, 404-405, 413 ETEC. See Enterotoxigenic E. coli Exopolysaccharides (EPSs), 202, 205 Exported proteins, targeting, 136, 153-154 "Exteins," 115 Ezrin, 354, 776
FACS. See Fluorescence-activated cell sorting F-actin, 138-139 Factor for inversion stimulation. See FIS
FbpA protein, 592 Fbp protein, 696 fhiiB gene, 633, 650-651 FhaB protein, 634 fluiC gene, 633 FHA protein, 633-635 Fibronectin, 580, 724-725 Filamin, 233 Fim2 protein, 632-633 Fim3 protein, 632-633 jimA gene, 103 finiB^^ene. 103,633 FimB protein, 103 Fimbriae Borcletella spp., 632-634 Haemophilus influenzae, 688 Fimbrial gene expression, in E. coli, 98-99, 101, 103, 115 Fimbrin, 776 finiC gene, 633 finiD gene, 633 FimD protein, 633-634 fmiE gene, 103 FimE protein, 103 .///;? genes. 101-103,292,302 Fire blight, 183-184 FIS, 93-94 JlaA gene, 20, 525 FlaA protein, 525 flaS gene, 20, 525 FlaB protein, 525 Flagella, H. pylori, 524 Flagellins, 20 FlbA protein, 525 FlgM protein, 105 FlhA protein, 194,525 Fluorescence-activated cell sorting (FACS), 155 FNR protein, 81 F plasmids, conjugal transfer systems, 55 Fur protein, 81-82 FyuA/Psn-Irp. 232. 236
G
i^acA gene, 209 GacA protein, 205 G-actin, 138-139 GalE protein, 292 GAMBIT. See Genomic analysis and mapping by /// vitro transposition
810 gapA gene, 24 GAPDH, 738 Gastric adenocarcinoma, Helicobacter pylori, 511,515-516,517-518 Gastric epithelial dysplasia, Helicobacter pylori, 511 Gastric lymphoma, Helicobacter pylori, 511, 518-519 Gastric ulcer, Helicobacter pylori, 511, 540 Gastritis, Helicobacter pylori, 511 Gastrospirillum hominis, 519 Gel electrophoresis, 135 Gelsolin, 776-777 Gene expression, virulence genes. See Virulence gene expression Genetic screens, 136-137, 149-150 coordinate regulation screens, 136, 154 host mimicry screens, 136, 154-156 in vitro, 136, 150-156 in vivo, 156-159 large-scale screening, 136, 150-153 recombination-based in vivo expression technology (RIVET), 137, 156-157 signature-tagged mutagenesis, 137, 157-159 targeting exported proteins, 136, 153-154 Genetic selections, pathogenicity factors, 159-164 Genetic switches, 98-99 fimbrial gene expression in E. coli, 98-99, 101, 103 Genome walking, pathogenicity factors, 165-166 Genomic analysis and mapping by in vitro transposition (GAMBIT), pathogenicity factors, 137, 166-167 Gentamicin protection assay, 345 gfp genes, host mimicry screens, 155 Globotriaosylceramide, 410 Glomerulonephritis, 720, 742 Glutaredoxin, 88 gnd gene, 24 Gonococcal conjunctivitis, 564 Gonorrhea, 561, 564 gpIV protein, 50 Gram-negative bacteria cell-density-dependent gene regulation, 110, 112 compartmentalization, 44 conjugal transfer systems, 55 plant pathogens, 181 -184 attachment factors, 201-202 exopolysaccharides, 202 host antimicrobial peptides and, 204-205 iron uptake, 203-204
INDEX
necrogenic, 181-184 toxins, 202-203 virulence regulation, 205-206 secretion system, 44, 55 Gram-positive bacteria cell-density-dependent gene regulation, 110, 112 compartmentalization, 44 secretion system, 44 Granulomatosis infantisepticum, 654 Group A streptococci, 719, 739 Group B streptococci, 725, 727 Group C streptococci, 718 Group G streptococci, 718 GyrA protein, 115
Haemophilus avium, 678 Haemophilus ducreyi, 696 Haemophilus galanarium, 678 Haemophilus influenzae, 705 adhesins, 692 capsular polysaccharide, 682-684 colonization, 679, 682, 699, 701 epidemiology, 679-680 fimbriae, 688 genome, 676-677, 697 hifA and hifB genes, 101, 102 history, 676-677 IgA protease, 47, 693-694 iron uptake, 694-696 lipid A, 687 lipopolysaccharide, 684-688 microbiology, 677-679 outer-membrane proteins, 691-693 pathogenesis, 699-705 pathogenicity islands, 3 peptidoglycan, 697 pili, 688-691 population genetics, 15, 19, 680-682 virulence factors, 682-699 virulence genes, 697-699 Haemophilus influenzae infections, 676, 705 bacteremia, 702-704 Brazilian purpuric fever (BPF), 681 epidemiology, 679-680 influenza, 676 local disease, 701-702 meningitis, 676 otitis media, 696, 701-702 urogenital and neonatal sepsis, 681-682
INDEX
Haemophilus parainfluenzae, 678 Haemophilus spp., 616-611 microbiology, 677-679 population genetics, 15, 19 vaccine, 676 HAI-1,89-90 HAI-2, 89-90 Harpins, 194-196 HasA protein, 54 Has system, Serratia marcescens, 54 HbpA protein, 695-696 HcrV protein, 194 Heat-labile toxin, Escherichia coli, 392, 470 Heat-stable toxin, Escherichia coli, 392-393 hel gene, 695 Helicobacter acinonyx, 520 Helicobacter bills, 520 Helicobacter bizzozeronii, 520 Helicobacter canis, 520 Helicobacter cholescystus, 520 Helicobacter cinaedi, 520 Helicobacter felis, 520 Helicobacter fennelliae, 520 Helicobacter heilmanii, 510,512,519-520 Helicobacter hepaticus, 519-520 Helicobacter muridarum, 520 Helicobacter mustelae, 520 Helicobacter nemestrinae, 520 Helicobacter pametensis, 520 Helicobacter pullorum, 520 Helicobacter pylori, 510, 520 acid resistance, 526-527 adherence, 528-529 apoptosis and, 532 atrophic gastritis, 511,515, 540 bacteria-host equilibrium, 538-539 cytokine production, 531 development of clinically evident disease, 539-542 epidemiology, 510-512 gastric acid production and, 527-528 gastric colonization, 524-529 gastric inflammation and, 529-532 genome, 520-521 infection, 510 Lewis B receptor, 144 lipopolysaccharides, 537-538 microbiology, 519-521 motility, 524-526 pathogenicity islands, 3, 530-531 population genetics, 15, 19-20, 521-524 resistance to host immune defenses, 536-537 stomach and, 512-516 urease, 526-527
811 VacA protein, 47 vacuolating cytotoxin, 532-535 virulence genes, 8-9 Helicobacter pylori infection, 541-542 acute, 516 development of clinically evident disease, 539-542 duodenal ulcer, 511, 540 epidemiology, 510-512 gastric adenocarcinoma, 511,515-516, 517-518 gastric epithelial dysplasia, 511 gastric lymphoma, 511,518-519 gastric ulcer, 511, 540 gastritis, 511 host factors, 540-541 intestinal metaplasia, 511, 515 peptic ulcer disease, 516-517 persistence of, 536-539 Helicobacter rappini, 520 Helicobacter rodentium, 520 Helicobacter salmonis, 520 Helicobacter spp. microbiology, 519, 520 population genetics, 15, 19-20,521-524 Helicobacter suncus, 520 Helicobacter troguntum, 520 Heme, 695 Hemolysins, 53-54, 142 enterohemorrhagic E. coli (EHEC), 413 uropathogenic E. coli (UPEC), 419, 422 Vibrio cholerae, 461 Hemolytic and uremic syndrome (HUS) Escherichia coli, 408 Shigella dysenteriae, 341-372 Hia, 692 hifA gene, 101-102 HifA protein, 689-690 hifBgtne, 101-102,689 HifB protein, 689 hifC gene, 689 HifC protein, 689 hifD gene, 689 HifD protein, 689-690 hifE gene, 689-690 HifE protein, 690 HilA protein, 91, 292, 298-300 Histidine protein kinases, 89 hit operon, 696 HlyA protein, 53-54 HlyB protein, 53-54 HlyD protein, 53
812 HMWl protein, 691 HMW2 protein, 691 H-NS, 92-94 Hop, 196 Host mimicry screens, pathogenicity factors, 136, 154-156 Hp9(), 403 HpaA protein, 525 HR. See Hypersensitive response /7rr genes, 191, 194 hmiA gene, 195 /z/y? genes, 191, 194,205 HrpG protein, 205 HrpL protein, 205 Hrppilus, 196 Hrp proteins, 57 Hrp system, 185, 194-197,205 HrpWharpin, 195 HrpZ harpin, 195 HrpZ protein, 194 HS-GAG, 577, 580 hspA gene, 521 HspA protein, 525 HspB protein, 525 HSPGs, 577, 580 Humoral immunity Bordetelki infections, 641 neisserial infection, 597-598 HU protein, 93-94 HUS. See Hemolytic and uremic syndrome Hypermutation, 114 Hypersensitive response (HR), 189-190
icd gene, 24 iceA gene, 521 ICE protein, 363-364 /c\Vy4 gene, 356 IcsA protein enteroinvasive E. coli (EIEC), 397-398 Shigella spp., 356-358 IgA protease as autotransporter, 47 Haemophilus influenzae, 41, 693-694 Neisseria spp., 590-592 IHF protein, 93, 94, 103 IL-lp, Neisseria spp. and, 595 IL-6 Neisseria spp. and, 594-595 Streptococcus spp., 727
INDEX
IL-8 Helicobacter pylori, 530-531 Neisseria spp. and, 594 Immune response Bordetella infections, 641 Neisseria spp., 594-599 Salmonella infections, 276-280 Streptococcus pyogenes, 732-736 Vibrio cholerae. 489-490 Immunological methods, pathogenicity factors, 136, 144-145 IncN plasmids, conjugal transfer systems, 55 Infectious focus assay, 345 Influenza, 676 inlA gene, 761 InlAprotein, 759, 761,781 InlBprotein, 760, 763, 781 InlC2 protein, 781 InlC protein, 760 InlD protein, 781 InlE protein, 781 Integrins, 233, 349, 354 "Inteins," 115 Internalin, 758-763, 781 Internal in A, 346 Intestinal metaplasia, Helicobacter pylori, 511.515 Intimin, 405-406 Invasin (Inv) enteroinvasive E. coli (EIEC), 397 Shigella spp., 346 Yersinia spp., 231-233, 235-236, 246 In vitro screens, pathogenicity factors, 136, 150-156 //; I7\Y; expression technology (IVET), pathogenicity factors, 137, 156, 163-164 In vivo screens, pathogenicity factors, 156-159 Inv/Spa proteins, 57 I pa A protein, 304 ipaB gene, 349 IpaB protein, 208, 304, 306, 349, 363 ipaC gene, 349 IpaC protein, 304, 306, 349 ipaD gene, 349 IpaD protein, 304, 306, 349 Ipa protein, enteroinvasive E. coli (EIEC), 397 Ipa-vinculin interaction, 355 Iron uptake Bordetella spp., 647 Haemophilus influenzae, 694-696 Neisseria spp., 592-594 plant pathogens, 203-204 Yersinia spp., 232, 236-237 IVET. See In vivo expression technology
INDEX
JNK pathway, 289, 596
KatF protein, 300 katG gene, 88 Killed whole cell vaccine, cholera, 490-^91 Kinin peptides, 737 Klebsiella oxytocci, piil system, 49 KorB protein, 93
Lad repressor, 86 lac operon, 86 Lactoferrin, 372, 592, 695 /<:/cZ genes, host mimicry screens, 155 LacZ protein, 45 LamB protein, 45 LAMP-2, 287 LAMPs. See Lysosomal-associate membrane proteins LAP, 287 LAPs. See Lysosomal-associated proteins Large-scale screenings, pathogenicity factors, 136, 150-153 LasI protein, 110 LasR protein, 110 LbpA protein, 592 LbpB protein, 592 LcrD protein, 194,240 LcrF protein, 105 IciG gene, 249 LcrG protein, 245, 248 LcrQ protein, 105 LcrV protein, 59, 242, 246, 248 LEE, 411-412 Legionella pneumophila pathogenicity factors, 148 "thymineless death," 162-163 LemA/GacA system, 205-206 Ler protein, 307 /ef/Xgene, 115 Lewis B antigens, 528 lex2 locus, 686 lid locus, 685-686
813 Light microscopy, 140 Lipid A Haemophilus influenzae. 687 Helicobacter pylori, 537-538 Salmonella infections and, 296-297 Shigella spp., 338 Lipoarabinomannan, 139 Lipooligosaccharide, 567-569 Lipopolysaccharide (LPS) Bordetella spp., 628, 630 Haemophilus influenzae, 684-688 Helicobacter pylori, 537-538 Salmonella infection, 277-278, 295 5/z/;c,W/^/spp., 338, 370-371 Vibrio cholerae, 465 Lipoteichoic acid (LTA), 725 Listerella hepatolytica, 752 Listeria grayi, 752 Listeria innocua, 752-773 Listeria ivanovii, 752 Listeria monocytogenes, 752, 787 actin, 770-771 ecology, 752-753 entry into mammalian cells, 759-767 history, 752 infections. See Listeriosis internalin, 758-763, 781 intra- and intercellular spreading, 770-781 intracellular survival and growth, 161-110 motility, 770-780 pathogenesis, 755-758 population genetics, 15, 20-21 PrfA boxes, 783-784 serovars, 753 signal transduction, 764-767 virulence factors, 780-781 virulence gene regulation, 81, 782-787 virulence genes, 756-758 Listeria seeligeri, 752 Listeria spp., 752 ActA, 359 population genetics, 15, 20-21 Listeria welshimeri, 752 Listeriolysin, 760, 764-765 Listeriolysin O (LLO). 161-16^ Listeriosis, 787 animal models, 754-755, 787 central nervous system infection, 753 during pregnancy, 753-754 epidemiology, 753-755 neonatal infections, 754 Live vaccines, cholera, 491-493 LLO. See Listeriolysin O LOS, 567-569
814
INDEX
//7/gene, 292, 302 LPS. See Lipopolysaccharide LPXTG motif, 722-723 LRP protein, 88, 94 LT, Escherichia coli, 392, 473 LTA. See Lipoteichoic acid LTIIB, 474 LuxN protein, 91 LuxO protein, 91 LuxQ protein, 91 LuxR family, 312 LuxR-LuxI type, 206 LuxR protein, 110 LY294002, 766 Lyme disease, 16 Lysosomal-associated proteins (LAPs), 284 Lysosomal-associate membrane proteins (LAMPS), 284 LysR protein, 80
M Mabs. See Monoclonal antibodies Macrophages cytotoxicity of Salmonellae on, 284-286, 309-310 Shigella spp. and, 361-364 Macropinocytosis, Salmonella, 281-282 MafA protein, 586 MafB protein, 586 Magnesium transporters, Salmonella spp., 297-298 MALT, 518 MALT lymphomas, 518-519 MAP, 289 MAP-kinase pathway, 766 MCP, 575 mdh gene, 24 Membrane fusion protein, 53 Mena, 774, 776 Meningitis E. coli, 426 Haemophilus influenzae, 676 Listeria spp., 753-754 Meningococcal capsule, 561, 569-570, 597 Meningococcal disease, 22-23, 565-566 Methicillin-resistant Staphylococcus aureus (MRSA), population genetics, 25 MetL protein, 292
mgtA gene, 297 MgtA protein, 297 mgtB gene, 297 MgtB protein, 297 mgtC gene, 297 MgtC protein, 292, 297 MHC, 740 Microarrays, pathogenicity factors, 137, 169 MKKK, 596 MLEE. See Multilocus enzyme electrophoresis MLST. See Multilocus sequence typing Monoclonal antibodies (MAbs), 136, 144-145 Motility Bordetella spp., 646-647 Listeria monocytogenes, 770-780 Mpl, 780 M protein. Streptococcus spp., 725, 728-730, 733 MRSA. See Methicillin-resistant Staphylococcus aureus MSHA, 480-481 Mucin, 699 Multi-antibiotic-resistant Salmonella spp., 312-313 Multilocus enzyme electrophoresis (MLEE), population genetics by, 10-28, 680-681 Multilocus sequence typing (MLST), population genetics by, 12-13 Murein hydrolase, 142 Mutagenesis, 152 mutS gtnt, 114 mviA gene, 91 Mxi/Spa proteins, 57 Mycobacterium leprae, RecA protein, 115 Mycobacterium spp., population genetics, 15, 21-22 Mycobacterium tuberculosis adherence to host cells, 145 pathogenicity factor, 139 population genetics, 15, 21-22 RecA protein, 115 myfA/psaA gene, 234 myfA/psaA operon, 234 MyfA/PsaA protein, 234 myfB/psaB gene, 234 MyfB/PsaB protein, 234 MyfC/PsaC protein, 234 myfE/psaE gene, 234 Myffi/PsaE protein, 234 myff/psaF gene, 234 MyfF/PsaF protein, 234 Myf/Ph6 antigen. Yersinia spp., 232, 234-236 Myositis, 720
815
INDEX
Natural killer cells. Neisseria spp. and, 596 Necrogenic plant pathogens, 181-182, 184-185 brute-force-type, 181-182, 184,200-201 stealth-type, 181-182, 184, 188-199 ovrgene, 190-191, 196, 198-199 Avr proteins, 196, 198-199 gene-for-gene interactions, 190-193 harpins, 194-196 hrc gene, 191, 194 hrp gene, 191, 194 HR plant defense syndrome, 189-190 Hrp system, 194-197 pilin, 196 Necrotizing fasciitis, 720 Neisseria flava, 586 Neisseria gonorrhoeae adhesin, 143 apoptosis, 589-590 CEACAM receptors, 581 genetic difference from A^. meningitidis, 146 humoral immunity, 597 Iga 1 protease, 590 IgA protease, 47 IHF, 93 infections, 560-561,564 morphology and physiology, 560 opacity protein genes, 98, 101 pile gene, 102 pilin gene, 98, 100-101 porins, 586 Neisseria lactamica, 598 Neisserial infections, 560-566 humoral immunity, 597-598 immune response, 594-599 vaccines, 598-599 Neisseria meningitidis CEACAM receptors, 581 genetic difference from N. gonorrhoeae, 146 humoral immunity, 597 Igal protease, 590 infection, 560-561, 565-566 meningococcal capsule, 561, 569-570, 597 opacity protein genes, 98, 101 ope outer membrane protein gene, 101 population genetics, 15, 22-23 porins, 586 vaccines, 598 Neisseria spp., 559-560 adhesins, 143, 568-569, 585-586 colonization, 561-563, 570-586
DNA transformation, 566-567 Igal protease, 590-592 immune response, 594-599 infections. See Neisserial infections iron acquisition, 592-594 lipooligosaccharide, 567-569 meningococcal capsule, 561, 569-570, 597 morphology and physiology, 560 Opa-mediated interactions, 576-585 Opc-mediated interactions, 585 pilus biogenesis, 570-576 population genetics, 15, 22-23 PorB, 586-590 surface structures, 567-570 vaccines, 598-599 Neonatal sepsis, 426 Nonspecific acid phosphatase. Salmonella spp., 298 Nontyphoidal salmonellosis, 269-270, 271, 273 N protein, 206-207 NtrB/NtrC proteins, 82-83 N-WASP, 360
O Obsessive-compulsive disorder syndrome, streptococcal infection and, 721 OHHL, 110 ompC gene, 87, 372 ompF gene, 87 OmpR/EnvZ, 82, 84, 86, 89, 292, 301 OmpRprotein, 89, 91,292 omprgene, 5-6, 485, 488 ompU gene, 488 ompU/ompT gene, 482 OMV vaccine. Neisseria spp., 598-599 Opacity protein genes, 98, 101 Opa protein, 576-585 Ope protein, 585 Ophthalmia neonatorum, 564 ospC gene, 16 Otitis media, Haemophilus influenzae, 696, 701-702 Outer-membrane proteins, Haemophilus influenzae, 691 -693 Oxidative stress response, virulence gene regulation, 87-88 oxyS gene, 88
816
pl25FAK, 354 PAF, 767 pagA gene, 295 PagC protein, 311 pagP gene, 295 pagS gene, 298 PAKl protein, 596 Pap, 42(M21 PapG protein, 421 Papl protein, 104 Pappili, 103-105 pap system, 104 Pathogenesis-related proteins, 190 Pathogenicity, 134-135; See also Virulence Pathogenicity factors, identification of, 135, 169-170; See also Virulence factors biochemical strategies chemical modification screens, 136, 139-141 classical approaches, 135-136, 138-139 differential display, 136, 147-149 immunological methods, 136, 144-145 receptor/1 igand affinity screens, 136, 143-144 reverse genetics, 136, 149 subtractive hybridization. 136, 145-147 zymography, 136, 141-143 genetic screens, 136-137, 149-150 coordinate regulation screens, 136, 154 host mimicry screens, 136, 154-156 in vitro, 136, 150-156 in vivo, 156-159 large-scale screening, 136, 150-153 recombination-based in vivo expression technology (RIVET), 137, 156-157 signature-tagged mutagenesis, 137, 157-159 targeting exported proteins, 136, 153-154 genetic selections, 159-160 complementation approaches, 137, 161162 direct selections, 137, 160-161 in vivo expression technology (IVET). 137, 156, 163-164 nongrowing bacterial mutants, 137, 162163 genomic approaches, 165 computational screens, 137, 167-168 genome analysis and mapping by in vitro transposition (GAMBIT), 137, 166-167 genome walking, 165-166
INDEX
microarrays, 137, 169 transcriptional profiling, 137, 168-169 plant pathogens, 181-211 Pathogenicity islands (Pai), 3-4, 5 Escherichia coii, 3-4, 93, 423 Helicobacter pylori, 3, 530-531 Salmonella spp., 3-4, 297, 302-303, 308 Yersinia spp., 237-238 Paxillin. 354, 636 phgP gene, 295 PCFs, 394 PC-PLC. See Phosphatidylcholine-phospholipase C PCR. See Polymerase chain reaction Pectic enzymes. 200-201, 205-206 /?c^/'gene, 292 Pelvic inflammatory disease (PID), 564 Penicillin, selection for nongrowing bacterial mutants, 162-163 Peptic ulcer disease, 516-517 Peptidoglycan. Haemophilus influenzae, 697 Pertactin. 631-632 Pertussis. 621 Pet protein, 416 PGI2. 767 pH 6 antigen, 234-236 Phagolysosomal fusion. Salmonella, 283-284 Phenotypic modulation, Bordetella spp., 646-650 phoA genes, host mimicry screens, 155 phoN gene, 298 PhoP/PhoQ system. Salmonella, 83-84, 290-291,293-294,298 PhoPprotein, 84, 291,298 PhoPQ regulon, 154,291-292 PhoQprotein, 84, 291 Phosphatidylcholine-phospholipase C (PC-PLC), 760. 768-770 Phosphatidylinositol-phospholipase C (PI-PLC), 760, 768-769 Phosphoinositides, 767 Phosphorylation, transcription factor modulation, 82-86 Photohacterium fischeri, cell-density-dependent regulation, 110-113 PI 3-kinase, 765 PIC protein, 417 PID. See Pelvic inflammatory disease pile gene, 102 Pile protein, 570 PilD peptidase, 571 PilD protein. 156
INDEX
pilE gene, 93, 566 PilE protein, 570 PilF protein, 570 Pili, 429 Haemophilus influenzae, 688-691 Neisseria spp., 570-576 regulation of, 481-489 type I pili, 420, 425, 427 type IV pili, 478^79 Pilin, 196,574 Pilin gene, 93, 98, 100-101 PilQ protein, 570 PilQ proteins, 50 Pilus. See Pili PI-PLC. See Phosphatidylinositol phospholipase PIVETl, 164 Plant pathogens, 180-181 avirulence phenotype, 196 Gram-negative, 181-184 attachment factors, 201-202 exopolysaccharides, 202 host antimicrobial peptides and, 204—205 iron uptake, 203-204 necrogenic, 181-184 toxins, 202-203 tumorigenic, 181-182 virulence regulation, 205-206 Hrp system, 194-197,205 necrogenic, 181-182, 184-185 brute-force-type, 181-182, 184,200-201 stealth, 181-182, 184, 188-199 plant surveillance system, 206-209 secretory system type 11,200-201 type III, 189-199 type IV, 186-189 siderophores, 203-204 tumorigenic, 181-182, 185 secretory system, 186-189 virulence proteins, 180 Plants gene-for-gene interaction, 190-191, 192-193 hypersensitive response, 189-190 nonpathogenic bacteria of, 184-185 pathogenesis, 179-211 pathogens of. See Plant pathogens PLAP, 469 Plaque assay, 345 Plasmin, 738 Platelet-activating factor, 767 pleS gene, 209 Ply protein, 138 PMNs. See Polymorphonuclear leukocytes
817 pmrAB genes, 295 PmrAB proteins, 292 PmrA protein, 84, 295 PmrB protein, 84 pnirE gene, 295 pmrF gene, 295 Pneumolysin, 138 Polymerase-chain reaction (PCR), 135 Polymorphonuclear leukocytes (PMNs), 728 PopA protein, 195 Population genetics, 9-13 Bo relet ella spp., 14-16 Borrelia spp., 16-17 by DNA sequencing, 12 by multilocus enzyme electrophoresis, (MLEE), 10-28 by multilocus sequence typing (MLST), 12-13 Eseherichiacoli, 15, 17-18 Haemophilus spp., 15, 19,680-682 Helicobacter spp., 15, 19-20,521-524 Listeria spp., 15, 20-21 Mycobacterium spp., 15, 21-22 Neisseria spp., 15, 22-23 Salmonella spp., 9, 15, 23-25 Shigella spp., 9, 15, 18 Staphylococcus spp., 15, 25-26 Streptococcus spp., 15, 26 V/7?/7V;spp., 15,27-28 PorA protein, 586 PorB protein, 586-590, 599 Porins, 525 Neisseria spp., 586-590 regulation of, 82-86 P pili, 429 Pregnancy, listeriosis during, 753-754 Prepilin peptidase, 167 Prevotella melanginogenica, hemolysin, 142 PrfA boxes. Listeria monocytogenes, 783-784 prfA gene, 782, 784-785 PrfA protein, 81, 167, 760, 782-786 PrgHIJK proteins, 292 PrhA protein, 205 prl genes, 46 Profilin, 776 Promoters, 77-78 Prostaglandin 12, 767 Prostaglandins, cholera toxin and, 468-469 Proteases, zymography to identify, 142 Protein F, 722, 724-725 Protein phosphorylation, transcription factor modulation, 82-86 Protein splicing, 115 Proteus vulgaris, hemolysins, 54
INDEX
proU gene, 87, 96 PR proteins, 190 Pseudo-focal adhesion, 354 Pseudomonas aeruginosa cell-density-dependent gene regulation, 112 GacA protein, 205 gonococcal pili, 570-571 plant pathogenesis, 209-210 Pseudomonas fluorescens, as plant pathogen, 184 Pseudomonas solanacearum, virulence proteins, 57 Pseudomonas syringae avrE gene, 199 Hrp system, 205 plant pathogenesis, 181-184, 195, 198 Pseudopilins, 471 Psoriasis, 721 Ptdlns, 767 ptl genes, 637 Ptl proteins, 531 Pto gene, 207 ptxA-E genes, 637 ptxA gene, 651 Ptx protein, 637-638 PulC protein, 50 PulD protein, 50 PulE protein, 50 PulF protein, 50 PulK protein, 50 PulL protein, 50 Pullulanase, 49, 51 PulM protein, 50 PulN protein, 50 pul system, Klebsiella oxytoca, 49 purA gene, 163 pur gene, 292 Putative effector proteins. Salmonella spp., 306-307 putP gene, 24 Pyelonephritis, 418 pYV plasmid, 238
Quorum sensing, 89, 206, 210, 312
Rac protein, 352, 776 Radioiodination, 141
Ralstonia solanacearum exopolysaccharides, 202, 205 harpins, 195 Hrp system, 205 plant pathogenesis, 182-183 PopA protein, 195 Rare codons, 115 Rckprotein, 293, 311 RDA, 147 RecA protein, 115,409,574 Receptor activity-directed affinity tagging, 528 Receptor/1 igand affinity screens, pathogenicity factors, 136, 143-144 Recombination-based in vivo expression technology (RIVET), 137, 156-157 Regulatory networks, 86-87 Regulatory proteins, 77-79 families, 79-82 modular nature of, 89-92 oxidative stress, 87 Regulon, defined, 76 Reiter's syndrome. Salmonella spp., 271 ReTagging, 144 Reverse genetics, identification of pathogenicity factors, 136, 149 rfbT gene, 466 /? genes, 191,207-210 RhlR protein, 112 Rheumatic fever, 720-721, 742 Rhll protein, 112 Rhizobium meliloti, virulence gene expression, 79 Rhomboencephalitis, Listeria spp., 753 Rho protein, 288, 352, 354 "Rice water stool," 463-464 RIVET. See Recombination-based in vivo expression technology RNA polymerase, 77-78 rpml gene, 207 rpoA gene, 652 RpoH protein, 78 RpoN protein, 78-79 rpoS gene, 112 RpoS protein, 78, 88, 91, 206, 293, 300 R proteins, 206-207 rps2 gene, 207 rrnB PI promoter, 79 RssB protein, 91 RtxA, 461-462
Salmonella abortusovis, 310-311 Salmonella arizonae, 267, 268
INDEX
Salmonella bongori, 23, 25, 308 Salmonella choleraesuis, 266, 268, 310 Salmonella-contmn'mg vacuoles (SCVs), 282-284 Salmonella dublin, 268, 306-307, 310 Salmonella enterica pathogenicity islands, 3 ^ population genetics, 15, 23-25 taxonomy, 267 Salmonella enteritidis, 268, 270, 311 Salmonella gallinarum, 268, 310 Salmonella hadar, 268 Salmonella hartford, 268 Salmonella infections, 268 cellular responses to, 288-289 chronic carrier state, 271-272 disease course, 212-214 enteric fever, 268-269 factors required for, 308-310 gastroenteritis, 269-270 immunology, 276-280 inbred mouse enteric fever model, 274-276 lipid A and, 296-297 nontyphoidal salmonellosis, 269-270, 271,273 typhoid fever, 266-267, 268-269, 273, 312 vaccines, 314-316 Salmonella marina, 268 Salmonella paratyphi, 268-269 Salmonella spp., 266 acid tolerance response (ATR), 300-301 adhesion factors, 302 antibiotic-resistant, 312-314 antimicrobial peptides and, 278-279, 295-296 bacterial-mediated endocytosis, 286-288 cancer therapy based on, 316 Cya/Crp regulon, 301-302 cytotoxicity on macrophages, 284-286, 309-310 effector proteins, 306-307 history, 206-207 infections. See Salmonella infections invasion of epithelial cells, 302-307, 336 mutator strains, 114 nonspecific acid phosphatase, 298 pathogenicity islands, 3-4, 297, 302-303, 308 PhoP/PhoQ regulon, 83-84, 290-291, 293-294, 298
819 population genetics, 9, 15, 23-25 RpoS protein, 300 secretory system, 303-306 serotypes, 267-268 taxonomy, 267 toxins, 310 transcriptional regulators, 290-302 translocated proteins, 306-307 vaccines, 314-316 virulence, in vitro models, 280-290 virulence factors, 290-312 virulence plasmids, 310-312 Salmonella Stanley, 268 Salmonella typhi, 266-269, 310 antibiotic resistance, 312 infection, 336 virulence plasmids, 311 Salmonella typhimurium, 267-268 antibiotic resistance, 313 effector proteins, 306 flagellar biosynthesis in, 105 HilA protein, 91,298-300 inbred mouse enteric fever model, 274-276 MviA protein, 91 OmpR/EnvZ, 82, 84, 86, 292, 301 pathogenicity factors, 148 PhoP/PhoQ system, 83-84 phoPQ regulon, 154 population genetics, 23 porin expression, 82 regulatory proteins, 82-84, 92 resistance to antimicrobial peptides, 204 secretory system, 57, 59 SopE protein, 307 TlpA protein, 109 vaccines, 314 virulence plasmids, 311 sap genes, 204-205 Sap system, 204 SAR proteins, 190 "Scaffolding" proteins. Shigella spp., 354 Scarlet fever, 720 SCAR protein, 360 SCP, 731 SCPA, 731 Screening chemical modification screens, 136, 139-141 computational screens, 137, 167-168 genetic screens, 136-137, 149-150 coordinate regulation screens, 136, 154 host mimicry screens, 136, 154-156 in vitro, 136, 150-156 in vivo, 156-159 large-scale screening, 136, 150-153
820 recombination-based in vivo expression technology (RIVET), 137, 156-157 signature-tagged mutagenesis, 137, 157-159 targeting exported proteins, 136, 153-154 receptor/1 igand affinity screens, 136, 143-144 SCVs. See Salmonella-contammg vacuoles SEC. See Septic E. coli SecA protein, 46-47 SecB protein, 46-47, 54 SecD protein, 46 SecF protein, 46 sec genes, 46 Sec proteins, 45-46, 54 Secretins, 49-50 Secretory system, 43-47 Agrohacteniim tiimefaciens, 186-189 Bordetelki spp., 639 Escherichia coli, 403, 405 plant pathogens, 185-189 SalmoneUci spp., 303-306 Shigella spp., 348-350 Type I, 44, 52-54 Type II, 44, 49-52, 185, 200-201 Type III, 44-45, 57-61, 185, 189-199, 240-242 Type IV, 44, 55-57, 185-189 Type V, 44, 47-49 Yersinia spp., 240-242 Ysc secretion system, 240-242, 252, 348 SecYEG complex, 46 SecY protein, 46 Seizures, Shigella spp., 341 Septic E. coli (SEC), 389, 426-428 Septicemia, Shigella spp., 341 Sereny test, 343 Serine protease, enterohemorrhagic E. coli (EHEC), 413 Serratia tnarcescens, 4 7 ^ 8 , 54 sfa operons, 104 S-fimbrial adhesins, 427 Shiga-like toxins. See SLTs Shiga toxin (Stx), 341, 371-372, 409-411 Shigella boydii, 337 infection, 338 population genetics, 9 Shigella dysenteriae, 336-337 hemolytic and uremic syndrome (HUS), 341,372 infection, 338, 340-341 LPS, 338
INDEX
population genetics, 9 toxin. 371 Shigella enterica, invasion gene, 6 Shigella fle.xneri, 337 IHF, 93 infection, 338, 340 invasion gene, 6 LPS, 338 OmpR/EnvZ, 84 pathogenicity islands, 3 ^ plaques, 758 population genetics, 9 VirP proteins, 81 virulence gene regulatory cascade, 106-109 virulence genes, 348-350 virulence proteins, 57, 92 Shigella infection. See Shigellosis Shigella sonnei, 9, 18, 337-338 Shigella spp. actin-based intracellular motility, 356-361 adhesins, 366 bacteriology, 337 cell-to-cell spreading, 355, 360-361 epithelial cell invasion, 345-355, 366-368 escape into cell cytoplasm, 355 IcsA protein, 356-358 infection. See Shigellosis intracellular motility, 355-361 invasive phenotype in vitro expression, 345-365 in vivo expression, 365-369 lipopolysaccharide, 338, 370-371 macrophages and, 361-364 natural hosts, 339 01m phenotype, 355 pathogenesis, 342-343 pathogenicity, 345-369 population genetics, 9, 15, 18 secretory system, 348-350 siderophores, 372 somatic antigen, 338 toxins, 341,371-372 virulence, 5-6 virulence genes, 369-373 Shigellosis, 336 complications, 341-342 histopathology, 342-343 incidence, 339 intestinal inflammation during, 368 models animal models, 343-344 cellular models, 344-345 mortality, 339 symptoms, 340-341
INDEX
Siderophores Bordetella spp., 647 Haemophilus influenzae, 695 plant pathogens, 203-204 Shigella spp., 372 Yersinia spp., 236 Sigma-32, 77-78 Sigma-38, 77-78 Sigma-54, 77-79 Sigma-70, 77-78 Signal recognition particle (SRP), 46 Signal transduction enterohemorrhagic E. coli (EHEC), 413 Listeria monocytogenes, 764-767 Signature-tagged mutagenesis (STM), pathogenicity factors, 137, 157-159 SipB protein, 304, 306 SipC protein, 304 Sip/SspBCD protein, 293, 304, 306 SLS. See Streptolysin S SLTs, 409 SlyA protein, 293 Small GTPases, Shigella spp., 351-353 sodA gene, 87-88 Soft rot, 183-184 SopB protein, 293, 306-307 SopD protein, 293, 307 SopE protein, 293, 307 Southern bacterial wilt, 183 SoxR protein, 87 SoxRS system, 88 SoxS protein, 87 SP12 effector proteins, 308-309 Spacious phagosome (SP), Salmonella, 281-282 SpaPQRS proteins, 293 SPE-A, 741 SpeB protein, 731 sjieB gene, 731 SPE-C, 741 5P//gene, 291,298 SPII pathogenicity island, 298, 303 SPI2 pathogenicity island, 308 SPI3 pathogenicity island, 293 SPI4 pathogenicity island, 293 SPI5 pathogenicity island, 293 SprE protein, 91 SptP protein, 293, 307 SpvABCD protein, 293 .syn-genes, 81, 86,92 SpvR protein. Salmonella typhimurium, 81 src gene, 353 Src protein, 354 SRP. See Signal recognition particle
821 SSA, 741 SsaBCDE.G-V proteins, 293 ssDNA tester fragments, 146 SseBCD proteins, 293 SSH. See Suppressive subtractive hybridization Ssps, 304 SsrAB proteins, 293 ST, Escherichia coli, 392 STa enterotoxins, 393 Staphylococcus aureus cell-density-dependent gene regulation, 112-113 MRSA, 25 murein hydrolase, 142 pathogenicity islands, 3 population genetics, 15, 25-26 Staphylococcus spp., population genetics, 15, 25-26 STb enterotoxins, 393 Stealth-type necrogenic plant pathogens, 181-182. 188-199, 194 ^/iTgene, 190-191, 196, 198-199 Avr proteins, 196, 198-199 gene-for-gene interactions, 190-191, 192-193 harpins, 194-196 /z/rgene, 191, 194 ///y^gene, 191, 194 HR plant defense syndrome, 189-190 Hrp system, 194-197 pilin, 196 Stereotypical events, gene expression control, 97 STM. See Signature-tagged mutagenesis Stochastic genetic prcx:ess, gene expression control, 98 Stomach Helicobacter pylori and, 512-516, 529-532 histology and physiology, 512-513 Stomach cancer, Helicobacter pylori, 511, 516-518,540 stpA gene, 93 StpA protein, 92-93, 94 "Strangles,'' 718 *'Strep throat," 720 Streptococcal infections acute glomerulonephritis, 720, 742 immunopathological-based diseases, 720-721,742-743 nonsuppurative sequelae, 721 pathogenesis, 718-743 rheumatic fever, 720-721, 742 scarlet fever, 720 toxic shock syndrome, 720-739 toxin-mediated disease, 720, 739-742
822 Streptococcus dysgalactiae, 118 Streptococcus equi, 718 Streptococcus iniae, 118 Streptococcus mutans, 718 Streptococcus pneumoniae, 15, 26, 138, 718, 725, 727 Streptococcus pyogenes, 743 adherence, 721-725 adhesins, 143,725 antigenic mimicry, 742-743 capsule, 730-731 host cell signaling, 727-728 immune response, 732-736 immunopathological-based diseases, 720-721,742-743 invasion and multiplication, 719-720, 728-729 M protein, 725, 728-730, 733 multiplication, 725-727 pathogenesis, 718-743 population genetics, 15, 26 superantigens, 740-742 toxin-mediated disease, 720, 739-742 toxins, 720, 739-742 Streptococcus spp., 717-718 classification, 718 infections. See Streptococcal infections population genetics, 15, 26 toxins, 720, 739-742 viridans group, 718 virulence genes, 736 Streptolysin S (SLS), 732 Stx. See Shiga toxin Subtractive hybridization, pathogenicity factors, 136, 145-147 Subunit vaccine, cholera, 491 Suicide plasmid insertion-duplication mutagenesis, 152 Superantigens, 740-742 Suppressive subtractive hybridization (SSH), 147 Switches, genetic, 98-99, 115 Syc cytosolic chaperones. Yersinia spp., 245-246 SycD protein, 59 SycE protein, 59 SycH protein, 59 Syc proteins, 240 Syringomycin, 205 Systemic acquired resistance proteins. See SAR proteins
INDEX
Tabtoxin, 205 Talin, 233, 354, 776 Tazl protein, 89 TbpA protein, 592, 599 TbpB protein, 592, 599 TCP, 478^81 TcpA protein, 462, 479-480, 483 tcp genes, 462 TcpH protein, 483, 487 tcpl gene, 482 tcp operon, 483 TcpP protein, 483, 488 TCT, Bordetella spp., 630-631 T-DNA, 186, 188,637 "Thymineless death," 162-163 Ti plasmid, 186, 188 Tir protein, 403, 405, 406-407 tlpA gene, 109 TlpA protein, 109 TNF-a, Neisseria spp. and, 594, 595 InphoA mutagenesis, 153 TolC protein, 53-54, 293 TonB/ExbB system, 696 toxA gene, 209 Toxic megacolon, 341 Toxic shock syndrome (TSS) Staphylococcus aureus, 25 Streptococcus pyogenes, 720, 739 Toxin-coregulated pilus. See TCP Toxins Bordetella pertussis, 55 Bordetella spp., 635-636, 637-638 dermonecrotic toxin, 635-636 Escherichia coli, 389, 391-394 phytotoxins, 202-203 Salmonella spp., 310 5/2/^^//« spp., 341,371-372 Streptococcus pyogenes, 720, 739-742 Streptococcus spp., 720 Vibrio cholerae, 48, 138, 461^63, 467^78 Yersinia spp., 232, 238 ToxRprotein, 91,483, 487 ToxRregulon, 481-489 ToxR/ToxT cascade, 483^84 ToxS protein, 481, 483, 487 /ojcr gene, 483-384 toxT-tcpJ gene, 482 tra genes, 55 Transcription, initiation of, 77-79 Transcriptional profiling, pathogenicity factors, 137, 168-169
823
INDEX
Transcriptional regulators, Salmonella spp., 290-302 Transcription factors, 79 covalent modification, 82-86 Vibrio cholerae, 485-488 Transferrin, 372, 592, 695 Translational modulation, 115 Translocase proteins, 306 Translocated intimin receptor. See Tir protein Translocated proteins. Salmonella spp., 306-307 Transposon insertions, 152 Traveler's diarrhea, 390 trb gene, 186 Tropomyosin, 776 TSS. See Toxic shock syndrome TTSS. See Type III secretory system Tuberculosis, 21-22 Tumorigenic plant pathogens, 181-182, 185-189 TveA protein, 245 tyeA gene, 249 TyeA protein, 248 Type I pili, 420, 425, 427 Type IV pili, 478-479 Type I secretory system, 44, 52-54 Type II secretory system, 44, 49-52, 185 plant pathogens, 200-201 Type III secretory system, 44-45, 57-61, 185 Bordetella bronchiseptica, 639 Escherichia coli, 403, 405 plant pathogens, 185, 189-199 Salmonella spp., 303-306 Ysc system, 240-242, 252, 348 Type IV secretory system, 44, 55-57, 185 Agrobacterium tumefaciens, 186-189 plant pathogens, 185-189 Type V secretory system, 44, 47-49 Typhoid fever, 266-269, 273, 312 Typhoid vaccines, 314-316 U ugd gene, 295 Ulcers, Helicobacter pylori, 511,516-517, 540 Urease, H. pylori, 526-527 Urinary tract infections (UTIs), 418 Uropathogenic E. coli (UPEC) disease, 389, 418 hemolysins, 419, 422 pathogenicity islands, 423 virulence factors, 389, 418-423 virulence gene regulation, 423-425 UTIs. See Urinary tract infections
vacA gene, 19-20, 521, 523, 533-534 VacA protein, 525, 532-535 vacB gene, 370 Vaccines Bordetella spp., 641 cholera, 490-493 Haemophilus spp., 676 Neisseria spp., 598-599 pertussis, 621 Sahnonella-bsLsed, 314-316 Vacuolating cytotoxin. See VacA protein vapD gene, 521 VASP, 774, 776, 778 vcpD gene, 471 VcpD protein, 471 VDACs, 587 Verotoxins (VTs), 409 Vi antigen, 310 Vibrio cholerae, 493-494 bacteriology, 458^63 biotypes, 459-461 cholera. See Cholera El Tor strain, 459-460, 464, 480, 491^92 hemolysin, 461 host susceptibility, 464 immune response, 489-490 infection. See Cholera lipopolysaccharide, 465 pathogenicity islands, 3 population genetics, 15, 27 prepilin peptidase, 167 serotype 0139, 140 serovars and serotypes, 459, 466-467 surface biology, 465-467 TCP, 478 toxin, 48, 138, 461^62, 463, 467-478 ToxR protein, 91 transcription factors, 485-488 vaccines, 490-493 virulence, 5 virulence factors, 461-463 virulence gene regulation, 81 Vibrio fischeri, 110 Vibrio harveyii, H9,9\, 110 Vibrio mimicus, 458 Vibrio parahaemolyticus, 458 Vz77nV;spp., 15,27-28,458 Vibrio vulnificus, 458 Villin, 776 Vinculin, 354-355, 359, 774, 776 VirA protein, 89-90, 188
824 VirBI protein, 56 VirB3 protein, 56 VirB4 protein, 56 VirB6 protein, 56 VirB8 protein, 56 VirBIO protein, 56 VirBI I protein, 56 virBgem, 106-109, 186 VirB protein, 56, 106 VirDI protein, 188 VirD2 protein, 55-56, 188 VirD4 protein, 56 VirE2 protein, 55-56, 188 v'//Fgene, 106-109,370 VirF/LcrF, 244 VirFprotein, 56, 81, 105-106 VirG protein, 188, 240, 397-398 Vir proteins, 531 17/7? gene, 370 Virulence. See also Pathogenicity genetics of, 2-8 plant pathogens, 181-211 Salmonella spp., in vitro model, 280-290 Virulence factors. See also Pathogenicity factors Borcletella spp., 628-639 Escherichia coli DAEC, 417^18 EAEC, 415-^17 EHEC, 409-414 EI EC, 396-398 EPEC, 400-407 ETEC, 390-395 SEP, 426-428 UPEC, 418-425 Haemophilus influenzae, 682-699 Listeria monocytogenes, 780-781 Salmonella spp., 290-312 Shigella spp., 369-373 Vibrio cholerae, 461 -463 Yersinia spp., 231 Virulence gene expression, 76. 79-117 Virulence gene regulation, 76, 116-117 adaptive mutation, 114 antisense RNA, 115-116 cell-density-dependent regulation, 110-113 contact-dependent gene regulation, 105 DNA structure and, 94-97 Escherichia coli EHEC, 414 EIEC, 398 EPEC, 407-408 ETEC, 395-396 UPEC, 423-425
INDEX
fimbrial gene expression in E. coli, 101-103 genome structure and, 92-94 Listeria monocytogenes, 81, 782-787 oxidative stress response, 87-88 pap pilus gene transcription, 103-105 plant pathogens, 205-206 protein splicing, 115 raretRNAs, 114 regulatory networks, 86-87 regulatory proteins, 77-79 families, 79-82 modular nature of, 89-92 oxidative stress, 87 S. flexneri regulatory cascade, 106-109 stereotypical and stochastic events in, 97-101 thermometer protein from S. typhimurium, 109-110 transcription factors, 77-86 translation modulation, 115 Yersinia spp., 244-245 Virulence genes absence of suppressor locus and, 5 allelic differences between homologous genes, 6 Borcletella spp., 650-658 classification of, 7 differential regulation of same genes, 6 expression. See Virulence gene expression Haemophilus influenzae, 697-699 Listeria monocytogenes, 756-758 recovery of, 8-9 regulation. See Virulence gene regulation Shigella flexneri, 348-350 Shigella spp., 369-373 species- or strain-specific, 2-5 Streptococcus spp., 736 Virulence plasmids. Salmonella spp., 310-312 Virulence proteins, 43 Vitronectin, 201,578 i7^'.v gene, 646 VsmI protein, 112 VsmR protein, 112 VTs. See Verotoxins W WASP, 360 WAVE. 360 Western blot analysis, 135 Whooping cough, 621 Whooping cough vaccine, 621 Wiskott-Aldrich syndrome protein, 136 Wortmannin. 766
INDHX
Xanthomomis campesths exopolysaccharides, 202 Hrp system, 205 plant pathogenesis, 182, 184 XcpQ protein, 50
yaclA gene, 235 YadA protein. Yersinia spp., 232, 235-236, 246 yajC genes, 46 YajC protein, 46 YbtA protein, 237 YerA protein, 59 Yersiniabactin, 236 Yersinia enterocolitica, 228 adhesive factors, 231-234, 246 infection route, 228-229 iron uptake, 232, 236-237 pathogenicity islands, 237 pYV plasmid, 238 virulence gene regulation, 105, 210 Yop proteins, 242-243 Yersinia infection, 228-230 Yersinia pestis, 228 adhesion factors, 246 infection route, 228-229 iron uptake, 237 pathogenicity islands, 3-4, 237 virulence gene regulation, 105 Yersinia pseudotuberculosis, 228 adhesive factors, 231-234, 246 infection route, 228-229 iron uptake system, 236 virulence gene regulation, 105 Yersinia spp., 228 adhesive factors, 231-236, 246 enterotoxin, 232, 238 host defense mechanism against, 230-231 infection route, 228-229 iron acquisition, 232, 236-237 pathogenicity islands, 237-238 regulatory proteins, 81 Syc cytosolic chaperones, 245-246 virulence, 5 virulence factors, 231, 252 virulence gene regulation, 244-245 virulence plasmid, 238-252
825 virulence proteins, 57-60 Yop proteins. 57-60, 240, 242-244, 246-252 Ysc secretion system, 240-242, 252, 348 YmoA protein, 244 YopB protein, 58-59, 242, 246, 248, 306, 348 YopD protein, 57-59, 242, 246, 248 YopE-Cya hybrid, 247 yopE gene, 248 YopE protein, 57-59, 242, 246-251, 307, 348 u;/? genes, 81, 105,244 YopH protein, 59-60, 138, 242, 246, 248, 250-252, 307, 348 YopJ/Pprotein, 208, 242, 251 YopJ protein, 306 YopK protein, 59 YopK/YopQ, 248 YopM protein, 242, 246 yopN gene, 249 YopN protein, 59, 60, 242, 245 YopO protein, 246 YopO/YpkA, 242, 250-251 YopP protein, 246 Yop proteins, 57-60, 240, 242-244, 246-252, 348 YopP/YopJ, 242, 251 YopQ/YopK, 242 YopR protein. 240, 242 YopTprotein, 242, 246, 251 Yop virulon. 238. 240 YscB protein. 59 YscC protein, 58, 240 YscD protein, 58, 240 YscF protein, 58 ysc genes, 240 YscG protein, 58 Yscl protein, 58 Ysc J protein, 58 YscK protein, 58 YscL protein, 58 YscM 1 protein, 242 YscM/LcrQ proteins, 240 YscM protein, 105 YscN protein, 240 YscO protein, 58, 240 YscP protein, 58, 240, 242 Ysc proteins, 57, 58 YscQ protein, 58 YscR protein, 58, 240 Ysc secretion system, 240-242 YscS protein, 58 YscT protein, 58 YscU protein, 58, 240 Yst protein, 232, 238
826
ZOP, 726 Zymography, pathogenicity factors, 136, 141-143 Zyxin, 774
INDEX
