Legionella Pneumophila: Pathogenesis and Immunity (Infectious Agents and Pathogenesis)

Legionella pneumophila: Pathogenesis and Immunity

INFECTIOUS DISEASES AND PATHOGENESIS Series Editors: Mauro Bendinelli, University of Pisa Herman Friedman, University of South Florida College of Medicine Recent volumes in this series: IN VIVO MODELS OF HIV DISEASE AND CONTROL Edited by Herman Friedman, Steven Specter, and Mauro Bendinelli INFECTIOUS DISEASES AND SUBSTANCE ABUSE Edited by Herman Friedman, Catherine Newton, and Thomas W. Klein CHLAMYDIA PNEUMONIAE INFECTION AND DISEASE Infection and Disease Edited by Herman Friedman, Yoshimasa Yamamoto, and Mauro Bendinelli DNA TUMOR VIRUSES Oncogenic Mechanisms Edited by Giuseppe Barbanti-Brodano, Mauro Bendinelli, and Herman Friedman ENTERIC INFECTIONS AND IMMUNITY Edited by Lois J. Paradise, Mauro Bendinelli, and Herman Friedman HELICOBACTER PYLORI INFECTION AND IMMUNITY Edited by Yoshimasa Yamamoto, Herman Friedman, and Paul S. Hoffman HERPESVIRUSES AND IMMUNITY Edited by Peter G. Medveczky, Herman Friedman, and Mauro Bendinelli HUMAN RETROVIRAL INFECTIONS Immunological and Therapeutic Control Edited by Kenneth E. Ugen, Mauro Bendinelli, and Herman Friedman MICROORGANISMS AND AUTOIMMUNE DISEASES Edited by Herman Friedman, Noel R. Rose, and Mauro Bendinelli LEGIONELLA PNEUMOPHILA: PATHOGENESIS AND IMMUNITY Edited by Paul Hoffman, Herman Friedman, and Mauro Bendinelli

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Legionella pneumophila: Pathogenesis and Immunity

Paul Hoffman Department of Microbiology Medicine, Division of Infectious Diseases and International Health, UVA Health System, University of Virginia, Charlottesville, VA, USA

Herman Friedman College of Medicine, University of South Florida, Tampa, FL, USA

Mauro Bendinelli University of Pisa, Italy

Paul Hoffman University of Virginia, Charlottesville, VA, USA Herman Friedman University of South Florida, Tampa, FL, USA Mauro Bendinelli University of Pisa, Pisa Italy

ISBN-13: 978-0-387-70895-9

e-ISBN-13: 978-0-387-70896-6

Library of Congress Control Number: 2007926437 © 2008 Springer Science⫹Business Media, LLC All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science⫹Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper. 9 8 7 6 5 4 3 2 1 springer.com

Dedication We dedicate this book to the memory of Dr. Herman Friedman, editor and friend, who passed away on August 25, 2007. As scholar, scientist and teacher, he contributed broadly to the disciplines of medical microbiology and immunology, but he will be best remembered for his books, mentorship of young scientists and for his seminal contributions to our knowledge of the immunobiology of Legionnaires’ disease.

Contents

Preface and Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii 1. Legionella pneumophila Pathogenesis: Lessons Learned from Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CHRISTEL CAZALET and CARMEN BUCHRIESER 1. Genomics of Legionella pneumophila . . . . . . . . . . . . . . . . . . . . . . . 1.1. General Features and Organization of the L. pneumophila Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Host–Pathogen Interaction—Specific Features of the L. pneumophila Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Eukaryotic-Like Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Putative Virulence Factors as Deduced Prom Sequence Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Secretion Machineries—Presence of Particular Many and a Wide Variety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Metabolism as Deduced from the Genome Sequence . . . . . . . . . . . . 4. Clues to Adaptation—Regulation and Signal Transduction . . . . . . . 5. Comparative Genomics of Legionella pneumophila . . . . . . . . . . . . . 5.1. The Specific Gene Complements of L. pneumophila Paris, Lens and Philadelphia . . . . . . . . . . . . 5.2. Genomic and Pathogenicity Islands . . . . . . . . . . . . . . . . . . . . . 5.3. Plasmids of Legionella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.4. Variability at the Gene Level . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Comparative Genomics— Legionella, Coxiella and Other Intracellular Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.1. Genome Comparison of Legionella pneumophila and Coxiella burnetii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. Genome Redundancy—Gene Family Expansion in Legionella . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Comparative Genomics of Legionella pneumophila and the Genus Legionella —a Perspective . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 1 1 3 3 11 13 14 15 17 17 18 20 22 23 23 24 25 26 vii

viii

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2. Iron Assimilation and Type II Protein Secretion . . . . . . . . . . . . . . . . . . NICHOLAS P. CIANCIOTTO

33

1. Iron Assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The Importance of Iron for Legionella pneumophila . . . . . . . . 1.2. Legionella Siderophores . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Ferrous Iron Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4. The L. pneumophila Iron Acquisition/Assimilation Locus . . . . 1.5. Cytochrome C Maturation and Iron Assimilation . . . . . . . . . . . 1.6. Legionella Ferric Reductases . . . . . . . . . . . . . . . . . . . . . . . . . . 1.7. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Type II Protein Secretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Type II Protein Secretion in Gram-Negative Bacteria . . . . . . . 2.2. Discovery of Type II Secretion in Legionella pneumophila . . . 2.3. Factors Secreted by the Type II System of L. pneumophila . . . 2.4. Role of Type II Secretion in Pathogenesis . . . . . . . . . . . . . . . . 2.5. Role of Type II Secretion in Environmental Survival . . . . . . . . 2.6. Concluding Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

33 33 34 37 38 38 39 39 40 40 41 42 43 44 45 45

3. The Dot/Icm Type IVB Secretion System . . . . . . . . . . . . . . . . . . . . . . . JASON J. LEBLANC and JOSEPH P. VOGEL

49

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Dot/Icm Type IV Secretion System . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Structural Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Adaptor/Chaperone Complexes . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Regulation of Effector Export . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Genetic Regulation of the Dot/Icm System . . . . . . . . . . . . . . . . . . . 4. Possible Effector Proteins and Associated Functions . . . . . . . . . . . . 4.1. RalF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. The Sid Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. LidA and SidM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. LepA and LepB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.5. Ylf and the Vips . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Temporal Modulation of the LCV Surface . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49 50 52 53 54 54 55 55 56 56 57 57 58 59 59

4. Life Cycle, Growth Cycles and Developmental Cycle of Legionella pneumophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . RAFAEL A. GARDUÑO

65

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Life Cycle of L. pneumophila as Currently Understood . . . . . . 2.1. Attachment and Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65 66 67

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2.2. Post-Internalization Events and Intracellular Growth . . . . . . . . 2.3. End of Replication and Exit . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. What Role Do Ciliates Play in the Life Cycle of L. pneumophila? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Growth Cycles of L. pneumophila . . . . . . . . . . . . . . . . . . . . . . . 4. The Developmental Cycle of L. pneumophila . . . . . . . . . . . . . . . . . 4.1. Legionella pneumophila Development Along the Extracellular Growth Cycle . . . . . . . . . . . . . . . . . . . 4.2. MIF: The Result of L. pneumophila Intracellular Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. The Central Role of MIFs in the Study of L. pneumophila Virulence . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

68 69 69 70 72 73 76 77 78 79

5. Legionella in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . BARRY S. FIELDS

85

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. The Distribution of Legionella as a Function of Various Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Natural Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Man-Made Environments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Interaction with Other Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Association with Biofilms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

85

6. Regulation of the Legionella pneumophila Life Cycle . . . . . . . . . . . . . RACHEL L. EDWARDS and MICHELE S. SWANSON

95

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Life Cycle of L. pneumophila . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Broth Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Amino Acid Availability Governs Differentiation . . . . . . . . . . . . . . 4.1. Pht Family of Transporters . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Stringent Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Evidence that Other Factors Trigger Differentiation . . . . . . . . . . . . 5.1. Acetyl-phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Transcriptional Control of Differentiation via Sigma Factors . . . . . 6.1. RpoS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.2. RpoN and FleQ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6.3. FliA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Post-Transcriptional Control of Differentiation . . . . . . . . . . . . . . . . 7.1. LetA/LetS Two-Component System . . . . . . . . . . . . . . . . . . . . . 7.2. Carbon Storage Regulatory (Csr) System . . . . . . . . . . . . . . . . . 7.3. Hfq and Small RNAs (sRNAs) . . . . . . . . . . . . . . . . . . . . . . . . .

95 95 97 98 98 100 101 101 102 103 103 103 104 104 105 106

85 87 88 90 90 91

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Contents

8. Regulation of the Dot/Icm Type IV Secretion System . . . . . . . . . . . 107 9. Genomic Methods of Studying Differentiation . . . . . . . . . . . . . . . . . 108 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 7. Microbial Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 PAUL S. HOFFMAN 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Intracellular Growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Morphology, Ultrastructure, and Surface Proteins . . . . . . . . . . . . . 3.1. Envelope Structure and Lipopolysaccharide (LPS) . . . . . . . . 3.2. Outer Membrane Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Secreted Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Motility and Chemotaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Respiratory Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Central Intermediary Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Biosynthetic Capacity and Amino Acid Auxotrophies . . . . . . . . . . 7. Protection from Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . 8. Heat Shock Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Stationary Phase Genes and Cell Wall Assembly . . . . . . . . . . . . . . 10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

113 114 115 116 116 117 118 119 119 123 124 126 127 128 128

8. Legionnaires’ Disease—Clinical Picture . . . . . . . . . . . . . . . . . . . . . . . . 133 THOMAS J. MARRIE 1. 2. 3. 4.

5. 6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Epidemiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Clinical Features of Legionnaires’ Disease . . . . . . . . . . . . . . . . . . . . Diagnosis of Legionella Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Routine Laboratory Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Specific Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pontiac Fever . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Nosocomial Legionnaire’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

133 133 137 140 140 140 141 143 143 144 144

9. Legionella pneumophila: Innate and Adaptive Immunity . . . . . . . . . . . 151 HERMAN FRIEDMAN, CATHERINE NEWTON and THOMAS KLEIN 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Legionella Immunogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Innate Immunity to L. pneumophila . . . . . . . . . . . . . . . . . . . . . . . . . Adaptive Immunity to L. pneumophila Infection . . . . . . . . . . . . . . .

151 152 153 156

Contents

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5. Immune Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Discussion and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

157 164 166 167

10. Nonspecific Stimulation of Immunity Against Legionella . . . . . . . . . . 173 JAMES ROGERS, AMAL HAKKI and HERMAN FRIEDMAN 1. 2. 3. 4. 5. 6. 7. 8. 9.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Stimulation of Immunity by Natural Products . . . . . . . . . . . . . . . . . Antibacterial and Antiviral Effects of Epigallocatechin Gallate . . . . Modulation of Cytokines by Natural Products . . . . . . . . . . . . . . . . . The Effects of EGCG on Cytokine Production by Legionella-Infected Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . The Effects of EGCG on Co-stimulatory Molecule Expression by Legionella-infected Dendritic Cells . . . . . . . . . . . . . The Effects of EGCG on Chemokine Production by Legionella-infected Dendritic Cells . . . . . . . . . . . . . . . . . . . . . . . Immunomodulatory Effects of Natural Products on TLR Expression and Signal Transduction . . . . . . . . . . . . . . . . . . Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

173 174 174 175 176 177 178 179 181 182

11. Interaction of Legionella pneumophila with Amoeba . . . . . . . . . . . . . . 185 MAËLLE MOLMERET, MARINA SANTIC, and YOUSEF ABU KWAIK 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Legionellae: A Facultative Intracellular Pathogen of Free-Living Amoebae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. The Role of Amoebae in Persistence of Legionella in the Environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. The Role of Amoebae in Pathogenesis of Legionella . . . . . . . . . . . . 5. Entry of L. pneumophila into Protozoa . . . . . . . . . . . . . . . . . . . . . . . 6. Intracellular Trafficking and Multiplication of L. pneumophila . . . . 7. Role of the dot/icm Genes in Evasion of the Endocytic Pathway . . . 8. Egress of L. pneumophila from Amoebae . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

185 187 188 189 190 191 192 194 196

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

Preface and Introduction

The summer of 2006 marked the 30th anniversary of the outbreak of Legionnaires’ disease, an acute pneumonia that occurred among veterans who had gathered in Philadelphia, Pennsylvania, in 1976 to celebrate the bicentennial of the founding of the United States. Ironically, the nation and world were braced for an epidemic of swine influenza, which never materialized and no one expected the emergence of a new bacterial pathogen, since most in the medical field sincerely believed that all the pathogens of humans were now known. This new bacterial agent of Legionnaires’ disease, Legionella pneumophila (named in memory of the deceased veterans) was a harbinger of diseases to come (emerging pathogens) that has included HIV/AIDS, SARS, Lyme disease, hamburger disease, and many others. The further discovery that L. pneumophila was ubiquitous to aquatic environments worldwide and resided as an intracellular parasite of amoeba and protozoa provided a link between natural environments and human disease. Thus, environmental monitoring, especially of potable water, cooling towers, and related sources, has become a major focus in efforts to control the spread of this disease. Fortunately, as noted in the 1976 epidemic and holds true today, the disease is not spread from human to human. The remarkable ability of L. pneumophila to multiply in alveolar macrophages, as if they were amoebae, has further contributed to our understanding of the disease and stimulated much research in the area of cellular microbiology of pathogenesis. Today, Legionnaires’ disease is both sporadic (community acquired) and epidemic (explosive point sources such as cooling towers) and is on the rise worldwide – attributed to a greater dependence on technology and air conditioning together with an increasingly susceptible population due to medical technology. Despite all the advances in medicine, Legionnaires’ disease continues to have a high mortality rate, not uncommon when pneumonia is involved. Advances in understanding pathogenesis and immunity have benefited greatly from the availability of three genome sequences for L. pneumophila and one for close relative Coxiella burnetii. Both Legionella and Coxiella produce survival forms and display a developmental cycle which contribute to pathogenesis, persistence, and resistance to biocides in natural environments. There have been many major developments in the Legionella field ranging from strategies to treat infections to how xiii

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Preface and Introduction

this organism interacts with innate immune mechanisms to promote infections. This book represents the first collection of chapters written by experts in each of the specialty areas in this field. Unlike past books, which have been proceedings from international meetings, we have provided an opportunity for each of the authors to present “state of the art” detailed descriptions of specific areas that permit interesting reading for the non-expert and a solid reference for those researchers active in this field. The authors of each of the chapters in this book are internationally recognized experts concerning Legionella infections. The first chapter by Drs. Christel Cazalet and Carmen Buchreiser from the Institute Pasteur, Paris, France, concerns the molecular biology and genetics of L. pneumophila pathogenesis. The authors describe in-depth laboratory investigations of the genetic differences between the numerous strains of these opportunistic bacteria and provide important new information how these microbes interact with environmental factors and their host. The next chapter by Dr. Nicholas Cianciotto, Northwestern University Medical School, Chicago, Illinois, stresses important physiological aspects of Legionella–host interactions, especially the important role of iron assimilation and the nature and mechanism of protein secretion by these bacteria. Drs. J. Le Blanc, Dalhouse University, Halifax, Nova Scotia from Canada, and Joseph P. Vogel from St. Louis, Missouri, then describe the metabolic activity of these ubiquitous microbes, especially the nature and mechanism of their secretion system. Dr. Rafael Garduño, Dalhousie University, Halifax, Nova Scotia Canada, then discusses pathogenesis and immunity to that bacterium, including some unique features of the life as well as developmental cycle of L. pneumophila in a host, both human and protozoa in their natural habitat. Dr. Barry Fields, Centers for Disease Control and Prevention, Atlanta, Georgia, then presents detailed observations how Legionella are widely dispersed in the environment, especially freshwater habitats. Drs. Rachel Edwards and Michele Swanson, University of Michigan, Ann Arbor, Michigan, describe how the life cycle of these bacteria is regulated and information necessary to control the presence of the bacteria, especially in the environment, and treatment modalities with antibiotics which abrogate bacterial replication and/or disease progression. Although no effective vaccine has yet been developed, information concerning interaction of these Legionella with the human host and how they infect host cells, as well as various antigenic structures of the bacteria important for infectivity and replication, provide a foundation necessary for developing protective vaccines which can induce adaptive immunity. Dr. Paul Hoffman, University of Virginia, provides new information about the interesting unique physiology of L. pneumophila and other species of these bacteria. He compares different aspects of their physiology in the human host vs. replication in amoeba and protozoa. Dr. Thomas J. Marrie, Walter C. Mackenzie Health Science Center, Edmonton, Alberta, Canada, then describes medical and clinical aspects of Legionnaire’s disease which first attracted attention in the epidemic pneumonia outbreak in a hotel in Philadelphia during the annual convention of Pennsylvania State Legionnaires after which the disease is named. His chapter describes clinical aspects of the

Preface and Introduction

xv

infection in individuals showing respiratory symptoms. It is also now widely recognized that many individuals have subclinical exposure to Legionella, as evident by high titers of specific humoral antibody and marked in vitro blastogenic responsiveness of their blood lymphocytes to Legionella antigen. Drs. H. Friedman, C. Newton, and Thomas W. Klein, University of South Florida, discuss in detail the exploding new knowledge concerning immunity to L. pneumophila in terms of newer information about innate and adaptive specific immune responses to these ubiquitous opportunistic intracellular bacteria which primarily cause serious infection in immunocompromised individuals but which have their natural habitat single cell protozoa. They apparently interact with human hosts because of their ubiquitous presence in warm lakes, airconditioning cooling towers, circulating warm water in plumbing, and drinking water in institutions such as hospitals. The next chapter by Drs. J. Rogers, A. Hakki, and H. Friedman, also from the University of South Florida, is concerned with information how nonspecific immune stimulators modify host susceptibility to infection by opportunistic bacteria such as Legionella. This chapter reviews nonspecific modulators of both innate and adaptive immune mechanisms which impact the host immune response to an opportunistic pathogen like Legionella. Plant-derived nonspecific immunoenhancing substances are being investigated worldwide and various small molecular weight substances used for centuries as “folk medicine” to ameliorate infections have been shown clinically to significantly enhance host resistance to ubiquitous microbial infections. Drs. Maelle Molmeret and Yousef Abu Kwaik, University of Louisville, Louisville, Kentucky, then describe in detail information concerning L. pneumophila interactions with amoeba in vitro. Knowledge about such interactions is important to understand the nature and mechanism whereby these bacteria interact with phagocytic cells of the human host and the many biochemical and physiological features of legionellae common to their uptake and regulation in different cell types. The editors as well as authors of individual chapters believe the many ongoing studies of host immunity to ubiquitous opportunistic pathogens like L. pneumophila will undoubtedly lead to more effective control methods to prevent or ameliorate infections by such microorganisms, which still account for many thousands of infections annually in the United States alone. Many details concerning the nature and mechanism of interactions between host and opportunistic intracellular organisms like Legionella, which preferentially infect phagocytic cells, are being clarified and provide important knowledge directly related to mechanisms of host resistance to other important intracellular opportunistic pathogens, including Mycobacteria which cause tuberculosis. We thank Ms. Ilona Friedman for continuing excellent editorial assistance correlating and assisting in preparation of manuscripts for this book in the series. Paul S. Hoffman Herman Friedman Thomas W. Klein

Contributors

Carmen Buchrieser • Unité de Génomique des Microorganismes Pathogegens, Institut Pasteur, Paris, FRANCE Christel Cazalet • Unité de Génomique des Microorganismes Pathogegens, Institut Pasteur, Paris, FRANCE Nicholas P. Cianciotto • Department of Microbiology-Immunology, Northwestern University Medical School, Chicago, IL Rachel L. Edwards • Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI Barry S. Fields • Centers for Disease Control and Prevention, Atlanta, GA Herman Friedman • Department of Molecular Medicine, University of South Florida, College of Medicine, Tampa, FL Rafael A. Garduño • Department of Microbiology and Immunology and Department of Medicine, Dalhousie University, Halifax, Nova Scotia, CANADA Amal Hakki • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL Paul S. Hoffman • Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia Health Systems, Charlottesville, VA Thomas W. Klein • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL Yousef Abu Kwaik • Department of Microbiology and Immunology, University of Louisville, Louisville, KY Jason J. LeBlanc • Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, CANADA Thomas J. Marrie • Department of Medicine, University of Alberta, Edmonton, Alberta, CANADA

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Contributors

Maelle Molmeret • Department of Microbiology and Immunology, University of Louisville, Louisville, KY Catherine Newton • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL James Rogers • Department of Molecular Medicine, University of South Florida College of Medicine, Tampa, FL Marina Santic • Department of Microbiology and Parasitology, University of Rijeka, Rijeka, CROATIA Michele S. Swanson • Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI Joseph P. Vogel • Department of Molecular Microbiology, Washington University, St. Louis, MO

1 Legionella pneumophila Pathogenesis: Lessons Learned from Genomics Christel Cazalet and Carmen Buchrieser

The last years have seen a giant step in the genomics of Legionella pneumophila. The establishment and publication of the complete genome sequences of three clinical L. pneumophila isolates in 2004 paves now the way for major breakthroughs in understanding the biology of L. pneumophila in particular and Legionella in general (Cazalet et al. 2004; Chien et al. 2004). This chapter starts with a description of the three genome sequences of L. pneumophila strain Paris, L. pneumophila strain Lens and L. pneumophila strain Philadelphia 1 and then highlights the characteristic features and common traits of the Legionella genomes. Emphasis is given on putative virulence and Legionella life cycle–related functions. In the second part, we focus on the comparison of these three complete genome sequences, in order to learn about the plasticity of the Legionella genomes and the possible mechanisms involved. In the third part we compare the Legionella genomes with those of their closest relative, Coxiella burnetii, to try to trace the genetic basis of their common features. Finally, other Legionella species, for which genomic data are available, are discussed briefly and future perspectives in Legionella genomics are presented.

1. Genomics of Legionella pneumophila 1.1. General Features and Organization of the L. pneumophila Genomes The complete genome sequences of three strains of L. pneumophila (Paris, Lens and Philadelphia 1) are available at the time of writing (Cazalet et al. 2004; Chien et al. 2004). Legionella pneumophila has a single, circular chromosome, 3,503 610 base pairs (bp) (Paris), 3,345 687 bp (Lens) and 3,397 754 bp (Philadelphia 1) in size, with an average G ⫹ C content of 38% (Table 1.1; Figure 1.1. Strains Paris and Lens each contain a plasmid, 131.9 kb and 59.8 kb in size, respectively. In strain Philadelphia 1 no plasmid was identified. The genomes contain each ⬃3000 genes distributed fairly evenly between the two strands (⬃57% on the leading strand) and accounting for ⬃88% of the potential coding capacity. No function 1

2

Christel Cazalet and Carmen Buchrieser

TABLE 1.1. General features of the three sequenced Legionella pneumophila genomes.

Size of the chromosome (bp) G ⫹ C content Total number of protein-coding genes Average length of protein-coding genes Number of rRNA operons (16S-23S-5S) Number of tRNA genes Percentage coding Plasmid1 Number of strain specific genes Number of orthologous genes

L. pneumophila Paris

L. pneumophila Lens

L. pneumophila Philadelphia

3,503,610

3,345,687

3,397,754

38.3% (37.4%) 3077 (141)

38.4% (38.4%) 2931 (57)

38% 2953

331*

333*

338*

3

3

3

43 87.9% (92%) 1 (131.9 kb) 313 (125)

43 88% (83.7%) 1 (59.8 kb) 259 (44)

43 89.8 – 335

2500

2500

2500

*codons 1kilobase-pairs

() plasmid related

could be predicted for 42.1% of the L. pneumophila Paris, 44.1% of the L. pneumophila Lens and for 28% of the L. pneumophila Philadelphia 1 genes. A high proportion of the predicted genes (21% for strain Paris, 20.4% for strain Lens, 17% for strain Philadelphia 1) are unique to the genus Legionella. Comparative analysis of the genome structure of the three L. pneumophila genomes showed high colinearity, with only few translocations, duplications, deletions or inversions (Figure 1.2). Principally, the genomes contain three large plasticity zones, where the synteny is disrupted: a 260kb inversion in strain Lens with respect to strains Paris and Philadelphia 1, a 130kb fragment which is partly similar partly different and which is inserted in a different genomic location in strains Paris and Philadelphia 1 but which is truncated in strain Lens and the chromosomal region carrying the Lvh type IV secretion system (Figure 1.2). The Lvh type IV secretion system, previously described in strain Philadelphia (Segal et al. 1999) is encoded by a 36 kb region (strain Paris) or a 45 kb region (strain Philadelphia 1) that can be either carried on a multi-copy plasmid or be integrated into the chromosome. Further diversity on the genome level is present as deletions and insertions of several smaller regions were identified in each strain, as well as regions where the gene content is variable. Finally, also variations at the gene level are present as discussed later. The sequences and their analysis are accessible at http://genolist.pasteur.fr/LegioList; http://genome3.cpmc.columbia.edu/⬃legion/project/.

1. Legionella pneumophila Pathogenesis

3

0 10

3.0

8

1

9

0.5 2

Legionella pneumophila strain Paris

3

3 503 610 bp

1.0

2.5 4

7 2.0

65

1.5

FIGURE 1.1. Circular genome map of L. pneumophila strain Paris and the specific genes with respect to strain Lens and Philadelphia. From the outside—circle 1: Strain Paris genes on the ⫹ and – strands, respectively. Red line, inversion in strain Lens. Numbers indicate their position: 1 lvh-lvr type IV secretion system (lvrABC, lvhB2B3B4B5, lvrD, lvhB6B8B9B10B11D4, lvrE); 2 dot/icm type IV secretion system (icmTSRQOMLKEGCDJBF); 3 mip, 4 lspA, 5 lspDE, 6 htrA, 7 lspFGHIJK, 8 enhABC, 9 dot/icm type IV secretion system (icmVWX and dotABCD), 10 momp; circle 2: specific genes of strain Paris with respect to strain Lens; circle 3: specific genes of strain Paris with respect to strain Philadelphia; circle 4: G/C bias (G ⫹ C/G-C) of strain Paris; circle 5: G ⫹ C content of strain Paris with ⬍32.5% G ⫹ C short length, between 32.5% and 44.1% medium length and with ⬎44.1% G ⫹ C long lines. The scale (Mb) is indicated on the outside, origin of replication at position 0.

2. Host–Pathogen Interaction—Specific Features of the L. pneumophila Genomes 2.1. Eukaryotic-Like Proteins Protozoa are essential for the growth of Legionella. The interaction of L. pneumophila with aquatic protozoa seems to have generated a pool of virulence traits during evolution, which allow Legionella to infect also human cells. This is reflected in the genome sequence of this pathogen, as it codes an unexpected

Christel Cazalet and Carmen Buchrieser A

B

L. pneumophila

Lens

2

1

2

L. pneumophil a Philadelphia

4

2 4

3 2

3

L. pneumophila

Paris

L. pneumophila

Paris

FIGURE 1.2: Synteny plot of the chromosomes of strains Paris and Lens (A) and strains Paris and Philadelphia (B). Inversions between the genomic sequences are represented in blue. Genome-wide synteny is disrupted by a 260 kb inversion 1 and a 130 kb plasticity zone in strain Paris 2 as well as by some smaller clusters. The insertion 2 is also present in strain Philadelphia but in a different chromosomal location as well as the lvh secretion system 3. Synteny is also disrupted by an insertion of a 65 kb pathogenicity island in strain Philadelphia 4. Plots were created with the mummer software package developed by Tigr (http://www.tigr.org).

high number and variety of eukaryotic-like proteins, which are probably able to interfere in all different steps of the infectious cycle by mimicking functions of eukaryotic proteins. The large number and wide variety of eukaryotic-like proteins present in the L. pneumophila genomes is to date unique for a prokaryotic genome. Half of these proteins show the strongest similarity to eukaryotic proteins and the others contain motifs known to be implicated in protein–protein interactions mostly or uniquely present in eukaryotes, suggesting that L. pneumophila may have developed specific mechanisms to cross-talk with its eukaryotic hosts. It is thus likely that these proteins confer to L. pneumophila the capacity to subvert host functions and to modulate them to its advantage. When comparing the three sequenced genomes, it becomes evident that most of these proteins should fulfill important functions, as nearly all are conserved in the three sequenced genomes (Tables 1.2A and 1.2B). Recently, analysis of the gene content of 180 strains by DNA–DNA hybridization showed that the eukaryotic-like genes identified during sequence analysis are with small exceptions, conserved in all the strain tested (Cazalet et al. 2005a). These results support the hypothesis that they are required for intracellular growth and that they may play a role in virulence. Some of these proteins are predicted to have a role in different regulatory pathways, others may be secreted into the eukaryotic cell and thus be implicated

34 39

lpp0321

lpp1157

RNA binding protein precursor pyruvate decarboxylase

38 36 39

36 41 39 39 36 39 40

lpp2832

plpp0050

lpp0634

lpp0965

lpp2748 lpp2128

lpp0489 lpp0955 lpp0578 lpp0379 lpp1033

DegP protease

phytanoyl-coA dioxygenase sphingosine-1-phosphate lyase glucoamylase cytokinin oxidase phytanoyl coA dioxygenase Hypothetical protein ectonucleoside triphosphate diphosphohydrolase (apyrase)

39

38

lpp1522

thiamine biosynthesis protein NMT-1 nuoE NADH dehydrogenase I chain E hypersensitive induced response protein hypothetical protein

39

lpp0702

exoA exodeoxyribonuclease III

G–C(%) 38

L.p. Paris lpp1647

Predicted product purC

lpl0465 lpl0925 lpl0554 lpl0354 lpl1000

lpl2621 lpl2102

lpl0935

lpl0618

–

lpl2701

lpl1461

lpl1162

–

lpl0684

L.p.Lens lpl1640

39 39 37 40 39

36 41

40

39

–

38

39

39

–

39

G–C(%) 39

lpg0422 lpg0894 lpg0515 lpg0301 lpg0971

lpg2694 lpg2176

lpg0903

lpg0584

–

lpg2785

lpg1565

lpg1155

lpg0251

lpg0648

L.p.Phila lpg1675

39 40 37 40 40

36 40

40

39

–

37

40

40

37

39

G–C(%) 40

(Continued)

(P42042 Arxula adeninivorans) (NP_484368.1 Nostoc sp.) (EAA70100.1 Gibberella zeae) (CAD21525.1 Taenia solium) nucleoside phosphatase signature conserved (Q9MYU4 Sus scrofa)

(AAN17462.1 Hordeum vulgare subsp. Vulgare) (XP_306643.1 Anopheles gambiae) (NP_189431.2 Arabidopsis thaliana) (XP_372144.1 Homo sapiens) (NP_775139.1 Rattus norvegicus)

Best Hit BLASTp (AAR06292.1 Nicotania tabacum) (EAA20230.1 Plasmodium yoelii yoelii) (AAL07519 Solanum tubeosum) (AAB16855.1 Arabidopsis thaliana) (AAC64375.1 Botryotinia fuckeliana) (BAA25988.1 Homo sapiens)

TABLE 1.2A. Proteins with the highest similarity score to eukaryotic proteins and their distribution in the three sequenced strains.

lpp1127

lpp1167 lpp2626 lpp1439

uridine kinase ser/thr proteine kinase domain serine threonine protein kinase

36 41

lpp1665 lpp1959

Ca2 ⫹ -transporting ATPase

39 34

lpp2468 lpp1824

33 32 36

37

38

35

lpp2747

lpp0358

39

lpp1880

hypothetical protein

35

lpp2134

38

lpp3071

SAM dependent methyltransferase ectonucleoside triphosphate diphosphohydrolase (apyrase) SAM dependent methyltransferase cytochrome P450 nuclear membrane binding protein uracyl DNA glycosylase chromosome condensation 1-like

34

lpp2923

6-pyruvoyl-tetrahydropterin synthase zinc metalloproteinase

G–C(%)

L.p. Paris

Predicted product

TABLE 1.2A. (Continued)

lpl1173 lpl2481 lpl1545

lpl1131

lpl0334

lpl1659 lpl1953

lpl2326 –

lpl2620

lpl1869

lpl2109

lpl2927

lpl2777

L.p.Lens

34 32 35

37

38

36 38

39 –

35

39

36

38

35

G–C(%)

lpg1165 lpg2556 lpg1483

lpg1126

lpg0282

lpg1700 lpg1976

lpg2403 –

lpg2693

lpg1905

lpg2182

lpg2999

lpg2865

L.p.Phila

34 32 36

38

39

37 43

38 –

36

40

36

38

35

G–C(%)

Best Hit BLASTp

(EAA36774.1 Giardia lamblia) Pattern of chromosome condensation regulator conserved (EAA20288.1 Plasmodium yoelii yoelii) (AAB81284.1 Paramecium tetraurelia) (AAM09314.2 Mus musculus) conserved domain conserved domain

(CAE70887.1 Caenorhabditis briggsae) (EAA20288.1 Plasmodium yoelii yoelii) (NP_487786.1 Nostoc sp.) (NP_082559.1 Mus musculus)

(NP_703938.1 Plasmodium falciparum) (AAF56122.1 Drosophila melanogaster) (BAC98835.1 Bombyx mori)

L.p. Paris enhC (lpp2692) lidL(lpp1174)—EnhC paralog lpp1310—EnhC paralog lpp2174—EnhC paralog – ralF (lpp1932) lpp0267 lpp2626 lpp1439 lpp2065 lpp0037 plpp0098 lpp2058 lpp0750 lpp2061 lpp2270 lpp0503 lpp1905 lpp1683 lpp2248 lpp0202 lpp0469 lpp2517 lpp1100 lpp0126 lpp0356 lpp2522 lpp0547

41 40 45 34 38 32 36 37 38 37 38 35 39 34 38 35 33 39 38 38 36 48 39 38 39 40

G–C% 39 38

L. p.Lens enhC (lpl2564) lidL (lpl1180)—EnhC paralog lpl1307—EnhC paralog lpl1303—EnhC paralog lpl1059—EnhC paralog ralF lpl1919 lpl0262 lpl2481 lpl1545 lp2055 lpl0038 – lpl2048 lpl0732 lpl2051 lpl2242 lpl0479 – lpl1682 lpl2219 – lpl0445 lpl2370 – lpl0111 – lpl2375 lpl0523 41 39 45 34 39 32 35 37 39 – 38 35 39 34 36 – 34 39 – 38 37 – 39 – 39 41

G–C(%) 39 39

L.p.Philadelphia enhC (lpg2639) lidL (lpg1172)—EnhC paralog lpg1356—EnhC paralog lpg2222—EnhC paralog lpl1062—EnhC paralog ralF (lpp1950) lpg0208 lpg2556 lpg1483 lpg2214 lpg0038 – – lpg0695 – lpg2322 lpg0436 – lpg1718 lpg2300 – lpg0403 lpg2452 – lpg0112 – lpg2456 lpg0483 41 41 44 35 38 32 36 42 38 – – 36 – 35 37 – 34 39 – 39 37 – 39 – 40 42

–C(%) 39 39

(Continued)

4 sel-1 domains 3 sel-1 domains 7 sel-1 domains sec7 domain ser/thr kinase domain ser/thr kinase domain ser/thr kinase domain ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ⫹ SET ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat

Motif identified 21 sel-1 domains 6 sel-1 domains

TABLE 1.2B. Legionella pneumophila proteins-encoding domains preferentially found within eukaryotic proteins and their distribution in the three sequenced strains.

G–C% 34 35 40 38 39 36

34

43 39 35

L.p. Paris – – – – – lpp2082

lpp2486

– lpp0233 lpp2887

TABLE 1.2B. (Continued)

– lpl0234 –

–

L. p.Lens lpl1681 lpl2344 lpl2058 – – lpl2072

– 39 –

–

G–C(%) 34 35 40 – – 36

lpg2224 lpg0171 lpg2830

–

L.p.Philadelphia – – lpg2128 lpg0402 lpg2131 lpg2144

43 40 35

–

–C(%) – – 37 38 39 37

Motif identified ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat ankyrin repeat F-box domain ⫹ ankyrin repeat F-box domain ⫹ coiled-coil F-box domain F-box domain 2 U-box domains

1. Legionella pneumophila Pathogenesis

9

in different stages of the intracellular cycle of Legionella like in invasion, trafficking into host cell, modulation of host-cell functions and in evasion. Proteinmotifs predominantly found in eukaryotes, which were identified in the L. pneumophila genomes, are ankyrin, Sel-1 (TPR), U-box and F-box motifs. In Legionella ankyrin repeat proteins might play an important role in the intracellular life cycle as they form the most prominent family of proteins with eukaryotic-like domains. It is also the most heterogeneous protein family of the eukaryotic like proteins, as only twelve ankyrin repeat proteins are shared by all three isolates. In addition, strain Paris, Lens and Philadelphia 1 encode each six, four or two specific ankyrin proteins, respectively (Table 1.2B). Large families of ankyrin repeat proteins are so far only identified in C. burnetii (Seshadri et al. 2003) and Wolbachia pipitentis (Wu et al. 2004) and recently in Rickettsia felis (Ogata et al. 2005), all intracellular bacteria. Ankyrin proteins are multifunctional and involved in many cellular pathways, thus the prediction of the function of the Legionella ankyrin proteins is difficult. They might be involved in the interaction with the host cytoskeleton (Batrukova et al. 2000) or in targeting proteins to the plasma membrane or to the endoplasmic reticulum (HryniewiczJankowska et al. 2002). These protein motifs are frequently found in eukaryotic proteins, but their distribution is rather limited in viruses and bacteria, in both of which they appear to be linked with pathogenicity. Another interesting finding is the presence of F-box and U-box domain containing proteins in the Legionella genomes. One could speculate that these proteins may allow L. pneumophila to interfere with the ubiquitin machinery of eukaryotic cells. Three F-box proteins, one of which is specific for each strain, are present in strains Paris and Philadelphia 1, strain Lens contains two F-box proteins (Table 1.2B). Strain Philadelphia 1 and Paris encode the only U-box protein identified in a prokaryotic genome to date. Ubiquitination is a protein modification generally used by cells to tag proteins that are destined for proteasomal degradation. Ubiquitin is a highly conserved 76 amino acid polypeptide that can be covalently attached to a lysine residue of the target protein by an E3-ubiquitin ligase, which is primarily responsible for providing substrate specificity to ubiquitin conjugation. Several classes of ubiquitin ligases have been described based on the presence of specific domains, one of which carries U-box or F-box domains (Hershko and Ciechanover 1998; Heyninck and Beyaert 2005). Thus the L. pneumophila U-box and F-box proteins might modulate the eukaryotic ubiquitination machinery. F-box proteins assembled into SCF ubiquitin ligase complexes determine which substrate will be targeted for ubiquitination and subsequent proteolysis by the proteasome. Thus it is tempting to assume that L. pneumophila is able to tag proteins for ubiquitination, which then in turn are degraded by the proteasome. Four proteins with homologies only to eukaryotic proteins were identified for the first time in a prokaryotic genome: a sphingosine-1-phosphate lyase, a sphingosine kinase and two secreted apyrases, which are conserved in all three L. pneumophila genomes (Table 1.2A). In eukaryotes, sphingosine-1-phosphate lyase and sphingosine kinase are part of the sphingomyelin degradation pathway.

10

Christel Cazalet and Carmen Buchrieser

Sphingosine kinase phosphorylates sphingosine, a catabolite of ceramide, into sphingosine-1-phosphate, which is cleaved irreversibly by sphingosine1-phosphate lyase. As the balance of both ceramide and sphingosine-1-phosphate is important for inducing authophagy or apoptosis (Reiss et al. 2004), the presence of this protein in the Legionella genomes suggests that Legionella may influence the autophagy machinery, which recognizes Legionella phagosomes for lysosome delivery (Amer and Swanson 2005; Swanson and Fernandez-Moreira 2002). Recently, proteins homolog to sphingosine-1-phosphate lyase have been identified in other prokaryotic genomes like Symbiobacterium thermophilum, Erythrobacter litoralis, Burkholderia thailandensis and Burkholderia pseudomallei. Enzymes not identified in any other prokaryote to date are two apyrases (Table 1.2A). These enzymes have been isolated in authophagy vacuoles (Biederbick et al. 1999) and may thus influence the L. pneumophila phagosome by decreasing the concentration of NTPs and NDPs. Serine/threonine protein kinases (STPKs) are known to be essential virulence factors in other bacteria. They were shown to be, e.g., translocated into the host cells where they counteract the host defense by interfering with eukaryotic signal transduction pathways (Barz et al. 2000; Hakansson et al. 1996) or to be secreted into macrophage phagosomes, inhibiting phagosome–lysosome fusion and promoting intracellular survival (Walburger et al. 2004). The L. pneumophila strains encode three eukaryote-like STPKs (Table 1.2B). Multiple sequence comparisons of kinase domains from L. pneumophila strain Paris and other prokaryotic and eukaryotic STPKs cluster two of these STPKs (Lpp2626/Lpl2481/Lpg2556 and Lpp1439/Lpl1545/Lpg1483) within the group of eukaryotic STPKs, closest to STPKs from Entamoeba histolytica (Cazalet et al. 2004). This suggests that the L. pneumophila STPK may also modulate eukaryotic signal transduction mechanisms as well as host-cell trafficking pathways. The importance and specificity of these proteins for L. pneumophila is underlined by the results obtained from genome comparisons. In order to verify whether the presence and wide variety of these eukaryotic-like proteins is Legionella specific the motif search was conducted on 11 additional bacterial genomes. For these comparisons we selected C. burnetti, the prokaryote, evolutionary the most closely related to L. pneumophila, other intracellular pathogens like Shield flexneri and Listeria monocytogenes, other bacterial pathogens involved in pulmonary infection like Chlamydia pneumoniae, Pseudomonas aeruginosa or Mycobacterium tuberculosis, bacteria also present in the aquatic environment and capable of colonizing amoebae like Salmonella typhimurium and Escherichia coli K12 as reference genome (Table 1.3). Compared to L. pneumophila, none of these genomes encodes the same variety and number of proteins-containing motifs found preferentially in eukaryotes. With the exception of the ankyrin repeat family which is the most abundant in Wolbachia pipientis (Wu et al. 2004) and serine-threonine protein kinases, which are more abundant in M. tuberculosis (Cole et al. 1998), Legionella is the organism with the highest number of proteins with eukaryotic-like domains (Table 1.3).

1. Legionella pneumophila Pathogenesis

11

TABLE 1.3. Distribution of eukaryotic-like domains in the genome of different pathogens.

L. pneumophilaParis L. pneumophilaLens L. pneumophilaPhiladelphia 1 Coxiella burnetii Chlamydia pneumoniae Pseudomonas aeruginosa Escherichia coli Listeria monocytogenes Salmonella typhimurium Shield flexneri Mycobacterium tuberculosis Bartonella henselae Wolbachia pipientis Rickettsia prowazekii

F-box proteins 3 2 2

Ser/thr protein kinases 3 3 3

Sec7 domain 1 1 1

Ankyrin domain 20 17 13

SET domain 1 1 1

U-box proteins 1 0 1

0 0

1 2

0 0

16a 0

0 1

0 0

0

2

0

3

1

0

0 0

0 1

0 0

0 0

0 0

0 0

0

0

0

0

0

0

0 0

0 8b

0 0

1 0

0 0

0 0

0 0 0

0 0 0

0 0 1

0 23c 3

0 0 0

0 0 0

a

13 reported in Seshadri et al. (2003) 11 described in Cole et al. (1998) c 23 reported in Wu et al. (2004) b

Thus the genome sequence reflects the co-evolution of Legionella with its eukaryotic hosts, e.g., freshwater amoebae, and suggests that the different eukaryotic-like proteins are important for the intracellular life cycle of L. pneumophila, conferring probably in part its capacity to subvert host functions and to modulate them to its advantage.

2.2. Putative Virulence Factors as Deduced from Sequence Analysis In addition to the eukaryotic-like proteins, proteins with homology to virulence factors of other organisms were identified through sequence analysis. The three sequenced L. pneumophila strains encode a protein homologous to the enteropathogenic E. coli (EPEC) virulence regulator BipA. These three Legionella proteins (lpp2875, lpl2737, lpg2822) are identical and show 68% similarity to the E. coli BipA protein. The Legionella bipA gene has 43% GC content, which is significantly higher than that of the rest of the chromosome (38%), indicating perhaps horizontal gene transfer. However, the BipA protein is a member of the ribosomebinding GTPase superfamily that is widely distributed in bacteria and plants. In EPEC, BipA is thought to function high up in diverse regulatory cascades. It was

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shown to regulate the espC pathogenicity island and a type III secretion system specified by the locus of enterocyte effacement (LEE) and flagella-mediated cell motility (Farris et al. 1998; Grant et al. 2003). In L. pneumophila neither a homolog of the EspC toxin nor a type III system are present; however, BipA may be involved in regulation of flagellum expression. In a global gene expression analysis of L. pneumophila in its natural host Acanthamoeba castellani, we showed that bipA gene expression is growth phase dependent as its expression is between two- and threefold decreased in stationary phase with respect to exponential growth (Brüggemann et al. 2006). This is in accordance with a recent report about gene expression in Pseudomonas putida (Yuste et al. 2006). As flagella expression is also growth phase dependent, the hypothesis that BipA is involved in this complex regulation is possible. A gene encoding a putative inner membrane efflux protein similar to the virulence factor MviN is present in the three strains with strain Philadelphia 1 encoding two orthologs (lpg1087, lpg2635). Although present in both pathogenic and non-pathogenic strains, absence of this protein in S. typhimurium results in reduced virulence in a mouse typhoid disease model (Carsiotis et al. 1989; Rudnick et al. 2001) suggesting that it may be involved in virulence of L. pneumophila. Another interesting finding is the presence of three homologs of the factor for inversion stimulation Fis, which exists in only one copy in other genomes like C. buenetii, E. coli or Salmonella species. This DNA-binding protein is involved in many different processes including modulation of transcription. Recently this protein has been implicated in the control of virulence gene expression in E. coli and S. typhimurium (Keane and Dorman 2003; Kelly et al. 2004; Osuna et al. 1995) and biofilm formation in enteroaggregative E. coli (Sheikh et al. 2001). As the concentration of the Fis protein varies tremendously between exponential and stationary phase due to the stringent response, these proteins could be implicated in differential expression of virulence genes during the Legionella cell cycle. Legionella pneumophila strain Paris encodes a specific protein, which bears hallmarks of type-V secretion systems or autotransporters. It is composed of three major domains: an N-terminal leader peptide for secretion across the inner membrane, a C-terminal domain that forms a pore in the outer membrane thus processing the passenger domain to the cell surface (Desvaux et al. 2004). Like the E. coli autotransporters AIDA-I and Ag43, the L. pneumophila autotransporter contains hemagglutinin repeats, which usually confer the protein a function in cell-to-cell aggregation (Benz and Schmidt 1992; Klemm et al. 2004). However the known RGD interaction motif is absent in the L. pneumophila autotransporter. Thus, another interaction motif might exist and confer a role in adherence to mammalian cells and/or autoaggregation during biofilm formation. This autotransporter was probably acquired through horizontal gene transfer as it is suggested by its GC content of 41% higher than the average of 38% and the presence of many remnants of insertion sequences upstream.

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2.3. Secretion Machineries—Presence of Particular Many and a Wide Variety Legionella pneumophila has a rather exceptional number and wide variety of secretion systems for efficient and rapid deliverance of effector molecules into the phagocytotic host cell underlining the importance of protein secretion for this pathogen. The two major ones, known to be involved in virulence are the Dot/Icm type-IV secretion system and the lsp type II secretion system. Legionella pneumophila and C. burnetii are the only bacterial pathogens known to encode a type-IVB secretion system similar to the Tra/Trb system of IncI plasmids. This type-IVB secretion system is called icm (intracellular multiplication) (Marra et al. 1992) and/or dot (defective organelle trafficking) (Berger and Isberg 1993) system. It is required for intracellular growth in human macrophages as well as in amoebae and for intracellular trafficking. This macromolecular complex is encoded by 25 genes located on two genomic regions that are conserved and located in the same chromosomal location in strains Paris, Lens and Philadelphia 1. Morozova and colleagues (Morozova et al. 2004) showed by sequence comparison and DNA/DNA hybridization that the organization of this region is conserved among 18 L. pneumophila and 17 other Legionella species tested. Hybridization studies using a Legionella macro-array carrying these 25 genes further proved their presence and conservation among 180 tested L. pneumophila strains of all serotypes and different origins (Cazalet et al. 2005a) underlining again that the Dot/Icm type IV system is essential for the life as well as pathogenicity of Legionella. Different substrates of the Dot/Icm secretion system have been identified like RalF, which is required for the localization of ARF on phagosomes containing L. pneumophila (Nagai et al. 2002), LidA implicated in establishing a replicative niche (Conover et al. 2003; Derre and Isberg 2005), LepA and LepB involved in the release of Legionella from the vacuole after intracellular multiplication (Chen et al. 2004). Recently a large number of candidate effector proteins, designated SidA-G (substrate of Icm/Dot transporter) using a two-hybrid screen, and subsequent screening using a Cre/loxP-based system were identified (Luo and Isberg 2004). Interestingly, most of the Sid proteins have one or more paralogs present in the L. pneumophila genomes, and each of the three sequenced genomes have a slightly different array of Sid proteins. Furthermore, many effector proteins show no sequence similarity to proteins from other organisms, which indicates the uniqueness of the mechanism by which L. pneumophila subverts eukaryotic hostcell functions. The second type IV secretion system encoded by L. pneumophila is a type-IVA system similar to the Agrobacterium tumefaciens Vir system. It is dispensable for intracellular growth in both macrophages and amoebae (Segal et al. 1999), but it is implicated in host-cell infection by L. pneumophila at 30°C (Ridenour et al. 2003) and it can also mediate conjugational DNA transfer (Segal et al. 1999). To date L. pneumophila is the only intracellular pathogen known to encode a type-II secretion system. It is highly conserved in the three sequenced genomes

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(protein identities of 96–100%) and seems also to be conserved throughout the genus Legionella (Rossier and Cianciotto 2001). The lsp secretion system promotes the ability of L. pneumophila to infect both protozoan and macrophage hosts, to grow in the mammalian lung (Rossier and Cianciotto 2001) and to grow at low temperatures (Soderberg et al. 2004). Legionella pneumophila possesses several additional secretion systems like a type I secretion system called lss, which seems to be specific to L. pneumophila (Jacobi and Heuner 2003), a sec apparatus, which is complete with the exception of secG and a twin arginine translocation (TAT) pathway. In contrast to the usual organization of tatA, tatB and tatC in an operon, only the tatA and tatB genes are co-transcribed and tatC is in a different location of the L. pneumophila chromosome. This organization is conserved in all three genomes. In strains Paris, Lens and Philadelphia 1, the tatAB operon and the tatC gene are located 24 kb, 41 kb and 27 kb apart, respectively. However, the flanking regions are, despite the different distance between the tatAB and tatC genes, homologous in the three strains. The TAT secretion system is implicated in growth under low iron conditions, growth within macrophages in an Lsp-independent manner (Rossier and Cianciotto 2005). In addition to these conserved secretory pathways, strain Paris encodes a type-V secretion system specific to this strain.

3. Metabolism as Deduced from the Genome Sequence Legionella pneumophila is a quite fastidious organism to grow. The bacterium preferably utilizes proteinaceous substrates. This is reflected in the genome sequence, as a large set of oligopeptide and amino acid uptake and degradation systems is present. Legionella pneumophila requires arginine, isoleucine, leucine, valine, methionine, serine and threonine for growth. Genome analysis identified the biosynthetic pathways for all remaining amino acids but also for arginine. Thus, the auxotrophy for six amino acids but not for arginine is in agreement with the genome analysis. Together with many amino acid permeases, a broad range of secreted proteases for serine, cysteine and metal type ones are encoded by the genomes. In particular, besides the P. aeruginosa elastase homolog ProA three secreted paralogous metalloproteases are present. The presence of more than 40 peptidases in each genome suggests the breakdown of proteins to amino acids by Legionella. This suggests that besides free amino acids, proteins and peptides may also be an important source of nutriment for L. pneumophila. In contrast, although harboring the complete Embden–Meyerhof as well as the Entner–Doudoroff pathway, systems for sugar uptake seem to be rare; no PTSlike uptake systems are encoded by the genomes. However, some of the about 50 identified ABC-type transport systems might be involved in sugar uptake, since the bacterium possesses a few systems for the degradation of complex sugars, such as trehalase, polysaccharide deacetylase, glucoamylase, ␤-hexosaminidase and chitinases. The extracellular enzymes chitinase and a glucoamylase probably

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used for the degradation of sugar polymers are similar only to eukaryotic enzymes. The glucoamylase gene clusters encode enzymes of the glycolysis and a sugar permease, suggesting a significant role in catabolism. The pentose phosphate pathway and glycolysis are complete although the fructose 1–6 diphosphate aldolase is similar to eukaryotic enzymes. The key regulatory enzyme of glycolysis, phosphofructokinase (pfk) of L. pneumophila, seems to be bifunctional as it is not of the ATP-dependent but the PPi-dependent type. The widely distributed ATP-PFK catalyzes an irreversible catabolic reaction, the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, while the PPi-PFK, also called PPi-phosphofructokinase (PFP), catalyzes the same reaction in a reversible way and can thus function both in glycolysis and gluconeogenesis (Baptestea et al. 2003). The Legionella genomes also encode several enzymes for the utilization pathway for myo inositol, which may interfere with the host-cell signaling mediated by this intracellular messenger. The complete metabolic pathways for biotine, riboflavin, folate, nicotine amine and heme are present. However, the ubiquinone biosynthesis pathway in L. pneumophila is different to the one described for E. coli and may correspond to an uncharacterized biosynthetic pathway also present in P. aeruginosa. Legionella pneumophila employs an extensive aerobic respiratory chain consisting of NADH dehydrogenase, cytochrome-dependent succinate dehydrogenase, ubiquinol-cytochrome c reductase and four terminal oxidases, which guarantee the capacity to cope with changing oxygen tensions (an aa3 cytochrome, two bd-type quinol cytochrome and an o-type quinol cytochrome oxidase). The o-type oxidase, encoded by the cyo operon is present in strain Paris but is missing in strains Lens and Philadelphia 1. Systems involved in anaerobic respiration are apparently absent. Two ATP synthases are encoded by the genomes, an F0F1-type ATP synthase of ␥-proteobacteria present in all three genomes and a second one with highest similarity to systems of archaea and marine bacteria present only in strains Paris and Philadelphia 1. At least four sodium/proton antiporters seem to be involved in the interchangeability of the proton and the sodium gradient across the cytoplasmic membrane, raising the question if a sodium motive force is used for cellular activities. In this respect, the presence of a key component of sodium-type polar flagellar motors, MotY, as well as two significantly different MotAB gene clusters lead to the assumption that motility is powered by sodium as well as proton motive forces.

4. Clues to Adaptation—Regulation and Signal Transduction Pseudomonas aeruginosa and L. monocytogenes, which are both able to adapt to the host environment and to multiply and survive in a wide variety of environments outside a host contain large sets of regulatory proteins with 8.4% and 7.3% of the genome, respectively, dedicated to regulatory proteins (Stover, 2000 pp. 150; Glaser, 2001 pp. 149). In contrast, L. pneumophila is an intracellular

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pathogen that does not seem to be able to replicate in the environment outside the host. Consistent with this lifestyle the regulatory repertoire is rather small. Genome analysis identified 92, 79 and 106 transcriptional regulators in strains Paris, Lens and Philadelphia 1, respectively. This represents only a little more than 3% of the predicted genes. Each L. pneumophila genome encodes six putative sigma factors, the homologs of rpoD (the major sigma factor), rpoH, rpoS, rpoN, fliA and the ECF-type sigma factor rpoE. Furthermore one sigma 54 (RpoN) modulator, sigma factor-dependent enhancers (FleQ, FleR, PilR) and the anti-sigma factors FlgM and anti-sigma factor B are encoded by all three L. pneumophila strains. The number of two-component systems (13 histidine kinases and 13 response regulators) is also substantially lower compared to that of free-living bacteria like E. coli (35) or P. aeruginosa (100) but conserved in all three genomes. The most prominent classes of transcriptional regulators in L. pneumophila are LysR-type and phage-like repressors. The abundance of the latter class probably indicates that the genome is subject to continuously ongoing phage-mediated horizontal transfer. However, interestingly, in none of the three genomes sequenced, a complete prophage was identified. Regulators of the GGDEF/EAL family are abundant in L. pneumophila. The three strains encode 24, 21 and 21 regulators of this type, respectively (Table 1.4), TABLE 1.4. GGDEF/EAL regulators identified in the three L. pneumophila genomes. Paris lpp0029 lpp0087 lpp0127 lpp0219 lpp0220 lpp0299 lpp0351 lpp0352 lpp0440 lpp0809 lpp0891 lpp0942 lpp0952 lpp1114 lpp1170 lpp1311 lpp1425 lpp1475 lpp2071 lpp2324 lpp2355 lpp2477 lpp2695 lpp2708

Gene name Lens lpl0030 lpl0075 – lpl0219 lpl0220 lpl0283 – lpl0329 lpl0416 lpl0780 lpl0860 lpl0912 lpl0922 lpl1118 lpl1176 lpl1308 lpl1559 lpl1508 lpl2061 lpl1054 – – lpl2567b lpl2581

Philadelphia lpg0029 lpg0073 – lpg0155 lpg0156 lpg0230 – lpg0277 lpg0373 lpg0744 lpg0829 lpp0879 lpp0891 lpg1114 lpg1168 lpg1357 lpg1469 lpg1518 lpg2132 lpg1057 lpg1025 – lpg2642 lpg2655

Domains 3 PAS, GGDEF, EAL GGDEF, EAL EAL, COG1639 PAS, GGDEF EAL CHASE3, GAF, GGDEF EAL Response-Regulator, PAS, GGDEF GGDEF GGDEF GGDEF, EAL, HAMP GGDEF 2 PAS, GGDEF, EAL PAS, GGDEF, EAL GGDEF, EAL PAS, GGDEF, EAL EAL PAS, GGDEF, EAL GGDEF, EAL GGDEF, EAL GGDEF GGDEF 2 PAS, GAF, GGDEF, EAL GGDEF

TMa helices – 2 – – 1 1 – – 5 – 3 5 – – 9 7 2 13 9 4 5 1 – 6

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similar to many other bacteria, like Vibrio cholerae (41), P. aeruginosa (33), Wolinella succinogenes (26), and E. coli (19) (Aldridge et al. 2003; Baar et al. 2003). These regulators contain different N-terminal signaling domains and a C-terminal output domain, which is composed of two subdomains, GGDEF (DUF1) and EAL (DUF2) (Tal et al. 1998). Those domains modulate the intracellular level of cyclic-di-GMP, a recently discovered secondary messenger in the bacterial cell, which governs multicellular behavior such as the switch from sessility (e.g., in a biofilm) to motility (e.g., swarming) in the pathogenic organisms Salmonella enterica serovar Typhimurium, the nosocomial pathogen P. aeruginosa and the commensal species E. coli (Simm et al. 2004). Moreover, an EAL domain protein in S. enterica is involved in the resistance to oxidative stress and in the ability to kill macrophages (Hisert et al. 2005). Thus, EAL/GGDEF proteins could have a global role in governing the developmental differentiation of L. pneumophila.

5. Comparative Genomics of Legionella pneumophila 5.1. The Specific Gene Complements of L. pneumophila Paris, Lens and Philadelphia Legionella research is in a privileged situation, having complete genome sequences for three different isolates of the species L. pneumophila available. Whole genome comparisons allow now to define specific and common features. According to these comparisons, the L. pneumophila genomes, similar to many other bacterial species, consist of a conserved core part and a so-called “flexible gene pool.” The conserved core region is required for essential functions such as protein biosynthesis, the cell envelope and major metabolic pathways. In the flexible gene pool, plasmids as well as genomic and pathogenicity islands represent the major genetic elements. Furthermore, IS-elements and transposases were identified in the flexible gene pool as well as small DNA-pieces, termed “islets.” Comparison of strains Paris, Lens and Philadelphia 1 identified about 2500 genes as the conserved backbone and about 300 genes (⬃10%) in each genome as strain specific, constituting thus the flexible gene pool (Figure 1.3). Given the fact that the three analyzed strains belong to the same species and same serogroup, this diversity is quite high. Most of the strain-specific genes code proteins of unknown function; however, some proteins may be related to strain-specific niche adaptation or may code for specific virulence functions. The specific genetic equipment of strain Paris contains a number of regulators (two CsrA homologs, 11 transcriptional regulators), five additional ankyrin-repeat, one specific U-box domain protein and two eukaryotic-like proteins (Table 1.2A and Table 1.2B) as well as several restriction modification genes (DNA modification methylases, endonucleases), a specific

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Christel Cazalet and Carmen Buchrieser L. pneumophila Paris 3077* * : plasmids are not considered

313 (10%)

134

130 2500

L. pneumophila Philadelphia 2953

283 (10%)

257 (9%) 40

L. pneumophila Lens 2931 *

FIGURE 1.3. Diagram showing the core genome and the unique gene complement of strains L. pneumophila Paris, Lens and Philadelphia. Orthologous genes were defined by reciprocal best-match FASTA comparisons. The threshold was set to a minimum of 80% sequence identities and a ratio of the length of 0.75–1.33.

o-type cytochrome ubiquinol oxidase and a type V secretion system (autotransporter). Strain Lens contains four specific transcriptional regulators and three specific proteins with eukaryotic domains (Table 1.2A), two of which are ankyrin repeat proteins. Except four specific transcriptional regulators, Strain Philadelphia 1 codes two specific ankyrin repeat and one F-box domain protein. Strain Philadelphia 1 codes, in addition to the three peptide methionine sulfoxide reductase regulators msrA that are conserved in all strains, three additional orthologs msrA1,2,3 on its specific 65-kb pathogenicity island. msrA2 and msrA3 are homologous to msrA and msrB encoding antioxidant repair enzymes, characterized as virulence determinants in E. coli, Neisseria gonorrhea and Mycoplasma genitalium (Dhandayuthapani et al. 2001; Moskovitz et al. 1995; Skaar et al. 2002). In the same island the Philadelphia 1 specific magA gene, coding a protein putatively implicated in the maturation of the cyst-like mature intracellular form, is present (Hiltz et al. 2004). Furthermore, as mentioned before, each of the three sequenced genomes have a slightly different array of Sid proteins, candidate effectors of the Dot/Icm type IV secretion system (Luo and Isberg 2004).

5.2. Genomic and Pathogenicity Islands Like many bacterial species, the L. pneumophila genomes carry several pathogenicity island like regions. One example is the region encoding the lvh type-IV secretion system which is dispensable for intracellular growth in both macrophages and amoebae (Segal et al. 1999), but implicated in host-cell infection by L. pneumophila at 30°C (Ridenour et al. 2003) and which can also

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mediate conjugational DNA transfer (Segal et al. 1999). The lvh cluster itself is highly conserved in all three sequenced genomes. However, the flanking DNA regions are specific to each strain in size as well as in the genetic content. In strain Paris the lvh region is flanked by 11 kb and 22 kb, in strain Lens by 5.8 kb and 30 kb and in strain Philadelphia 1 by 14 kb and 30 kb. This region is inserted in the same tRNA in strains Lens and Paris but in an Arg tRNA in another chromosomal location in strain Philadelphia 1. The lvh region has in addition the particular capacity to exist in an integrated and an excised form as multi-copy plasmid of 36 kb in size in strain Paris and of 45 kb in strain Philadelphia 1. This was not observed in strain Lens; however, a 28bp perfect repeat is present in the flanking regions, suggesting that it is also able to excise. This element is probably of plasmid origin as it has a higher GC content (43%) than the rest of the chromosome (38%) and it carries some plasmid related genes. A similar element, contributing to phase variation that can also exist in a plasmid as well as integrated form, was previously identified in strain OLDA (Luneberg et al. 2001). The identification of the exact mechanism leading to the excision and integration of these elements will help to understand the adaptation and versatility mechanisms of Legionella. A second chromosomal region fulfilling the typical characteristics of pathogenicity islands is a 40 kb region encoding several efflux pumps induced in contact with the host cell. It is apparently dedicated to detoxification and proper metal ion balance within the bacteria (Rankin et al. 2002). In the three sequenced strains, the cluster coding the efflux pumps is preceded by genes homologous to lvrABC and is located between conserved regions encoding mostly proteins of unknown functions. Typically for pathogenicity islands these regions are interrupted by several insertions specific to each strain and are often flanked by integrase genes or insertion sequences. In strains Philadelphia 1 and Lens this large island of 135 kb and 87 kb, respectively, is located on the same chromosomal location and is flanked by a Thr tRNA gene. In strain Paris it is forming a large island of 130 kb present in a different chromosomal location and is flanked by a Met tRNA gene. The 65 kb pathogenicity island (LpPI-1), described previously as specific for strain Philadelphia 1 (Brassinga et al. 2003), is indeed absent from strains Paris and Lens. However, genome comparison by hybridization of 180 Legionella strains showed that this island is also present in other L. pneumophila strains as well as in strains of other Legionella species (Cazalet et al. 2005a). In strain Philadelphia 1, this island is inserted in a Val tRNA gene, it contains putative virulence factors already mentioned before, mobile elements and an additional cluster of genes with higher G ⫹ C content than the rest of the chromosome, encoding homologs of Tra proteins. The Tra region shows in average 55% similarity to the Tra proteins associated with the F plasmid of E. coli. The LpPI-1 tra gene homologs are arranged identically to those of the F plasmid, with the exception of traM, traY, and traX, which are missing in L. pneumophila. Comparative analysis of the borders of LpPI-1 in eight additional L. pneumophila strains lacking the core region of this island revealed pronounced genetic variability among

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Lys tRNA

Paris

Philadelphia Weakly similar to eukaryotic proteins Similar to bacteriophage proteins Conserved regions between the three genomes Integrase Regulator

FIGURE 1.4. Genetic organization of a putative pathogenicity island specific of L. pneumophila strain Paris and its chromosomal location as compared to strains L. pneumophila Lens and Philadelphia. The figure was created by using ACT (Artemis Comparison Tool) software (http://www.sanger.ac.uk/Software/ACT/).

the strains, suggesting that this region represents a “plasticity” or hypervariable zone (Brassinga et al. 2003). This hypothesis is reinforced by the comparison of this genomic region in the three sequenced strains. In strains Paris and Lens small insertions of 25 kb and 19.3 kb, respectively, which code mainly unknown proteins but also two ankyrin repeat proteins, a homolog of SidE, a regulatory protein and four transposases, are present. An islet of 11.3 kb inserted in a Lys tRNA gene is present only in strain Paris (Figure 4). Interestingly, this island contains an integrase, two phage-related genes and 14 genes coding proteins with some similarity to eukaryotic proteins. The G ⫹ C content of this region has a mosaic composition varying between 28 and 41% with the eukaryotic-like proteins showing mainly low G ⫹ C content of 30% in average. This region might be in favor of an acquisition of some eukaryotic-like proteins through horizontal gene transfers mediated by bacteriophages. Others, however, might have evolved by convergent evolution. Some of these pathogenicity/genomic islands of L. pneumophila are present in the three genomes at different locations or only in part. Figure 1.5 illustrates the genome rearrangements and plasticity in indicating these flexible regions with respect to their size and chromosomal location as compared to the three sequenced genomes.

5.3. Plasmids of Legionella Two of the three sequenced L. pneumophila strains carry plasmids that might play a role either in adaptation to the environment or in virulence as both encode putative virulence factors, mobile genetic elements and antibiotic resistance genes. In strain Paris a 132 kb plasmid and in strain Lens a 60 kb plasmid was identified, both of which seem to be single copy plasmids. These plasmids contain a 24 kb

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FIGURE 1.5. Regions of plasticity identified in the L. pneumophila genomes and their chromosomal locations in the three sequenced strains. Outer circle: Strain Paris; main specific regions are indicated in light grey; Central circle: Strain Lens, main specific regions are indicated in dark grey; Inner circle: Strain Philadelphia 1, main specific regions are indicated in grey. dot/icm, Dot/Icm type IV secretion system; lvh, Lvh type IV secretion system; efflux cluster; efflux pumps located on a plasticity zone of 130 kb (strain Lens), 135 kb (strain Philadelphia 1) and 87 kb (strain Lens).

and 30 kb gene cluster, respectively, encoding homologs of Tra proteins (Figure. 1.6). Furthermore several mobile elements like transposases and phage-related proteins are present and about half of the genes code proteins of unknown function. Both plasmids also encode a paralog of CsrA—a protein known to act as repressor of transmission traits of L. pneumophila and as activator of replication (Molofsky and Swanson 2003), revealing the question whether these plasmids are implicated in virulence. The plasmid identified in strain Paris carries many genes coding proteins probably conferring antibiotic resistances, like a betalactamase, two spectinomycin 3⬘ adenylyltransferases or an aminoglycoside 3⬘ phosphotransferase. Aminoglycoside phosphotransferase (3⬘)-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. The identified betalactamase is 93% similar to a previously identified D beta-lactamase determinant isolated from Legionella (Fluoribacter) gormanii ATCC 33297(T) where it was shown to transfer resistance to oxacillin, methicillin, penicillin G, ampicillin, carbenicillin, piperacillin and also for cefazolin and nitrocefin, oxyimino cephalosporins and aztreonam (Franceschini et al. 2001). Furthermore nine acetyltransferase genes,

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A

B

120

0

15

egion Tra-r

105

Plasmid Legionella pneumophila strain Paris 131.9 kb

30 45

Plasmid Legionella pneumophila strain Paris 59.8 kb

15

90 45

ion

reg

Tra 30 75

60

FIGURE 1.6. Circular map of the plasmid present in strain Paris (A) and strain Lens (B). For both plasmids from the outside—circle 1: Plasmid genes on the ⫹ and – strands, respectively. Black, Tra-region (traTLEKBVC, trbI, traWU, trbC, traNF, trbB, traHGDI); circle 2: G/C bias (G ⫹ C/G-C); 3: G ⫹ C content of the plasmid with ⬍32.5% G ⫹ C short length, between 32.5% and 44.1% medium length and with ⬎44.1% G ⫹ C in long lines.

two of which belonging to the GCN5-related N-acetyltransferase (GNAT) superfamily, are present which could either be implicated in detoxification, drug resistance or gene regulation. Interestingly, 6 kb of the Paris plasmid contain seven genes coding for proteins with 91–98% protein similarity to proteins coded by a plasmid previously identified in a L. longbeachae isolate. Most of the proteins code for unknown functions, but two code for a two-component system, lrpR/lskS, that was shown to be implicated in virulence of this species (Doyle and Heuzenroeder 2002). In contrast, the plasmid identified in strain Lens contains mainly genes coding for proteins of unknown functions. The role of specific plasmids in virulence of Legionella has not clearly been demonstrated, but a correlation between virulence in a mouse model and the presence of plasmids (Bezanson et al. 1994) and a higher persistence in the environment of strains containing plasmids (Brown et al. 1982) has been reported. Transfer of plasmids from a L. pneumophila donor to another L. pneumophila isolate or another Legionella species may be mediated by the Dot/Icm type-IV secretion system (Vogel et al. 1998). The identification, characterization and analysis of the functional role of Legionella plasmids might add to the understanding of versatility, adaptation and virulence mechanisms of this pathogen.

5.4. Variability at the Gene Level The plasticity of the Legionella genomes is not only due to insertions, deletions or rearrangements of genomic islands of different size or due to plasmids, but plasticity was also identified for single genes that seem particularly variable.

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An example for plasticity at the gene level is the rtxA gene. This gene was first described in strain L. pneumophila AA100 where it is located next to a gene named arpB. In strain Paris, the rtxA and arpB genes are fused and are followed by a region of about 17 kb of repetitive DNA organized in approximately 30 highly conserved tandem repeats. The same organization is observed in strain Lens; however, two kinds of repeats, which are completely different from those observed in strain Paris, are present. In strain Philadelphia 1, arpB is missing and a frameshift disrupts the fusion with the repeat region. The tandem repeats are also highly conserved but are again a new repeat type. Probably variations in the number and sequence of these repeats contributes to versatility and perhaps also to virulence of Legionella. Another example is the dotA gene that is part of the Dot/Icm type IV secretion system. The comparison of this system, in different Legionella species and L. pneumophila serogroups, reveals a complicated evolutionary history and identifies an extensive polymorphism and mosaic structure of the dotA gene due to several intragenic recombination events. DotA is a secreted protein implicated in survival within macrophages and inhibition of phagolysosome fusion by Legionella. Thus it is supposed that variability and rapid evolution of DotA creates antigenic variations for adaptation to certain environmental niches (Ko et al. 2003a, 2003b). Variation on the gene level is also seen in otherwise conserved regions among different Legionella species, as reported by Feldman and colleagues (Feldman and Segal 2004). The analysis of sequence variability of two essential components of the Dot/Icm system: icmQ, coding the pore-forming protein IcmQ and icmR, coding its regulator IcmR in 29 Legionella species revealed that the organization of this region is highly conserved, but that it contains a favorable site for gene variation. In the place where the icmR gene was expected to be located, other open reading frames that are non-homologous to each other or to any entry in the GenBank database were found (migAB in L. micdadei and ligB in L. longbeachae). This led to the identification of a hypervariable gene family named fir for functional homologs of icmR whereas icmQ is conserved. The only exception is a small variable region important for the interaction between the two proteins. Supposedly, the Fir proteins and the variable part of IcmQ coevolved enabling adaptation to the environment and survival in various protozoan hosts.

6. Comparative Genomics—Legionella, Coxiella and Other Intracellular Pathogens 6.1. Genome Comparison of Legionella pneumophila and Coxiella Burnetii Comparative genome analysis can be used to identify species-specific genes and gene clusters, and analysis of these genes can give insight into the mechanisms involved in specific host–pathogen interactions. We thus compared the genomes of

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L. pneumophila with that of the evolutionary most closely related species, C. burnetii. Coxiella burnetti is an obligate intracellular pathogen that, like Legionella, thrives on both macrophages and amoebae. Thus the comparison of these genomes should allow the identification of common pathways and mechanisms probably important for intracellular trafficking and the specificity of these organisms. Indeed, both organisms are vacuolar pathogens; they have a biphasic life cycle and alternate between a transmissive and a replicative phase and both of them are able to delay phagosome–lysosome fusion early in infection. However, different to C. burnetti, L. pneumophila replicates in a vacuole surrounded by endoplasmic reticulum. A global comparison identifies approximately 42% of the Legionella genes as shared with those predicted in Coxiella. As expected from their genetic and cell biological similarities some virulence factors are conserved in both organisms. Indeed, Coxiella encodes two homologs of the global regulator csrA that is important in Legionella for regulation of its biphasic life cycle, and the proteins enhABC, which are involved in entry of L. pneumophila into host cells. Interestingly, Coxiella and Legionella are the only two bacteria known to date to use a type-IVB secretion system (Dot/Icm in Legionella) for pathogenesis. The organization of the gene cluster is almost identical in both organisms and with the exception of the icmR gene the Dot/Icm secretion system of Legionella is conserved in Coxiella (Segal et al. 2005). However, interspecies complementation of L. pneumophila dot/icm mutants by homologous proteins of Coxiella showed that there exists only partial functional similarity between the two systems. Twentytwo effectors and potential effector proteins were identified in L. pneumophila. Surprisingly, none of them has a homolog in C. burnetii suggesting different mechanisms of subverting host functions for these two pathogens. In contrast to L. pneumophila, which shows high genome redundancy, C. burnetti seems to have undergone genome reduction as many intracellular pathogenic microorganisms, which are strictly host-adapted (Seshadri et al. 2003). However, some large protein families are conserved in both organisms. An example is the ankyrin protein family. Legionella pneumophila encodes 13–20 ankyrin repeat proteins and C. burnetii encodes 13, despite its smaller genome size of 1.99 Mb. Similarly, the recently identified Pht (phagosomal transporters) subfamily of MFS transporters (Sauer et al. 2005) has nine to eleven members depending on the sequenced strain of L. pneumophila, and C. burnetii encodes also nine transporters of this class. In Legionella, one of these MFS transporters, named PhtA, was shown to mediate the differentiation from transmissive to the replicative form. It was suggested that this transporter subfamily allows the vacuolar pathogens Legionella and Coxiella to assess phagosomal nutrient supply before entering the replicative phase (Sauer et al. 2005).

6.2. Genome Redundancy—Gene Family Expansion in Legionella Genome sequencing and comparison revealed expansion of some gene families as well as high functional redundancy in L. pneumophila. One of the largest classes

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of proteins are transporters like in many genomes; however, in Legionella multidrug efflux pumps for heavy metals and other toxic substances are highly represented. They could play an important role in adaptation to the environment and might also play a role in biocide resistance. Legionella also possess a prominent family of regulators with GGDEF-EAL domains (24 in strain Paris, 21 in strain Lens and 21 in strain Philadelphia 1), which are essential for virulence properties of many bacteria as they are involved in aggregation, biofilm formation or twitching motility. This protein family is also largely represented in many other bacteria; however, C. burnetii, the most closely related prokaryote to Legionella, encodes only one. Moreover, some virulence protein families contain several orthologs. For example, four, six and three (Paris, Lens, Philadelphia 1, respectively) paralogs of the major outer membrane protein Momp are encoded by Legionella. The metalloprotease ProA implicated in virulence has eight paralogs, and the enhanced entry protein EnhC has four paralogs. Furthermore three paralogs of the factor for inversion stimulation Fis as discussed in Section 2.2 are present in the three Legionella genomes. Similarly, many substrates of the Dot/Icm system such as the Sid proteins are part of large paralogous families with functional redundancy. This high functional redundancy of the L. pneumophila genomes provokes a question whose answer has both evolutionary and virulence implications: are all these paralogous proteins functionally redundant, or have they acquired sufficient specialization to enable adaptation to different niches like different protozoa that the bacteria may encounter? It seems that there is both specialization and redundancy in the functions of these proteins.

7. Comparative Genomics of Legionella pneumophila and the Genus Legionella—a Perspective Comparative genomics and related technologies are helping to unravel the molecular basis of the pathogenesis, host range, evolution and phenotypic differences of Legionellae. In Legionella research the area of genomics started with the determination of three complete genome sequences of clinical isolates of L. pneumophila. The availability of these three sequences opened the way to comparative and functional genomics and allows now to use new approaches like bioinformatics, micro-arrays and proteomics to gain functional information. Furthermore, comparison of pathogenic and apathogenic members of the genus Legionella should bring new insight into virulence functions important for disease in humans and into evolution of Legionellae. Thus further sequencing efforts are needed for in-depth comparative genomics. A second Legionella species is important for human disease, L. longbeachae, which accounts for 30.4% of community-acquired legionellosis cases in Australia and New Zealand (Yu, 2002 pp. 21). Other species like L. anisa, L. micdadei, L. dumofii or L. feeleii seem to be less virulent even though they relatively frequently colonize water distribution systems (Muder and Yu 2002), or have

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never been implicated in human disease like L. hackaliae. Recently, a first comparison of the completely sequenced L. pneumophila genomes with a partially sequenced L. anisa and L. longbeacheae isolate revealed a very high diversity, as only around 50% of the genome seems to be conserved among the three Legionella species (Cazalet et al. 2005b). Except these first preliminary comparisons, very little is known to date on the genomic level about the degree of the genetic diversity among different isolates of L. pneumophila of different serogroups or among different Legionella species. In particular, the relation between a specific gene content and virulence is unknown. However, this knowledge is a prerequisite for understanding differences in pathogenicity and the ecology of this organism and it is also the basis for the development of rational and accurate diagnostic tools. Importantly, additional sequence information will soon be available as a fourth L. pneumophila isolate, strain Corby, is being sequenced by the Institut for Molecular Biotechnology, IMB Jena, Germany. Furthermore, the determination of the genome sequences of L. hackeliae and L. mikdadei by the same Institution and that of an isolate of L. longbeachae by the Institut Pasteur, Paris, France, are nearing completion. The availability of these different genome sequences of Legionella will pave the way for in-depth comparative genomics and the identification of so far unknown virulence determinants.

Acknowledgments. We would like to thank many of our colleagues who have contributed in different ways to this research. In particular, P. Glaser for the many fruitful discussions, F. Kunst for his support and C. Rusniok for his help and never-ending patience with informatics problems or questions. This work received financial support from the Institut Pasteur, the Centre National de la Recherche and from the Consortium National de Recherche en Génomique du Réseau National des Genopoles, France. C. Cazalet is holder of a fellowship jointly financed by the Centre National de la Recherche, France, and VeoliaWater—Anjou Recherche.

References Aldridge P, Paul R, Goymer P, Rainey P, Jenal U (2003) Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus. Mol Microbiol 47:1695–708. Amer AO, Swanson MS (2005) Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol 7:765–778. Baar C, Eppinger M, Raddatz G, Simon J, Lanz C, Klimmek O, Nandakumar R, Gross R, Rosinus A, Keller H, Jagtap P, Linke B, Meyer F, Lederer H, Schuster SC (2003) Complete genome sequence and analysis of Wolinella succinogenes. Proc Natl Acad Sci USA 100:11690–11695. Baptestea E, Moreirab D, Philippe H (2003) Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase. Gene 318:185–191. Barz C, Abahji TN, Trulzsch K, Heesemann J (2000) The Yersinia Ser/Thr protein kinase YpkA/YopO directly interacts with the small GTPases RhoA and Rac-1. FEBS Lett 482:139–143.

1. Legionella pneumophila Pathogenesis

27

Batrukova MA, Betin VL, Rubtsov AM, Lopina OD (2000) Ankyrin: structure, properties, and functions. Biochemistry (Mosc) 65:395–408. Benz I, Schmidt MA (1992) AIDA-I, the adhesin involved in diffuse adherence of the diarrhoeagenic Escherichia coli strain 2787 (O126:H27), is synthesized via a precursor molecule. Mol Microbiol 6:1539–1546. Berger KH, Isberg RR (1993) Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7:7–19. Bezanson G, Fernandez R, Haldane D, Burbridge S, Marrie T (1994) Virulence of patient and water isolates of Legionella pneumophila in guinea pigs and mouse L929 cells varies with bacterial genotype. Can J Microbiol 40:426–431. Biederbick A, Rose S, Elsasser HP (1999) A human intracellular apyrase-like protein, LALP70, localizes to lysosomal/autophagic vacuoles. J Cell Sci 112:2473–2484. Brassinga AK, Hiltz MF, Sisson GR, Morash MG, Hill N, Garduño E, Edelstein PH, Garduño RA, Hoffman PS (2003) A 65-Kilobase Pathogenicity Island Is Unique to Philadelphia-1 Strains of Legionella pneumophila. J Bacteriol 185:4630–3637. Brown A, Vickers RM, Elder EM, Lema M, Garrity GM (1982) Plasmid and surface antigen markers of endemic and epidemic Legionella pneumophila strains. J Clin Microbiol 16:230–235. Brüggemann H, Hagman A, Jules M, Sismeiro O, Dillies M, Gouyette C, Kunst F, Steinert M, Heuner K, Coppée J, Buchrieser C (2006) Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell Microbiol 8(8):1228–1240. Carsiotis M, Stocker BA, Weinstein DL, O’Brien AD (1989) A Salmonella typhimurium virulence gene linked to flg. Infect Immun. 57:3276–3280. Cazalet C, Rusniok C, Bruggemann H, Zidane N, Magnier A, Ma L, Tichit M, Jarraud S, Bouchier C, Vandenesch F, Kunst F, Etienne J, Glaser P, Buchrieser C (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat Genet 36:1165–1173. Cazalet C, Jarraud S, Ghavi-Helm Y, Allignet J, Kunst F, Etienne J, Glaser P, Buchrieser C (2005a) Genetic diversity of the species Legionella pneumophila. In: Cianciotto NP (ed.) Legionella 2005. ASM Press, Chicago, USA. Cazalet C, Rusniok C, Bruggemann H, Jarraud S, Bouchier C, Vandenesch F, Kunst F, Etienne J, Glaser P, Buchrieser C (2005b) Comparative Genomics of the Causative Agent of Legionnaires’ Disease, Legionella pneumophila and Related Species XI International Congress of Bacteriology and Applied Microbiology (IUMS), San Francisco, 23–28 July. Chen J, de Felipe KS, Clarke M, Lu H, Anderson OR, Segal G, Shuman HA (2004) Legionella effectors that promote nonlytic release from protozoa. Science 303:1358–1361. Chien M, Morozova I, Shi S, Sheng H, Chen J, Gomez SM, Asamani G, Hill K, Nuara J, Feder M, Rineer J, Greenberg JJ, Steshenko V, Park SH, Zhao B, Teplitskaya E, Edwards JR, Pampou S, Georghiou A, Chou IC, Iannuccilli W, Ulz ME, Kim DH, Geringer-Sameth A, Goldsberry C, Morozov P, Fischer SG, Segal G, Qu X, Rzhetsky A, Zhang P, Cayanis E, De Jong PJ, Ju J, Kalachikov S, Shuman HA, Russo JJ (2004) The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305:1966–1968. Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE, 3rd, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T,

28

Christel Cazalet and Carmen Buchrieser

Jagels K, Barrell BG, et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544. Conover GM, Derre I, Vogel JP, Isberg RR (2003) The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol Microbiol 48:305–321. Derre I, Isberg RR (2005) LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway. Infect Immun 73:4370–4380. Desvaux M, Parham NJ, Henderson IR (2004) The autotransporter secretion system. Res Microbiol 155:53–60 Dhandayuthapani S, Blaylock MW, Bebear CM, Rasmussen WG, Baseman JB (2001) Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium. J Bacteriol 183:5645–5650. Doyle RM, Heuzenroeder MW (2002) A mutation in an ompR-like gene on a Legionella longbeachae serogroup 1 plasmid attenuates virulence. Int J Med Microbiol 292:227–239. Farris M, Grant A, Richardson TB, O’Connor CD (1998) BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells. Mol Microbiol 28:265–279. Feldman M, Segal G (2004) A specific genomic location within the icm/dot pathogenesis region of different Legionella species encodes functionally similar but nonhomologous virulence proteins. Infect Immun 72:4503–4511. Franceschini N, Boschi L, Pollini S, Herman R, Perilli M, Galleni M, Frere JM, Amicosante G, Rossolini GM (2001) Characterization of OXA-29 from Legionella (Fluoribacter) gormanii: molecular class D beta-lactamase with unusual properties. Antimicrob Agents Chemother 45:3509–3516. Glaser P, Frangeul L, Bluchrieser C, Rusniok C, Amend A, Baquero F, Berche P, Bloecker H, Brandt P, Chakraborty T, Charbit A, Chetouani F, Couve E, de Daruvar A, Dehoux P, Domann E, Dominguez-Bernal G, Duchaud E, Durant, Dussurget O, Entian KD, Fsihi H, Garcia-del Portillo F, Garrido P, Gautier L, Goebel W, Gomez-Lopez N, Hain T, Hauf J, Jackson D, Jones LM, Kaerst U, Kreft J, Kuhn M, Kunst F, Kurapkat G, Madueno E, Maitournam A, Vicente JM, Ng E, Nedjari H, Nordsiek G, Novella S, de Pablos B, Perez-Diaz JC, Purcell R, Remmel B, Rose M, Schlueter T, Slimoes N, Tierrez A, Vaquez-Boland JA, Voss H, Wehland J, Cossart P (2001) Comparative genomics of Listeria species. Science 294:849–852. Grant AJ, Farris M, Alefounder P, Williams PH, Woodward MJ, O’Connor CD (2003) Coordination of pathogenicity island expression by the BipA GTPase in enteropathogenic Escherichia coli (EPEC). Mol Microbiol 48:507–521. Hakansson S, Galyov EE, Rosqvist R, Wolf-Watz H (1996) The Yersinia YpkA Ser/Thr kinase is translocated and subsequently targeted to the inner surface of the HeLa cell plasma membrane. Mol Microbiol 20:593–603. Hershko A, Ciechanover A (1998) The ubiquitin system. Annu Rev Biochem 67:425–479. Heyninck K, Beyaert R (2005) A20 inhibits NF-kappaB activation by dual ubiquitin-editing functions. Trends Biochem Sci 30:1–4. Hiltz MF, Sisson GR, Brassinga AK, Garduño E, Garduño RA, Hoffman PS (2004) Expression of magA in Legionella pneumophila Philadelphia-1 is developmentally regulated and a marker of formation of mature intracellular forms. J Bacteriol 186:3038–3045.

1. Legionella pneumophila Pathogenesis

29

Hisert KB, MacCoss M, Shiloh MU, Darwin KH, Singh S, Jones RA, Ehrt S, Zhang Z, Gaffney BL, Gandotra S, Holden DW, Murray D, Nathan C (2005) A glutamate-alanine-leucine (EAL) domain protein of Salmonella controls bacterial survival in mice, antioxidant defence and killing of macrophages: role of cyclic diGMP. Mol Microbiol 56:1234–1245. Hryniewicz-Jankowska A, Czogalla A, Bok E, Sikorsk AF (2002) Ankyrins, multifunctional proteins involved in many cellular pathways. Folia Histochem Cytobiol 40:239–249. Jacobi S, Heuner K (2003) Description of a putative type I secretion system in Legionella pneumophila. Int J Med Microbiol 293:349–358. Keane OM, Dorman CJ (2003) The gyr genes of Salmonella enterica serovar Typhimurium are repressed by the factor for inversion stimulation, Fis. Mol Genet Genomics. 270:56–65. Epub 2003 Jul 30. Kelly A, Goldberg MD, Carroll RK, Danino V, Hinton JC, Dorman CJ (2004) A global role for Fis in the transcriptional control of metabolism and type III secretion in Salmonella enterica serovar Typhimurium. Microbiology. 150:2037–2053. Klemm P, Hjerrild L, Gjermansen M, Schembri MA (2004) Structure-function analysis of the self-recognizing Antigen 43 autotransporter protein from Escherichia coli. Mol Microbiol 51:283–296. Ko KS, Hong SK, Lee HK, Park MY, Kook YH (2003a) Molecular evolution of the dotA gene in Legionella pneumophila. J Bacteriol 185:6269–6277. Ko KS, Lee HK, Park MY, Kook YH (2003b) Mosaic structure of pathogenicity islands in Legionella pneumophila. J Mol Evol 57:63–72. Luneberg E, Mayer B, Daryab N, Kooistra O, Zahringer U, Rohde M, Swanson J, Frosch M (2001) Chromosomal insertion and excision of a 30 kb unstable genetic element is responsible for phase variation of lipopolysaccharide and other virulence determinants in Legionella pneumophila. Mol Microbiol 39:1259–1271. Luo ZQ, Isberg RR (2004) Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc Natl Acad Sci USA 101:841–846. Epub 2004 Jan 8. Marra A, Blander SJ, Horwitz MA, Shuman HA (1992) Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc Natl Acad Sci USA 89:9607–9611. Molofsky AB, Swanson MS (2003) Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication. Mol Microbiol 50:445–461. Morozova I, Qu X, Shi S, Asamani G, Greenberg JE, Shuman HA, Russo JJ (2004) Comparative sequence analysis of the icm/dot genes in Legionella. Plasmid 51:127–147. Moskovitz J, Rahman MA, Strassman J, Yancey SO, Kushner SR, Brot N, Weissbach H (1995) Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J Bacteriol 177:502–507. Muder RR, Yu VL (2002) Infection due to Legionella species other than L. pneumophila. Clin Infect Dis 35:990–998. Nagai H, Kagan JC, Zhu X, Kahn RA, Roy CR (2002) A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295:679–682. Ogata H, Renesto P, Audic S, Robert C, Blanc G, Fournier PE, Parinello H, Claverie JM, Raoult D, Galvao MA, Mafra C, Chamone CB, Calic SB, Zavala-Velazquez JE, Walker DH, Kelly PJ, Meads N, Theobald A, Fang R, Houhamdi L, Azad AF, Parola P, Sanogo OY, Lerdthusnee K, Zeaiter Z, Chauvancy G, Gonzalez JP, Miller RS, Telford SR, 3rd,

30

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Wongsrichanalai C, McDaniel P, Rolain JM, Franc M, Davoust B, La Scola B, Meconi S, Fenollar F, Roux V, Stuhl L, Maurin M, Richter J, Petridou J, Haussinger D, Enea M, de Lamballerie X (2005) The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite. PLoS Biol 3:e248. Epub 2005 Jul 5. Osuna R, Lienau D, Hughes KT, Johnson RC (1995) Sequence, regulation, and functions of fis in Salmonella typhimurium. J Bacteriol. 177:2021–2032. Rankin S, Li Z, Isberg RR (2002) Macrophage-induced genes of Legionella pneumophila: protection from reactive intermediates and solute imbalance during intracellular growth. Infect Immun 70:3637–3648. Reiss U, Oskouian B, Zhou J, Gupta V, Sooriyakumaran P, Kelly S, Wang E, Merrill AHJ, Saba JD (2004) Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis. J Biol Chem 279:1281–1290. Ridenour DA, Cirillo SL, Feng S, Samrakandi MM, Cirillo JD (2003) Identification of a gene that affects the efficiency of host cell infection by Legionella pneumophila in a temperature-dependent fashion. Infect Immun 71:6256–6263. Rossier O, Cianciotto NP (2001) Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila. Infect Immun 69:2092–2098. Rossier O, Cianciotto NP (2005) The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect Immun 73:2020–2032. Rudnick PA, Arcondeguy T, Kennedy CK, Kahn D (2001) glnD and mviN are genes of an essential operon in Sinorhizobium meliloti. J Bacteriol 183:2682–2685. Sauer JD, Bachman MA, Swanson MS (2005) The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. Proc Natl Acad Sci USA 102:9924–9929. Segal G, Russo JJ, Shuman HA (1999) Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila. Mol Microbiol 34:799–809. Segal G, Feldman M, Zusman T (2005) The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii. FEMS Microbiol Rev. 29:65–81. Seshadri R, Paulsen IT, Eisen JA, Read TD, Nelson KE, Nelson WC, Ward NL, Tettelin H, Davidsen TM, Beanan MJ, Deboy RT, Daugherty SC, Brinkac LM, Madupu R, Dodson RJ, Khouri HM, Lee KH, Carty HA, Scanlan D, Heinzen RA, Thompson HA, Samuel JE, Fraser CM, Heidelberg JF (2003) Complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci USA 100:5455–5460. Epub 2003 Apr 18. Sheikh J, Hicks S, Dall’Agnol M, Phillips AD, Nataro JP (2001) Roles for Fis and YafK in biofilm formation by enteroaggregative Escherichia coli. Mol Microbiol 41:983–997. Simm R, Morr M, Kader A, Nimtz M, Romling U (2004) GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53:1123–1134. Skaar EP, Tobiason DM, Quick J, Judd RC, Weissbach H, Etienne F, Brot N, Seifert HS (2002) The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. Proc Natl Acad Sci USA 99:10108–10113.

1. Legionella pneumophila Pathogenesis

31

Soderberg MA, Rossier O, Cianciotto NP (2004) The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures. J Bacteriol 186:3712–3720. Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M, Garber RL, Goltry L, Tolentino E, WestbrockWadman S, Yuan Y, Brody LL, Coulter SN, Folger KR, Kas A, Larbig K, Lim R, Smith K, Spencer D, Wong GK, Wu Z, Paulsen IT, Reizer J, Saier MH, Hancock RE, Lory S, Olson MV. (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–64. Swanson MS, Fernandez-Moreira E (2002) A microbial strategy to multiply in macrophages: the pregnant pause. Traffic 3:170–177. Tal R, Wong HC, Calhoon R, Gelfand D, Fear AL, Volman G, Mayer R, Ross P, Amikam D, Weinhouse H, Cohen A, Sapir S, Ohana P, Benziman M (1998) Three cdg operons control cellular turnover of cyclic di-GMP in Acetobacter xylinum: genetic organization and occurrence of conserved domains in isoenzymes. J Bacteriol 180:4416–4425. Vogel JP, Andrews HL, Wong SK, Isberg RR (1998) Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873–876. Walburger A, Koul A, Ferrari G, Nguyen L, Prescianotto-Baschong C, Huygen K, Klebl B, Thompson C, Bacher G, Pieters J (2004) Protein Kinase G from Pathogenic Mycobacteria Promotes Survival Within Macrophages. Science DOI: 10.1126/science.1099384. Wu M, Sun LV, Vamathevan J, Riegler M, Deboy R, Brownlie JC, McGraw EA, Martin W, Esser C, Ahmadinejad N, Wiegand C, Madupu R, Beanan MJ, Brinkac LM, Daugherty SC, Durkin AS, Kolonay JF, Nelson WC, Mohamoud Y, Lee P, Berry K, Young MB, Utterback T, Weidman J, Nierman WC, Paulsen IT, Nelson KE, Tettelin H, O’Neill SL, Eisen JA (2004) Phylogenomics of the Reproductive Parasite Wolbachia pipientis wMel: A Streamlined Genome Overrun by Mobile Genetic Elements. PLoS Biol 2:E69. Yu VL, Plouffe JF, Pastoris MC, Stout JE, Schousboe M, Widmer A, Summersgill J, File T, Health CM, Paterson DL, Chereshsky A (2002) Distribution of Legionella species and serogroups isolated by culture in patients with sporadic community-acquired legionellosis: an international collaborative survey. J Infect Dis 186:127–128. Yuste L, Hervas AB, Canosa I, Tobes R, Jimenez JI, Nogales J, Perez-Perez MM, Santero E, Diaz E, Ramos JL, de Lorenzo V, Rojo F (2006) Growth phase-dependent expression of the Pseudomonas putida KT2440 transcriptional machinery analysed with a genomewide DNA microarray. Environ Microbiol 8:165–77.

2 Iron Assimilation and Type II Protein Secretion Nicholas P. Cianciotto

1. Iron Assimilation 1.1. The Importance of Iron for Legionella pneumophila Iron is a key requirement for L. pneumophila replication (Reeves et al, 1981). As originally defined, the iron requirement for L. pneumophila was estimated to be 3–13 ␮M for minimal growth and ⬎20 ␮M for optimal growth (Reeves et al, 1983; Johnson et al, 1991; Mengaud and Horwitz, 1993). However, recent studies using chemically defined media indicate that the requirement is ⬍1 ␮M (James et al, 1995; Liles et al, 2000). In standard BCYE agar, iron is added in the form of ferric pyrophosphate, although ferric chloride, ferric nitrate, and ferrous sulfate can also be used. Upon incubation with 55FeCl3, L. pneumophila uptakes significant amounts of radiolabeled iron in an energy-dependent, protease-resistant process (Johnson et al, 1991). L. pneumophila also binds and uses hemin as a source of iron (O’Connell et al, 1996). As in other bacteria, L. pneumophila requires iron as a cofactor in enzymes, such as a superoxide dismutase and aconitase (Mengaud and Horwitz, 1993). Iron is especially relevant for Legionella pathogenesis. Much evidence signals that the ability of L. pneumophila to replicate in the mammalian host is dependent upon iron. First, human monocytes and macrophages treated with iron chelators do not support Legionella replication, and interferon-␥ inhibits bacterial growth by reducing available iron (Byrd and Horwitz, 2000; Viswanathan et al, 2000). Second, A/J mouse macrophages become permissive for Legionella infection following the addition of iron (Gebran et al, 1994). Third, a reduced ability of legionellae to establish intracellular infection is correlated with decreased levels of the receptor for transferrin, the key iron transport protein of the host cell (Byrd and Horwitz, 2000). Fourth, the introduction of iron into animals increases their susceptibility to infection. Fifth, legionellae grown under iron-deplete conditions exhibit reduced pathogenicity (James et al, 1995). The first genetic data on the importance of iron in Legionella was the identification of the transcriptional repressor Fur (Hickey and Cianciotto, 1994). L. pneumophila Fur is comparable in size and cross-reactive with Escherichia

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coli Fur. Its repressive activity is, as expected, highest in legionellae grown in iron-rich media (Hickey and Cianciotto, 1994, 1997). L. pneumophila Fur has an amino acid identity of over 54% and a similarity of over 72% to Fur from E. coli, Pseudomonas aeruginosa, and others (Hickey and Cianciotto, 1994). The promoter region of L. pneumophila fur contains sequences homologous to the Fur-binding site, suggesting that fur is auto-regulated in Legionella. The fur gene is conserved among Legionella species (Hickey and Cianciotto, 1994). Given an inability to insertionally inactivate L. pneumophila fur, Fur may be essential for Legionella aerobic growth. The importance of fur is substantiated by the identification of multiple iron- and Fur-regulated genes in L. pneumophila (Hickey and Cianciotto, 1997).

1.2. Legionella Siderophores 1.2.1. Early Negative Findings It was reported in 1983 that L. pneumophila does not make siderophores (Reeves et al, 1983). This conclusion was based largely on results from Arnow and Csáky assays, which detect catecholate and hydroxamate structures, respectively. Years later, the issue of Legionella siderophores was revisited using the CAS assay, which detects iron chelators independently of structure (Goldoni et al, 1991). CAS reactivity was detected in statically grown L. pneumophila cultures, suggesting the existence of a non-catecholate, non-hydroxamate siderophore. However, a later study determined that the CAS reactivity was due to the cysteine in the chemically defined growth medium (Liles and Cianciotto, 1996). When supernatants were derived from cultures generated with cysteine-free media, no siderophore activity was detected using the CAS-, Arnow-, or Csáky assays (Liles and Cianciotto, 1996; James et al, 1997). 1.2.2. The Discovery of Legiobactin The story of Legionella siderophores changed in 2000, when it was shown that L. pneumophila could excrete a high-affinity iron-chelator (Liles et al, 2000). Indeed, when grown with shaking at 37oC in a low-iron, chemically defined medium (CDM), L. pneumophila strains secrete a substance that is highly reactive in the CAS assay. Importantly, the siderophore activity is only seen when cultures are inoculated with bacteria that had been grown to log or early-stationary phase. Inocula derived from late-stationary phase cultures, despite growing in the CDM, do not elaborate CAS reactivity. L. pneumophila CAS reactivity was observed for virulent serogroup 1 strains 130b (also known as AA100 and Wadsworth), Philadelphia-1, and Oxford-4032E, as well as clinical and environmental isolates representing all nine of the other L. pneumophila serogroups tested (Liles et al, 2000). The nature of the CAS reactivity has been studied further in L. pneumophila strain 130b, and all data indicate that it represents a bona fide siderophore (Liles et al, 2000). First, the presence of CAS reactivity correlates with enhanced aerobic

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growth in an iron-deplete defined medium. Second, the chelating activity is subject to iron-repression; i.e., the addition of iron to the CDM decreases reactivity, whereas the removal of additional iron from the CDM cultures increases CAS reactivity. Third, the CAS reactivity is less than 1 kDa and is resistant to heat and proteases. Fourth, CAS reactions are rapid and intense. Fifth, CAS-positive supernatants facilitate the growth of wild-type legionellae in BCYE agar containing otherwise inhibitory concentrations of the iron chelator 2,2’-dipyridyl (DIP) (Allard et al, 2005). The iron-chelating activity in L. pneumophila supernatants is known as legiobactin (Liles et al, 2000). CAS-reactive supernatants remain negative in the Arnow, and Csáky assays, implying that legiobactin is not a typical catecholate or hydroxamate. In support of this notion, solvents used to extract common siderophores do not extract the Legionella siderophore (Liles et al, 2000). Legiobactin has been purified and its structure is nearly determined (K. A. Allard and N. P. Cianciotto, unpublished results). Representatives of most other Legionella species tested, including clinical and environmental isolates, also secrete CAS reactivity (Liles et al, 2000; Starkenburg et al, 2004). Although it cannot be concluded these species are producing legiobactin, their supernatants, like that of the L. pneumophila strains, are negative in the Arnow and Csáky assays. Some Legionella species do not display significant CAS reactivity (Starkenburg et al, 2004). However, these bacteria grow poorly in the CDM, because the medium is lacking an undefined growth factor other than iron. Thus, it is uncertain as to whether these legionellae lack a siderophore, since the absence of CAS reactivity could have been an indirect effect of minimal growth. 1.2.3. Genetics of Legiobactin Production The first gene to be examined as a possible promoter of legiobactin production was frgA, the ferric-regulated gene A identified in 1997 as an iron-regulated locus in strain 130b (Hickey and Cianciotto, 1997). Although FrgA is homologous with several hydroxamate synthetases, including enzymes involved in the biosynthesis of aerobactin in E. coli and Shigella species, alcaligin in Bordetella, and rhizobactin in Sinorhizobium (Hickey and Cianciotto, 1997), frgA mutants proved not to be defective for CAS reactivity (Liles et al, 2000). However, a L. pneumophila gene that is involved in legiobactin production has been recently uncovered. This gene, called lbtA for legiobactin gene A, encodes a product that is also homologous to multiple hydroxamate synthetases. Most importantly, several independently derived lbtA mutants have ca. 50% less CAS-reactive material when they are grown in iron-deplete CDM, indicating that LbtA is required for legiobactin production (Allard et al, 2005). The gene (lbtB) immediately downstream of lbtA is also required for full siderophore expression. Whereas LbtA is predicted to be involved in the biosynthesis of legiobactin, LbtB is likely involved in siderophore export, as it is a member of a family of transmembrane proteins associated with the export of small molecules such as siderophores. Several Fur boxes precede lbtA, suggesting that Fur mediates the iron regulation associated with legiobactin

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production. LbtA mutants show impaired growth in deferrated CDM that contains citrate, indicating that legiobactin facilitates extracellular growth in low-iron conditions. Southern blots show that lbtA is distributed among Legionella strains, a result compatible with the fact that many species produce CAS reactivity (Allard et al, 2005). 1.2.4. The Role of lbtA and frgA in Infection To start to gauge the role of siderophores in Legionella infection, the ability of the lbtA mutant to infect human U937 macrophages and Hartmannella vermiformis amoebae was determined. Although lbtA was expressed intracellularly, the mutant was unimpaired for infection of host cells, suggesting that LbtA and legiobactin are relevant but not required for intracellular infection (Allard et al, 2005). These data do not, however, indicate that siderophores are not needed intracellularly; the loss of legiobactin might be compensated for by another siderophore. Along those lines, frgA mutants are defective for growth in macrophages (Hickey and Cianciotto, 1997), raising the possibility that L. pneumophila encodes a siderophore that, unlike legiobactin, is necessary for optimal intracellular replication. Since frgA is absent from non-pneumophila strains (Hickey and Cianciotto, 1997), it is tempting to speculate that such a siderophore would be specific to L. pneumophila. 1.2.5. Summary and Current Models With the discovery of legiobactin and lbtA, we now, after years of disbelief, have strong evidence for the existence of a Legionella siderophore. It is probable that the novel influence of the bacterial inoculum on siderophore production is the main reason that legiobactin was not detected earlier. In retrospect, it is not surprising that L. pneumophila produces a siderophore, since many other aquatic bacteria produce this type of iron scavenger. Based upon the behavior of lbtA mutants, legiobactin is most critical for extracellular, rather than intracellular, growth. Thus, it is conceivable that the siderophore is vital for growth in aquatic environments such as biofilms. Although lbtA shares homology with enzymes involved in the biosynthesis of several well-studied hydroxamates, legiobactin may prove to be novel in structure, for several reasons. First, the observed CAS-reactivity is Csákyand Arnow-negative. Second, the L. pneumophila genome has not revealed additional genes (other than frgA) that would be predicted to promote the biosynthesis of a known hydroxamate. Although a biochemical activity associated with frgA has not been defined, the gene, based upon sequence data, is still a candidate siderophore gene. At present, the argument for frgA having a role in iron assimilation is that the gene is iron-regulated and required for optimal infection of macrophages. These data, coupled with the fact that LbtA mutants are not completely lacking CAS reactivity, suggests that L. pneumophila elaborates multiple siderophores. Whereas legiobactin is linked to extracellular growth, a second siderophore might be critical for intracellular infection. That L. pneumophila uses multiple siderophores in order to flourish in different niches is quite reasonable.

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1.3. Ferrous Iron Transport Since Fe2⫹ is present in the eukaryotic iron pool, presumably available to intracellular legionellae, the means by which L. pneumophila acquires and uses ferrous iron has been recently addressed. Toward that end, the feoB gene was identified and mutated in L. pneumophila strain 130b (Robey and Cianciotto, 2002). L. pneumophila FeoB shares 44% identity and 61% similarity with E. coli FeoB. Like its E. coli counterpart, Legionella FeoB has ten transmembrane domains, consistent with an inner membrane location. The gene’s promoter region has a putative Fur binding site, suggesting that L. pneumophila feoB is subject to iron regulation. feoB mutants exhibit decreased ferrous, but not ferric, radiolabeled iron uptake (Robey and Cianciotto, 2002). Growth of L. pneumophila in standard media is unaffected by the loss of FeoB. However, the feoB mutant has reduced growth on low-iron BCYE agar (Robey and Cianciotto, 2002). Compatible with its iron uptake defect, the mutant shows accelerated colonial growth on low-iron agar when plated around wells containing ferric but not ferrous iron salts. Additionally, the feoB mutant shows accelerated colonial growth when plated around wells containing legiobactin. The feoB mutant also has a growth defect in low-iron BYE broth (Robey and Cianciotto, 2002). This defect is exacerbated by the addition of DIP to the media, a result that can be reversed by additional supplementation with ferric chloride (Robey and Cianciotto, 2002). Together, these data indicate that L. pneumophila FeoB and Fe2⫹ transport are required for extracellular growth in low-iron conditions. Although aerobic conditions can result in a predominance of ferric iron, ferrous iron is present within Legionella cultures. FeoB might also transport Fe2⫹ generated in the periplasm, following reduction of acquired Fe3⫹ by a periplasmic ferric reductase (see below). The role of FeoB in intracellular infection has been assessed using U937 cells and H. vermiformis amoebae (Robey and Cianciotto, 2002). In the macrophage cell line, the feoB mutant exhibits a 10- to 15-fold reduced recovery compared to wildtype. Further evidence of the role of L. pneumophila FeoB in macrophage infection was seen using a cytopathicity assay. Whereas ⬎95% of the host cells are destroyed 72h after inoculation with wildtype bacteria, the viability of macrophages infected with FeoB-deficient bacteria does not decrease significantly over the assay period. A correlation between iron levels in macrophages and the growth defect observed in the feoB mutant has been obtained from infection assays using U937 cells that were depleted of iron by treatment with DIP. Indeed, the 15-fold growth defect seen with untreated cells increases to 105-fold in the presence of 50 ␮M DIP, respectively. When amoebal cultures were done in the presence of 50 ␮M DIP, the feoB mutant exhibited a 150-fold reduction in growth, relative to wildtype. Together, these results indicate that feoB and ferrous transport are important for the growth and survival of L. pneumophila in macrophages and amoebae (Robey and Cianciotto, 2002). The role of feoB in disease was examined using a competition assay and the A/J mouse model of pneumonia (Robey and Cianciotto, 2002). Bacteria were recovered from the

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lungs of mice following intratracheal inoculation with equal numbers of wildtype and mutant. At 3 days post-inoculation, the wildtype had outgrown the mutant by threefold, indicating that FeoB promotes in vivo growth. In summary, L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth. The infectivity defect of the feoB mutant indicates that ferrous iron is present and accessible in the L. pneumophila intracellular niche. That the feoB mutant is not completely defective for intracellular growth and only exhibits a modest virulence defect in mice implies that other uptake systems and/or iron sources can overcome decreased ferrous iron uptake. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.

1.4. The L. pneumophila Iron Acquisition/Assimilation Locus Following a screen for mutants that are hypersensitive to the iron chelator EDDA and/or resistant to streptonigrin, the L. pneumophila iraAB (iron acquisition/assimilation) locus was identified (Pope et al, 1996). Importantly, insertions within iraA result in a severe lag in intracellular growth and a slowed rate of replication such that macrophage monolayers infected with iraA mutants yield 1000-fold fewer bacteria (Pope et al, 1996; Viswanathan et al, 2000). This intracellular growth defect is exacerbated by treatment of the host cells with desferrioxamine, confirming that the iraA mutants are defective for intracellular iron acquisition (Pope et al, 1996; Viswanathan et al, 2000). The iraA gene encodes a 272-aa protein that shows sequence similarity to methyltransferases that use Sadenosylmethionine as a donor. The iraB gene encodes a 501-aa protein that is highly similar to the PTR2 family of di- or tripeptide transporters and is predicted to be an inner membrane protein with 12 membrane spanning domains. A mutant containing an insertion in iraB consistently shows reduced growth in iron-depleted BYE broth (a phenotype eventually lost by iraA mutants) but does not have a defect in macrophages (Viswanathan et al, 2000). Thus, L. pneumophila iraA is critical for intracellular infection, although it is still not clear how IraA promotes infection or serves as a dispensable facilitator of iron assimilation. In contrast, iraB is most critical for growth under low-iron extracellular conditions. IraB may be involved in a novel pathway that imports iron-loaded peptides as an iron source. iraAB is conserved among strains of L. pneumophila but largely absent from other legionellae (Viswanathan et al, 2000).

1.5. Cytochrome C Maturation and Iron Assimilation Mutations within the cytochrome c maturation (ccm) locus greatly diminish the ability of L. pneumophila to grow on low-iron media; e.g., ccmB, ccmC, and ccmF mutants have a greatly reduced efficiency of plating on BCYE agar lacking added iron (Viswanathan et al, 2002; Naylor and Cianciotto, 2004). Ccm mutants are also dramatically impaired for replication in macrophages, amoebae, and the lungs of A/J mice (Viswanathan et al, 2002; Naylor and Cianciotto, 2004). The mutants’

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intracellular infectivity defects are exacerbated by treatment of the macrophages with desferrioxamine but ameliorated somewhat by the addition of iron, indicating that L. pneumophila ccm is required for optimal intracellular iron assimilation (Viswanathan et al, 2002; Naylor and Cianciotto, 2004). Bacterial Ccm systems are generally hypothesized to mediate the transfer of heme into the periplasm and then the attachment of heme to apocytochrome c (Cianciotto et al, 2005). Indeed, L. pneumophila ccm mutants lack cytochrome c (Viswanathan et al, 2002). However, the observations made with L. pneumophila are the fourth example of a linkage between ccm genes and bacterial iron assimilation, complementing studies with Paracoccus, Pseudomonas, and Rhizobium species (Cianciotto et al, 2005). The manner in which ccm promotes iron assimilation is unclear, although, in the case of the other bacteria, ccm mutants have changes in siderophore expression. Since the L. pneumophila ccm mutants produce normal levels of CAS reactivity (Viswanathan et al, 2002), it is possible that the Legionella ccm locus facilitates growth in low-iron conditions by a novel mechanism. The infectivity defects of the L. pneumophila ccm mutants indicate, for the first time, that ccm can be necessary for growth within an intracellular niche.

1.6. Legionella Ferric Reductases Legionella pneumophila encodes a cytoplasmic and a periplasmic ferric reductase (Johnson et al, 1991; Poch and Johnson, 1993; James et al, 1997). The purified periplasmic and cytoplasmic enzymes respectively use glutathione and NADPH as favored reductants (Poch and Johnson, 1993; James et al, 1997). Both enzymes are expressed within iron-limited and iron-replete cultures and within virulent and avirulent organisms (Poch and Johnson, 1993; James et al, 1997). Although ferric citrate can serve as a substrate for the purified reductases, it need not be the most physiologically relevant substrate.

1.7. Concluding Remarks Legionella pneumophila uses multiple pathways for iron acquisition. It may produce several siderophores; i.e., there is biochemical and genetic evidence for legiobactin, and genetic data for the existence of an hydroxamate-like iron chelator. Based upon observations with other bacteria, it is possible that Ccm proteins influence Legionella siderophores. Once internalized, ferrisiderophores may be acted upon by the periplasmic ferric reductase, yielding ferrous iron that would be transported across the inner membrane by FeoB. Alternatively, a yet-to-be described transporter might deliver the ferrisiderophore across the inner membrane, whereupon the cytoplasmic ferric reductase would generate ferrous iron. L. pneumophila environments contain significant amounts of ferrous iron, and thus a Fe2⫹-uptake pathway may also feed into FeoB. As yet another pathway, the L. pneumophila iraAB locus may encode an inner-membrane transporter that imports iron-loaded peptides. In addition to utilizing ferric and ferrous iron, L. pneumophila can bind and use hemin, in part through the action of the Hbp

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protein (O’Connell et al, 1996). The existence of multiple iron uptake systems in L. pneumophila is quite compatible with the fact that the bacterium resides within such a variety of environments.

2. Type II Protein Secretion 2.1. Type II Protein Secretion in Gram-Negative Bacteria In Gram-negative bacteria, type II protein secretion (T2S) is one of five protein secretion systems that permit the export of proteins from within the bacterial cell to the extracellular milieu and/or into target host cells. An analysis of the many sequenced genomes reveals that T2S genes are common but not universal within Gram-negatives (Cianciotto, 2005). Recent functional studies indicate that T2S can promote the virulence of human, animal, and plant pathogens as well as the physiology of various environmental bacteria (Cianciotto, 2005). T2S can be viewed as a two-step process (Filloux, 2004). In the first step, proteins destined for export are translocated across the inner membrane by the Sec or Tat pathway and then N-terminally cleaved by signal peptidase. In the second step, the proteins are transported from the periplasm to the exterior through the action of a complex of proteins specifically dedicated to T2S (i.e., the secreton), ultimately resulting in the passage of the proteins through an outer membrane pore (i.e., secretin). From work in several model organisms, such as Klebsiella, Pseudomonas, and Vibrio, there is now a relatively good understanding of the components of the T2S apparatus and the mechanism of the secretion process (Filloux, 2004). It is also clear that the T2S apparatus bears an evolutionary relationship to the type 4 pilus apparatus (Peabody et al, 2003). From the analysis of approximately a dozen different genera, the T2S can be viewed as consisting of 12 core components; i.e., the outer membrane secretin (T2S D), a cytoplasmic ATPase (T2S E), an inner (trans)membrane protein (T2S F), the major (T2S G) and minor (T2S H, I, J, K) pseudopilins, facilitators of the ATPase attachment to the inner membrane that appear, along with T2S F, to form an inner membrane platform (T2S L, M), the pre-pseudopilin peptidase/methyltransferase (T2S O) that also acts upon type 4 prepilin, and a protein that may be involved in substrate recognition and/or secretin interactions (T2S C) (Peabody et al, 2003; Filloux, 2004). After translocation across the inner membrane, a protein destined for export is cleaved and its nascent N-terminus methylated by the pre-pseudopilin peptidase (also commonly known as PilD). The processed protein is then recognized for entry into the T2S apparatus by a mechanism that is not understood but likely involves tertiary structural determinants. The proteins ultimately exit the cell through the secretin pore in an energydependent process, with the pseudopilins forming a pilus-like structure that appears like a piston to push the proteins out through the secretin.

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2.2. Discovery of Type II Secretion in Legionella pneumophila The first indication that L. pneumophila has a T2S system was a 1998 report of a pilBCD operon, encoding the pre-pseudopilin peptidase T2S O (PilD), along with PilB and PilC proteins, which are strictly dedicated to type 4 pilus biogenesis (Liles et al, 1998). A subsequent report then showed that a mutation in the pilD gene of L. pneumophila serogroup-1 strain 130b reduced protein secretion as evidenced by the loss of proteins in mutant culture supernatants visualized by 1-dimensional SDS-PAGE (Liles et al, 1999). A later study in L. pneumophila strain Philadelphia-1 reported the presence of a locus, lspFGHIJK, predicted to encode homologs of the T2S FGHIJK proteins (Hales and Shuman, 1999). As was the case for mutation of pilD, mutation of lspGH resulted in loss of protein secretion as measured by 1-dimensional SDS-PAGE. Two further studies in L. pneumophila strain 130b reported the presence of genes encoding homologs of T2S DE (lspDE), C (lspC), and LM (lspLM) (Rossier and Cianciotto, 2001; Rossier et al, 2004). Together, these studies indicated that L. pneumophila has a complete and functional type II protein secretion system. This was confirmed when, in 2004, the complete genomes of L. pneumophila serogroup 1 strains Philadelphia 1, Paris, and Lens were published (Cazalet et al, 2004; Chien et al, 2004; Cianciotto, 2005). Southern hybridization analysis has demonstrated that T2S genes are also in other serogroups of L. pneumophila and other species of Legionella (Rossier et al, 2004). Genome sequencing of several strains has also revealed that L. pneumophila encodes Sec and Tat systems for translocating proteins across the inner membrane into the periplasm (Cazalet et al, 2004; Chien et al, 2004; De Buck et al, 2004; Rossier and Cianciotto, 2005). That L. pneumophila sec is essential for growth and necessary for protein secretion is unquestioned. Yet, recent studies have confirmed that the genes encoding the twin-arginine translocation system are expressed during L. pneumophila extra- and intracellular growth (De Buck et al, 2004) and are functional as judged by the negative impact that their mutation has on cytochrome c-dependent respiration and the processing of the cytochrome c reductase PetA (Rossier and Cianciotto, 2005). As expected, L. pneumophila also has and expresses the type I signal peptidase (LepB), which cleaves the N-terminus from proteins translocated across the inner membrane (Lammertyn et al, 2004). Thus, not only does L. pneumophila possess the full set of T2S-specific genes, but it also has the machinery for delivering proteins into the periplasm in order that they might then enter the secretion apparatus. A variety of T2S-specific mutants have been constructed and characterized. All mutants replicate normally within standard BYE broth incubated at 37°C (Rossier and Cianciotto, 2001; Rossier et al, 2004). They also have a typical efficiency of plating on standard BCYE agar at 37°C. However, the mutants do display a slightly altered colony morphology on the BCYE agar (Rossier and Cianciotto, 2001; Rossier et al, 2004).

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2.3. Factors Secreted by the Type II System of L. pneumophila Presently, at least 11 enzymes have been determined to be secreted via the L. pneumophila T2S system (Rossier et al, 2004; Banerji et al, 2005; Cianciotto, 2005). This conclusion is based upon the loss of enzymatic activities from the culture supernatants of lspDE, lspF, lspG, lspGH, or pilD mutants grown in standard BYE broth at 37°C (Hales and Shuman, 1999; Liles et al, 1999; Aragon et al, 2000; Rossier and Cianciotto, 2001; Rossier et al, 2004). The T2S-dependent proteins/activities include tartrate-sensitive and tartrate-resistant acid phosphatases, phospholipase A, phospholipase(s) C, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase (GCAT), mono-, di-, and triacylglycerol lipases, ribonuclease, and protease (Hales and Shuman, 1999; Liles et al, 1999; Aragon et al, 2000; Aragon et al, 2001; Flieger et al, 2001b; Rossier and Cianciotto, 2001; Aragon et al, 2002; Flieger et al, 2002; Rossier et al, 2004; Banerji et al, 2005). Because mutants specifically lacking the type 4 pilus are not defective for any of these activities, the altered secretion of the pilD mutants is due to the loss of T2S (Rossier and Cianciotto, 2001; Rossier et al, 2004). Several of the secreted enzymatic activities have now been identified in other species of Legionella (Flieger et al, 2001a; Rossier et al, 2004). In some cases, the structural genes (proteins) encoding T2S-dependent secreted activities have been identified. These include map (Map) for the tartrate-sensitive acid phosphatase (Aragon et al, 2001), plcA (PlcA) for phospholipase C activity (Aragon et al, 2002), plaA (PlaA) for the lysophospholipase A (Flieger et al, 2002), plaC (PlaC) for GCAT (Banerji et al, 2005), lipA (LipA) and lipB (LipB) for mono- and triacylglycerol lipases (Aragon et al, 2002), and proA/msp (ProA/Msp) the zinc metalloprotease (Hales and Shuman, 1999; Liles et al, 1999). Notably, mutations in any one of these genes do not completely abolish the corresponding activity, suggesting that L. pneumophila has more than one secreted phosphatase, phospholipase C, lysophospholipase A, lipase, and protease/peptidase and that the Legionella T2S system mediates the secretion of more than 11 proteins. Indeed, 2-dimensional SDS-PAGE analysis of wild type strain 130b and its lsp mutant derivatives indicate that more than 30 L. pneumophila proteins are T2S-dependent (DebRoy, S., and N.P. Cianciotto, unpublished results). Given the completion of the L. pneumophila genome, mass spectrometric analysis of protein spots extracted from 2-D gels is now allowing a direct determination of those secreted proteins that are linked to the T2S system. In addition to confirming the T2S-dependency of previously described proteins (e.g., ProA/Msp, Map, PlaA), this type of analysis is defining for the first time the genetic basis for other previously described enzymatic activities (e.g., ribonuclease) as well as uncovering new secreted enzymatic activities (e.g., chitinase, aminopeptidase) (DebRoy, S., and N.P. Cianciotto, unpublished results). The analysis of supernatants from proA/msp mutants is demonstrating that some secreted proteins are subject to cleavage and perhaps activation by the T2S-dependent ProA/Msp metalloprotease (Flieger et al,

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2002; Banerji et al, 2005). In some instances, the T2S-dependent effectors of L. pneumophila are closely related to secreted proteins of other bacteria; e.g., ProA/Msp is homologous with well-characterized proteases from a variety of Gram-negative organisms, and PlcA is highly related to a new type of phospholipase C found in Pseudomonas (Aragon et al, 2002). However, in the case of the Map, the L. pneumophila protein is most related to eukaryotic acid phosphatases (Aragon et al, 2001); the fact that L. pneumophila encodes a number of proteins that bear their greatest homology with eukaryotic proteins has more recently emerged from the annotation of the completed genome (Cazalet et al, 2004; Chien et al, 2004). By virtue of a twin-arginine in its signal peptide, PlcA was predicted to be a Tat substrate; indeed mutations in tatB diminish secreted phospholipase C activity (Rossier and Cianciotto, 2005).

2.4. Role of Type II Secretion in Pathogenesis Legionella pneumophila T2S mutants (i.e., strain 130b derivatives containing an insertion mutation in lspDE, lspF, lspG, lspK, or pilD) display a reduced ability to infect human macrophages, including the U937 cell line and peripheral blood mononuclear cell-derived macrophages (Liles et al, 1999; Polesky et al, 2001; Rossier and Cianciotto, 2001; Rossier et al, 2004). In the U937 cells, the lsp mutants were modestly defective, showing a tenfold reduced recoverability at 48 hours post-inoculation (Polesky et al, 2001; Rossier and Cianciotto, 2001; Rossier et al, 2004). In contrast, an lsp mutant appeared incapable of replication within macrophages derived from peripheral blood cells, indicating that the magnitude of the mutant phenotype is influenced by the nature of the target host cell. Indeed, when tested in the human HL60 cell line, an lspGH mutant behaved in a manner like wild type (Hales and Shuman, 1999). The reduced infectivity of pilD and lsp mutants was complemented by reintroduction of the corresponding gene (Rossier et al, 2004). That L. pneumophila mutants specifically lacking type 4 pili are not defective for macrophage infection indicates that the altered infectivity of the pilD mutant is due to the loss of T2S (Rossier et al, 2004). The T2S mutants do not appear to be defective for entry but rather lack the capacity to optimally replicate (Rossier et al, 2004). They are not impaired for the induction of apoptosis or a pore-forming activity linked to egress from host cells (Molmeret et al, 2002; Zink et al, 2002). Together, these data indicate that T2S promotes L. pneumophila infection of macrophages. Recently, it has been shown that lsp mutants are defective in an animal model of Legionnaires’ disease pneumonia (Rossier et al, 2004). More specifically, upon intratracheal inoculation into A/J mice, lspDE, lspF, and pilD mutants, but not pilus mutants, exhibit a reduced ability to grow in the mammalian lung, as measured by competition assays. Additionally, the lspF was defective in an in vivo growth kinetics assay; i.e., whereas wild type bacteria increased at least tenfold in the A/J mouse lung, the T2S mutant failed to show any evidence of replication. Examination of infected mouse sera revealed that type-II secreted proteins are expressed in vivo. Thus, the L. pneumophila type II secretion is an

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important virulence factor. The reduced survival of the lsp mutants in the A/J mouse lung is likely due, at least in part, to diminished growth in alveolar macrophages. However, that lspF mutant CFU do not increase in the mouse lungs, whereas they do, albeit not optimally, in vitro, raises the possibility that the lsp mutants are also defective for extracellular processes that are operative in the lungs, such as combating other aspects of the innate immune response. The various mutants that lack particular T2S effectors have also been tested for alterations in intracellular infection. However, map, plcA, plaA, plaC, lipA, lipB, and proA/msp mutants all grow normally within macrophages, indicating that the tartrate-sensitive acid phosphatase, PlcA phospholipase C, PlaA lysophospholipase A, PlaC GCAT, LipA and LipB lipases, and the zinc metalloprotease are not required for intracellular infection (Aragon et al, 2001, 2002; Flieger et al, 2002; Banerji et al, 2005). These results suggest that the T2S system secretes a yet-tobe-defined factor that is required for infection. Alternatively, there may be redundancy in the effectors, such that the loss of one enzyme can be compensated with a similar type of enzyme. Clearly, more work is needed in order to fully explain the role of T2S in pathogenesis. However, these various studies do represent the first documentation of a role for T2S in intracellular infection and in an animal model of disease.

2.5. Role of Type II Secretion in Environmental Survival Legionella pneumophila lsp and pilD mutants are severely defective for intracellular growth in fresh water amoebae, including H. vermiformis and Acanthamoeba castellanii (Hales and Shuman, 1999; Liles et al, 1999; Polesky et al, 2001; Rossier and Cianciotto, 2001; Rossier et al, 2004). Indeed, the mutants show little, if any, evidence of replication within the protozoan hosts. The reduced infectivity of the mutants is complemented by reintroduction of the corresponding intact gene, and thus these data clearly demonstrate that T2S is required for amoebal infection. As was the case for macrophage infection, studies using mutants lacking particular secreted enzymes have failed to identify the T2S effector(s) that is critical for protozoan infection. Nonetheless, given the absolute importance of protozoan infection for L. pneumophila survival in aquatic habitats, these data establish T2S as a highly significant promoter of L. pneumophila persistence in environmental niches. That T2S promotes protozoa infection may increase its relevance for pathogenesis, since infected protozoa have been hypothesized to be part of the infective dose (Brieland et al, 1996; Cirillo et al, 1999). Following upon the demonstration that pilD transcript levels are greater in legionellae grown at 30 vs. 37°C (Liles et al, 1998), it was shown, using a pilD::lacZ fusion strain, that pilD transcriptional initiation increases progressively as L. pneumophila strain 130b is grown at 30, 25, and 17°C (Söderberg et al, 2004). Legionella pilD mutants also had a dramatically reduced ability to grow in BYE broth and to form colonies on BCYE agar at the lower temperatures. Whereas strains specifically lacking pili were not defective for low-temperature growth, lsp mutants were greatly impaired for colony formation at 25, 17, and

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12°C (Söderberg et al, 2004). Indeed, the lsp mutants were completely unable to grow at 12°C. The growth defects of the pilD and lsp mutants on the yeast extract media was complemented by reintroduction of the corresponding intact gene. In a series of experiments that more closely mimic aquatic habitats, an lsp mutant displayed reduced survivability in tap water incubated at 25, 17, 12, and 4°C (Söderberg, M.A., and N.P. Cianciotto, unpublished results). Together, these data indicate that T2S promotes the growth of L. pneumophila at low temperatures. Interestingly, the lsp mutants displayed improved growth at 25°C when plated next to a streak of wild type but not mutant bacteria, implying that a secreted, diffusible factor promotes low-temperature growth (Söderberg et al, 2004). Indeed, growth stimulation could be seen using filter-sterilized supernatants from wild type. Mutants lacking the known acid phosphatase, lipases, phospholipase C, lysophospholipase A, or protease grow normally at 25°C, suggesting the existence of a critical, yet-to-be-defined exoproteins(s) (Söderberg et al, 2004). Recent 2-D SDS-PAGE comparisons of supernatants from cultures grown at different temperatures reveal proteins that are only expressed or hyperexpressed at low temperatures (Söderberg, M.A., and N.P. Cianciotto, unpublished results). In summary, these data document, for the first time, that L. pneumophila replicates at temperatures below 20°C and that a bacterial T2S system facilitates growth at low temperatures.

2.6. Concluding Comments The large amount of data obtained from studies with macrophages, experimental animals, protozoa, and low-temperature conditions show how the Legionella T2S system promotes bacterial growth both in a mammalian host and in environmental niches. Future work will assess whether the same or distinct T2S effectors are critical for growth in the disparate niches. Nonetheless, L. pneumophila Lsp already represents the best-characterized T2S system in terms of understanding the importance of the system in a bacterium’s physiology, pathogenesis, and ecology (Cianciotto, 2005).

Acknowledgments. Studies in the author’s laboratory are supported by NIH grants AI34937 and AI43987.

References Allard, K.A., Viswanathan, V.K., and Cianciotto, N.P. (2005) Genes lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin. J. Bacteriol. submitted for publication. Aragon, V., Kurtz, S., Flieger, A., Neumeister, B., and Cianciotto, N.P. (2000) Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila. Infect. Immun. 68: 1855–1863. Aragon, V., Kurtz, S., and Cianciotto, N.P. (2001) Legionella pneumophila major acid phosphatase and its role in intracellular infection. Infect. Immun. 69: 177–185.

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Aragon, V., Rossier, O., and Cianciotto, N.P. (2002) Legionella pneumophila genes that encode lipase and phospholipase C activities. Microbiol. 148: 2223–2231. Banerji, S., Bewersdorff, M., Hermes, B., Cianciotto, N.P., and Flieger, A. (2005) Characterization of the major secreted zinc metalloprotease-dependent glycerophospholipid:cholesterol acyltransferase, PlaC, of Legionella pneumophila. Infect. Immun. 73: 2899–2909. Brieland, J., McClain, M., Heath, L., Chrisp, C., Huffnagle, G., LeGendre, M., Hurley, M., Fantone, J., and Engleberg, C. (1996) Coinoculation with Hartmannella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires’ disease. Infect. Immun. 64: 2449–2456. Byrd, T.F., and Horwitz, M.A. (2000) Aberrantly low transferrin receptor expression on human monocytes is associated with nonpermissiveness for Legionella pneumophila growth. J. Infect. Dis. 181: 1394–1400. Cazalet, C., Rusniok, C., Bruggemann, H., Zidane, N., Magnier, A., Ma, L., Tichit, M., Jarraud, S., Bouchier, C., Vandenesch, F., Kunst, F., Etienne, J., Glaser, P., and Buchrieser, C. (2004) Evidence in the Legionella pneumophila genome for exploitation of host cell functions and high genome plasticity. Nat. Genet. 36: 1165–1173. Chien, M., Morozova, I., Shi, S., Sheng, H., Chen, J., Gomez, S.M., Asamani, G., Hill, K., Nuara, J., Feder, M., Rineer, J., Greenberg, J.J., Steshenko, V., Park, S.H., Zhao, B., Teplitskaya, E., Edwards, J.R., Pampou, S., Georghiou, A., Chou, I.C., Iannuccilli, W., Ulz, M.E., Kim, D.H., Geringer-Sameth, A., Goldsberry, C., Morozov, P., Fischer, S.G., Segal, G., Qu, X., Rzhetsky, A., Zhang, P., Cayanis, E., De Jong, P.J., Ju, J., Kalachikov, S., Shuman, H.A., and Russo, J.J. (2004) The genomic sequence of the accidental pathogen Legionella pneumophila. Science 305: 1966–1968. Cianciotto, N.P. (2005) Type II secretion: A protein secretion system for all seasons. Trends Microbiol. 13:581–588. Cianciotto, N.P., Cornelis, P., and Baysse, C. (2005) Impact of the bacterial type I cytochrome c maturation system on different biological processes. Mol. Microbiol. 56: 1408–1415. Cirillo, J.D., Cirillo, S.L.G., Yan, L., Bermudez, L.E., Falkow, S., and Tompkins, L.S. (1999) Intracellular growth in Acanthamoeba castellani affects monocyte entry mechansism and enhances virulence of Legionella pneumophila. Infect. Immun. 67: 4427–4434. De Buck, E., Lebeau, I., Maes, L., Geukens, N., Meyen, E., Van Mellaert, L., Anne, J., and Lammertyn, E. (2004) A putative twin-arginine translocation pathway in Legionella pneumophila. Biochem. Biophys. Res. Commun. 317: 654–661. Filloux, A. (2004) The underlying mechanisms of type II protein secretion. Biochim. Biophys. Acta-Mol. Cell. Res. 1694: 163–179. Flieger, A., Gong, S., Faigle, M., Northoff, H., and Neumeister, B. (2001a) In vitro secretion kinetics of proteins from Legionella pneumophila in comparison to proteins from non-pneumophila species. Microbiol. 147: 3127–3134. Flieger, A., Gong, S., Faigle, M., Stevanovic, S., Cianciotto, N.P., and Neumeister, B. (2001b) Novel lysophospholipase A secreted by Legionella pneumophila. J. Bacteriol. 183: 2121–2124. Flieger, A., Neumeister, B., and Cianciotto, N.P. (2002) Characterization of the gene encoding the major secreted lysophospholipase A of Legionella pneumophilaand its role in detoxification of lysophosphatidylcholine. Infect. Immun. 70: 6094–6106. Gebran, S.J., Newton, C., Yamamoto, Y., Widen, R., Klein, T.W., and Friedman, H. (1994) Macrophage permissiveness for Legionella pneumophila growth modulated by iron. Infect. Immun. 62: 564–568.

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Goldoni, P., Visca, P., Pastoris, M.C., Valenti, P., and Orsi, N. (1991) Growth of Legionella spp. under conditions of iron restriction. J. Med. Microbiol. 34: 113–118. Hales, L.M., and Shuman, H.A. (1999) Legionella pneumophila contains a type II general secretion pathway required for growth in amoebae as well as for secretion of the Msp protease. Infect. Immun. 67: 3662–3666. Hickey, E.K., and Cianciotto, N.P. (1994) Cloning and sequencing of the Legionella pneumophila fur gene. Gene 143: 117–121. Hickey, E.K., and Cianciotto, N.P. (1997) An iron- and fur-repressed Legionella pneumophila gene that promotes intracellular infection and encodes a protein with similarity to the Escherichia coli aerobactin synthetases. Infect. Immun. 65: 133–143. James, B.W., Mauchline, W.S., Fitzgeorge, R.B., Dennis, P.J., and Keevil, C.W. (1995) Influence of iron-limited continuous culture on physiology and virulence of Legionella pneumophila. Infect. Immun. 63: 4224–4230. James, B.W., Mauchline, W.S., Dennis, P.J., and Keevil, C.W. (1997) A study of iron acquisition mechanisms of Legionella pneumophila grown in chemostat culture. Curr. Microbiol. 34: 238–243. Johnson, W., Varner, L., and Poch, M. (1991) Acquisition of iron by Legionella pneumophila: role of iron reductase. Infect. Immun. 59: 2376–2381. Lammertyn, E., Van Mellaert, L., Meyen, E., Lebeau, I., De Buck, E., Anne, J., and Geukens, N. (2004) Molecular and functional characterization of type I signal peptidase from Legionella pneumophila. Microbiol. 150: 1475–1483. Liles, M.R., and Cianciotto, N.P. (1996) Absence of siderophore-like activity in Legionella pneumophila supernatants. Infect. Immun. 64: 1873–1875. Liles, M.R., Viswanathan, V.K., and Cianciotto, N.P. (1998) Identification and temperature regulation of Legionella pneumophila genes involved in type IV pilus biogenesis and type II protein secretion. Infect. Immun. 66: 1776–1782. Liles, M.R., Edelstein, P.H., and Cianciotto, N.P. (1999) The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol. Microbiol. 31: 959–970. Liles, M.R., Aber Scheel, T., and Cianciotto, N.P. (2000) Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila. J. Bacteriol. 182: 749–757. Mengaud, J.M., and Horwitz, M.A. (1993) The major iron-containing protein of Legionella pneumophila is an aconitase homologous with the human iron-responsive element-binding protein. J. Bacteriol. 175: 5666–5676. Molmeret, M., Alli, O.A., Zink, S., Flieger, A., Cianciotto, N.P., and Kwaik, Y.A. (2002) icmT is essential for pore formation-mediated egress of Legionella pneumophila from mammalian and protozoan cells. Infect. Immun. 70: 69–78. Naylor, J., and Cianciotto, N.P. (2004) Cytochrome c maturation proteins are critical for in vivo growth of Legionella pneumophila. FEMS Microbiol. Lett. 241: 249–256. O’Connell, W.A., Hickey, E.K., and Cianciotto, N.P. (1996) A Legionella pneumophila gene that promotes hemin binding. Infect. Immun. 64: 842–848. Peabody, C.R., Chung, Y.J., Yen, M.R., Vidal-Ingigliardi, D., Pugsley, A.P., and Saier, M.H., Jr. (2003) Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology 149: 3051–3072. Poch, M.T., and Johnson, W. (1993) Ferric reductases of Legionella pneumophila. Biometals 6: 107–114. Polesky, A.H., Ross, J.T., Falkow, S., and Tompkins, L.S. (2001) Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis. Infect. Immun. 69: 977–987.

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Pope, C.D., O’Connell, W., and Cianciotto, N.P. (1996) Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection. Infect. Immun. 64: 629–636. Reeves, M.W., Pine, L., Hutner, S.H., George, J.R., and Harrell, W.K. (1981) Metal requirements of Legionella pneumophila. J. Clin. Microbiol. 13: 688–695. Reeves, M.W., Pine, L., Neilands, J.B., and Balows, A. (1983) Absence of siderophore activity in Legionella species grown in iron-deficient media. J. Bacteriol. 154: 324–329. Robey, M., and Cianciotto, N.P. (2002) Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection. Infect. Immun. 70: 5659–5669. Rossier, O., and Cianciotto, N.P. (2001) Type II protein secretion is a subset of the PilDdependent processes that facilitate intracellular infection by Legionella pneumophila. Infect. Immun. 69: 2092–2098. Rossier, O., and Cianciotto, N.P. (2005) The Legionella pneumophila tatB gene facilitates secretion of phospholipase C, growth under iron-limiting conditions, and intracellular infection. Infect. Immun. 73: 2020–2032. Rossier, O., Starkenburg, S., and Cianciotto, N.P. (2004) Legionella pneumophila type II protein secretion promotes virulence in the A/J mouse model of Legionnaires’ disease pneumonia. Infect. Immun. 72: 310–321. Söderberg, M.A., Rossier, O., and Cianciotto, N.P. (2004) The type II protein secretion system of Legionella pneumophila promotes growth at low temperatures. J. Bacteriol. 186: 3712–3720. Starkenburg, S.R., Casey, J.M., and Cianciotto, N.P. (2004) Siderophore activity among members of the Legionella genus. Curr. Microbiol. 49: 203–207. Viswanathan, V.K., Edelstein, P.H., Pope, C.D., and Cianciotto, N.P. (2000) The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence. Infect. Immun. 68: 1069–1079. Viswanathan, V.K., Kurtz, S., Pedersen, L.L., Abu-Kwaik, Y., Krcmarik, K., Mody, S., and Cianciotto, N.P. (2002) The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element. Infect. Immun. 70: 1842–1852. Zink, S.D., Pedersen, L., Cianciotto, N.P., and Abu-Kwaik, Y. (2002) The Dot/Icm type IV secretion system of Legionella pneumophila is essential for the induction of apoptosis in human macrophages. Infect. Immun. 70: 1657–1663.

3 The Dot/Icm Type IVB Secretion System Jason J. LeBlanc and Joseph P. Vogel

1. Introduction As a fundamental strategy for virulence, a large number of pathogens possess specialized secretion systems that are used to deliver effector proteins into host cells, thus modulating host cell function. In Gram-negative bacteria, these specialized secretion systems include type III secretion systems (T3SSs) (Hueck, 1998) and type IV secretion systems (T4SSs) (Backert and Meyer, 2006; Christie and Vogel, 2000; Christie et al., 2005; Sexton and Vogel, 2002). T3SS components share similarities to flagellar biosynthetic proteins, whereas T4SSs are believed to be ancestrally related to bacterial conjugation systems (Sexton and Vogel, 2002; Backert and Meyer, 2006). Both T3SSs and T4SSs are multicomponent membrane complexes that can act as molecular syringes to directly inject bacterial proteins into target cells (Sexton and Vogel, 2002; Nagai and Roy, 2003; Backert and Meyer, 2006). T3SSs are present in many pathogens including a variety of Pseudomonas, Salmonella, Shigella, and Yersinia species (Hueck, 1998). T4SSs play equally important roles for the virulence of several pathogens including Brucella, Bordetella, Helicobacter pylori, and Legionella pneumophila (Backert and Meyer, 2006). T4SSs include both classical plasmid transfer systems and adapted conjugation systems used by pathogens (Christie and Vogel, 2000; Sexton and Vogel, 2002). The paradigm T4SS is generally considered to be the plasmid-encoded VirB system of Agrobacterium tumefaciens, which delivers both DNA and protein into plant cells (reviewed in Christie et al., 2005 and Christie and Vogel, 2000). Two additional well-characterized adapted conjugation systems include the Ptldependent secretion of pertussis toxin by Bordetella pertussis and the secretion of CagA by Helicobacter pylori (Backert and Meyer, 2006). T4SS-dependent virulence strategies are also thought to be used by intracellular pathogens such as Brucella spp. and Coxiella burnetii to generate a replication-permissive niche in host cells (Boschroli et al., 2002; Celli and Gorvel, 2004; Saver et al., 2005; Segal et al., 2005; Sexton and Vogel, 2002). The L. pneumophila T4SS, the focus of this chapter, is required for the intracellular survival and replication of this

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bacterium. Though the global architecture of the L. pneumophila T4SS has not yet been fully elucidated, the following sections will describe several key components of the macromolecular membrane complex, as well as several effector proteins that may be involved in the generation of a suitable niche for the intracellular growth of this organism.

2. Dot/Icm Type IV Secretion System At least two different T4SS have been identified in a number of L. pneumophila strains. The first T4SS, a type IVA secretion system, was designated Lvh for “Legionella Vir homolog” due to its similarities to the virB operon of A. tumefaciens. However, this system was shown to be dispensable for intracellular replication of L. pneumophila and is not present in several virulent strains (Christie and Vogel, 2000; Segal et al., 1999). The second T4SS, a type IVB secretion system, was named the Dot/Icm system and shares extensive similarity to the Tra/Trb components of the conjugal transfer apparatus of the IncI plasmids R64 and ColIb-P9 (Berger and Isberg, 1993; Christie and Vogel, 2000; Marra et al., 1992; Segal and Shuman, 1998; Sexton and Vogel, 2002; Vogel et al., 1998). The L. pneumophila type IVB secretion system is encoded by 26 genes designated dot for “defect in organelle trafficking” or icm for “intracellular multiplication” and is found on two loci in the L. pneumophila genome termed region I and II (Sexton and Vogel, 2002). The simultaneous discovery of the dot/icm genes by two laboratories (Berger and Isberg, 1993; Marra et al., 1992) resulted in some components having dual Dot and Icm designations (Table 3.1) (Sexton and Vogel, 2002). Since its discovery, the Dot/Icm T4SS has become the most extensively studied virulence factor associated with L. pneumophila pathogenesis. In contrast to the Lvh T4SS, the Dot/Icm T4SS is absolutely required for the virulence of L. pneumophila. Unlike wild-type L. pneumophila, phagosomes containing dot/icm mutants fail to evade the endocytic pathway and quickly acquire endosomal markers such as the small GTPase Rab5, the vacuolar H⫹-ATPase (V-ATPase), and the lysosomal-associated membrane protein (LAMP)-1 (Coers et al., 1999; Lu and Clarke, 2005; Swanson and Hammer, 2000). L. pneumophila is thought to avoid endocytic degradation by reprogramming host trafficking via the action of Dot/Icmtranslocated effector proteins, thus generating a safe niche termed the “replicative phagosome.” Dot/Icm-dependent alteration of the host endocytic pathway is a rapid event and occurs within minutes of bacterial internalization (Roy et al., 1998; Wiater et al., 1998). Unlike effectors injected in the host by the Salmonella T3SSs (Uchiya et al., 1999), Dot/Icm-dependent secretion does not result in a global effect on phagosome formation in the host cell (Coers et al., 1999; Horwitz and Maxfield, 1984). This result was based partially on the observations that phagosomes harboring Saccharomyces cerevisiae enter the endocytic pathway in a normal

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TABLE 3.1. Characteristics of the Legionella pneumophila Dot/Icm proteins. Proteina DotA

#aab 1048

Sizec 113

IncId TraY

DotB DotC DotD DotE/IcmC

377 303 163 194

42 34 18 20

TraJ TraI TraH TraQ

DotF/IcmG DotG//IcmE

269 1048

30 108

TraP TraO

DotH/IcmK

360

39

TraN

Peri/OM

DotI/IcmL DotJ/IcmM DotK/IcmN DotL/IcmO

212 94 189 783

23 11 21 87

TraM

IM IMh OM IM

DotM/IcmP DotN/IcmJ DotO/IcmB DotP/IcmD DotU DotV IcmF IcmQ IcmR IcmS IcmT IcmV IcmW IcmX

376 208 1009 132 261 180 973 191 120 114 86 151 151 466

43 24 112 14 30 20 111 22 13 13 10 18 17 51

TrbA TraT TraU TraR

TraL TrbC

TraK TraX TraW

Informatione Polytopic membrane protein VirB11-like Lipoprotein Lipoprotein Polytopic membrane protein VirB10-like

Lipoprotein VirD4-like/ Essential gene Essential gene Essential gene

Locationf IM Cyto OM OM IMh IM IM

IM Cyto/IM IM IM Accessory factor IM DotE homology IMh Accessory factor IM Pore forming molecule Cyto Chaperone for IcmQ Cyto Cyto IMh IM Cyto Peri

Functiong

ATPase

IM receptor Energy transducer Possible OM pore

Energy transducer

Chaperone Chaperone Pore? Chaperone Type IV adaptor

Type IV adaptor

a

Proteins are listed with the corresponding Dot or Icm designation. Number of amino acids. c Predicted size in kilo-Daltons. d Name of the IncI plasmid homolog. e Motifs or homology to the A. tumefaciens type IV secretion system. f Localization of the protein as determined experimentally (cyto is cytoplasm, IM is inner membrane, peri is periplasm, and OM is outer membrane). g Predicted function of the protein. h Localization of the protein by a variety of computer algorithms. b

manner in macrophages pre-infected with L. pneumophila (Coers et al., 1999). The lack of a global effect on trafficking by Legionella is likely due to retention of Dot/Icm-effectors on the cytoplasmic face of the Legionella-containing vacuole (LCV) (Bardill et al., 2005; Campodonico et al., 2005; Conover et al., 2003; Luo and Isberg, 2004; Nagai et al., 2002), thus allowing a more stealthy, ninja-like non-disruptive invasion of phagocytic cells.

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2.1. Structural Components The Dot/Icm system is a multiprotein structure that forms a channel spanning the bacterial inner and outer membranes (Coers et al., 1999). Although many of the Dot/Icm components have not been extensively characterized, a number of them have now been examined revealing their localization and putative roles in the formation/function of the secretion apparatus. The first to be examined, DotA, is a polytopic inner membrane protein that may act as a scaffolding protein (Roy and Isberg, 1997). DotB is a cytoplasmic, hexameric protein that associates with the inner membrane and possesses ATPase activity. DotB ATPase activity is crucial for the function of the Dot/Icm complex since mutations in the Walker A box motif (ATP-binding domain) result in a protein that cannot hydrolyze ATP or complement a dotB null mutant (Sexton et al., 2004b, 2005; Vogel et al., 1996, 1998). Current models suggest that DotB is an ATPase that acts as a molecular pump driving export of substrates through the Dot/Icm complex via hydrolysis of ATP while providing substrate specificity to the apparatus (Sexton et al., 2004b). IcmX was shown to be periplasmic protein, although its role in the apparatus remains unclear (Matthews and Roy, 2000). Interestingly, IcmX homologues have not been observed in other T4SSs. Five proteins, DotF/DotG/DotH/ DotC/DotD appear to form the core transmembrane complex for the L. pneumophila T4SS (Vincent et al., 2006). DotF and DotG are inner membrane proteins that interact with a subcomplex consisting of the putative outer membrane pore DotH and two associated lipoproteins DotC and DotD. Finally, a large number of the Dot/Icm proteins appear to be inner membrane proteins, including the type IV coupling protein DotL, and likely constitute the T4SS receptor for cytoplasmic substrates (see Fig. 3.1).

Outer membrane

DotH

Do

tC

tD

Do

tK

Do

Periplasm

DotF

DotA DotE DotI DotJ DotP DotV IcmT IcmV DotG

IcmW

DotL

M

DotU IcmF

Inner membrane

N IcmO IcmQ

Cytoplasm

DotB

IcmR ADP?

ATP? IcmS IcmW

FIGURE 3.1.

Substrates

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Other components of the Dot/Icm system include the proteins DotU and IcmF, which have orthologs in a wide range of Gram-negative bacteria (Sexton et al., 2004a; VanRheenen et al., 2004). DotU and IcmF are thought to stabilize the T4SS to maintain an active form of the apparatus or protect it from degradation, as dotU and icmF mutants displayed decreased stability of the DotH/DotG/DotF subcomplex (Sexton et al., 2004a; VanRheenen et al., 2004). Surprisingly, over-expression of DotH was sufficient to suppress the destabilization of the Dot/Icm apparatus in the absence of DotU and IcmF, restoring normal levels of DotG and DotF, and eliminating the intracellular growth defect in the dotU/icmF mutant. This suggests that DotH is a key component of this subcomplex. Interestingly, DotU was shown to be degraded when L. pneumophila enters stationary phase (VanRheenen et al., 2004), a period where the organism shows increased virulence (Molofsky and Swanson, 2004). Thus, IcmF and DotU may function together to stabilize the T4SS in exponentially growing bacteria ensuring that the translocon becomes active at the correct point in the cell cycle (Sexton et al., 2004b; VanRheenen et al., 2004). Interestingly two Dot/Icm components, DotO and DotH, were observed on the surface of L. pneumophila after interaction with macrophages (Watarai et al., 2001). Since DotO and DotH surface expression correlated with the formation of a fibrous structure on the surface of L. pneumophila, it was proposed that this structure may play a critical role in the function of the Dot/Icm complex (Watarai et al., 2001).

2.2. Adaptor/Chaperone Complexes L. pneumophila icmS and icmW mutants had a unique phenotype compared to other dot/icm mutant strains since the LCV associated with vesicles, but then quickly fused with the endocytic pathway (Coers et al., 2000; Zuckman et al., 1999). IcmS and IcmW were both shown to bind to a number of Dot/Icm-secreted proteins, and to interact with each other, suggesting that they function as an adaptor complex involved in substrate recognition by the Dot/Icm complex (Bardill et al., 2005; Coers et al., 2000; Ninio et al., 2005). By analogy to the T3SS chaperones (Hueck, 1998), type IV adaptor complexes might bind secreted substrates to prevent their aggregation or degradation preceding export. Vincent and Vogel (2006) recently reported that the product of the Legionella virulence gene A (lvgA) was also involved in adaptor functions, and proposed that IcmS exists in two separate complexes (IcmS/IcmW and IcmS/LvgA), presumably selecting for distinct classes of effectors (Vincent and Vogel, 2006). This was consistent with a previous report (Bardill et al. 2005) where SdeA was shown to be secreted by a Dot/Icm-dependent process involving both DotA and IcmS, whereas another Dot/Icm-secreted factor RalF was translocated by a DotA-dependent, but IcmSindependent, mechanism (Bardill et al., 2005). Interestingly, lvgA mutants were only impaired for growth in A/J mouse macrophages and not in other macrophages or a protozoan host (Vincent and Vogel, 2006). Finally, IcmR was also shown to be a chaperone for IcmQ (Duménil and Isberg, 2001). Phagosomes harboring icmR mutants display another unique characteristic in that they do not

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recruit vesicles but are able to evade endocytic maturation (Coers et al., 2000). The different phenotypes observed between icmS or icmW and icmR mutants suggest that evasion of the endocytic pathway and formation of the LCV likely involve multiple Dot/Icm-secreted proteins whose functions are required at different stages of the infection process (Coers et al., 2000).

2.3. Regulation of Effector Export Before releasing effectors through the Dot/Icm secretion apparatus, L. pneumophila must create a pore in the host plasma membrane. Based on its ability to form pores in membranes, the IcmQ protein may be responsible for this activity (Duménil et al., 2004). Consistent with this notion, IcmQ appeared to be localized on the bacterial surface shortly after contact with host cells (Duménil et al., 2004). IcmQ-mediated pore formation appears to be inhibited via an interaction with the chaperone-like molecule, IcmR, thus providing a molecular mechanism of regulation (Duménil et al., 2004). A similar contact-dependent pore-forming activity has been observed in Shigella flexneri as a mechanism to control the release of effectors from its T3SS (Hueck, 1998). A second mechanism of regulation may be provided by the activity of DotL, the Dot/Icm type IV coupling protein (Buscher et al., 2005). The dotL gene has been found to be essential for viability in the Lp02 strain of L. pneumophila, but not JR32 (Buscher et al., 2005). ⌬dotL lethality can be suppressed by inactivation of other dot/icm genes, suggesting that the absence of DotL may result in the formation of toxic membrane complex(es). Consistent with this hypothesis, the JR32 strain of L. pneumophila tolerates a dotL mutation, but displays defects in ion homeostasis resulting in hypersensitivity to salt. Therefore, DotL appears to prevent the unregulated flow of solute (and presumably effectors) through the complex, consistent with a “gatekeeping” mechanism (Buscher et al., 2005). Gatekeeper functions have also been reported for the IpaB and IpaD effectors of S. flexneri, where ipaB and ipaD mutants resulted in unregulated secretion of substrates from the T3SS (Hueck, 1998). DotL, in conjunction with the type IV adaptors, likely provides the major control of secretion by the L. pneumophila T4SS.

3. Genetic Regulation of the Dot/Icm System Temporal regulation of both the Dot/Icm apparatus and its substrates has been reported. Transcriptional control of the Dot/Icm T4SS has been proposed to be mediated in part by the ppGpp synthase RelA (icmP), the stationary phase sigma factor RpoS (icmP and icmR), LetA/S (icmP, icmR, icmT, dotA), and CpxA/CpxR two-component systems (icmR, icmV, and icmW) (Gal-Mor et al., 2002; Gal-Mor and Segal, 2003a, b; Shi et al., 2006; Zusman et al., 2002). However, none of these regulatory factors are required for intracellular growth in macrophages. Moreover, expression of most components of the Dot/Icm system appears to be constitutive when assayed by western analysis (Bardill et al., 2005; Vincent and Vogel, 2006).

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For example, DotL, IcmS, and IcmW are clearly constitutively expressed in all phases of growth (Bardill et al., 2005; Vincent and Vogel, 2006). In contrast, a number of Dot/Icm substrates including SdeC, SidC, LidA, and RalF were shown to be highly expressed during the transmissive phase of growth (Bardill et al., 2005; Luo and Isberg, 2004; Nagai et al., 2002). Furthermore, using microarray and real-time quantitative PCR technologies, Brüggemann et al. (2006) demonstrated that dotA was expressed at all times during the biphasic life cycle of L. pneumophila in Acanthamoeba castellanii, whereas effector molecules (ralF, sdbB, sidC, sdcA, sidG, sdeA, sdeB, sdeC, sdhB, vipD, vipE, lidA, and sidE) were up-regulated during the transmissive phase of growth. However, it should be noted that other putative effector molecules (sdeD, sdbA, sdbC, lepA, and lepB) showed no expression differences during the intracellular life cycle.

4. Possible Effector Proteins and Associated Functions The L. pneumophila Dot/Icm T4SS was originally shown to be competent for secretion by its ability to mediate conjugal transfer of the plasmid RSF1010 (Segal and Shuman, 1998; Vogel et al., 1998). Interestingly, the presence of this plasmid substantially inhibited intracellular replication of L. pneumophila (Segal and Shuman, 1998) and thus might compete with native L. pneumophila substrates for interaction with the Dot/Icm system. Using a variety of molecular methods, over 50 proteins have been identified as putative substrates of the L. pneumophila Dot/Icm system. The best characterized are RalF, SidA-H, LidA, LepA-B, YlfA, and the Vip proteins.

4.1. RalF RalF, the recruitment of ARF to Legionella-phagosome factor, was the first identified native Dot/Icm substrate (Nagai et al., 2002). RalF contains a Sec7 domain, which functions as a guanine nucleotide exchange factor (GEF) for the Arf family of GTPases (Amor et al., 2005; Nagai et al., 2002). Dot/Icm-translocated RalF recruits and activates the host GTPase ADP-ribosylation factor (Arf)-1 to the LCV (Nagai et al., 2002). Since Arf1 is a well-characterized regulator of vesicular traffic, it was suggested that RalF might aid in the formation of the replicative phagosome. This was supported by evidence indicating that interference of Arf1 activity abrogated LCV biogenesis (Kagan and Roy, 2002; Derré and Isberg, 2004; Kagan et al., 2004). Moreover, over-expression of RalF in a yeast model system caused drastic growth defects, whereas a yeast strain expressing a RalF mutant (E103A) lacking Arf-GEF activity was able to grow at a rate similar to that of wild-type yeast cells (Campodonico et al., 2005). As a result, it was surprising when it was determined that ralF mutants were fully capable of evading the endocytic pathway, forming a wild-type replicative phagosome, and replicating to normal levels in both protozoa and macrophages (Nagai et al., 2002). This phenomenon, where Dot/Icm substrates are not essential for intracellular survival

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and replication, is not restricted to RalF but appears to be applicable to most, if not all, substrates. This is likely due to the high level of redundancy achieved by L. pneumophila via the export of many substrates.

4.2. The Sid Family The Sid “substrate of Icm/Dot” family of proteins were identified via a combination of a two-hybrid screen and a bacterial protein translocation assay (Luo and Isberg, 2004). In the original report one Sid protein, SidC, was localized to the cytoplasmic face of the LCV within macrophages (Luo and Isberg, 2004). Weber et al. (2006) recently reported that SidC and its paralog SdcA were able to specifically bind to phosphatidylinositol(4) phosphate [PI(4)P] found on the LCV, thus providing an explanation for why the proteins may be retained on the LCV. SidC and SdcA possess coiled-coil motifs that may act similarly to synaptosomalassociated protein receptors (SNAREs) to mediate protein–protein interactions involved during uptake and LCV biogenesis (Weber et al., 2006). Experimental confirmation that the Sid proteins and their paralogs are translocated into hosts remains to be done. One family of Sid proteins, the SidE family (SdeA, SdeB, SdeC), was independently identified via their interaction with the type IV adaptor protein IcmS (Bardill et al., 2005). SdeA was shown to be secreted into host cells via two independent assays, adenylate cyclase fusions and immunofluorescence colocalization, and export was dependent on a functional Dot/Icm complex and the adaptor protein IcmS (Bardill et al., 2005). Though the SidE family was shown to be translocated into host cells in a Dot/Icm-dependent manner, a quadruple mutant lacking sidE and its paralogs showed wild-type growth kinetics in macrophages and only a modest intracellular growth defect in A. castellanii (Bardill et al., 2005).

4.3. LidA and SidM The lidA gene was identified by Conover et al. (2003) as an insertional mutant that exhibited decreased viability in the presence of a functional Dot/Icm complex, i.e., “lowered viability in the presence of dot.” LidA may interact with DotL and modify its gatekeeper functions. After contact with host cells, LidA is translocated across into the host cell and localizes on the cytoplasmic face of the LCV similar to SidC. Due to the slower kinetics between RalF and LidA, it was suggested that once secretion of LidA was engaged, other substrates could also to be secreted (Conover et al., 2003). Curiously, LidA was maintained on the LCV throughout the infection process and possessed a coiled-coil domain, suggesting that it might interact with host proteins involved in LCV biogenesis (Derré and Isberg, 2005; Kagan and Roy, 2002). Over-expression of LidA resulted in the redistribution of the endoplasmic reticulum–Golgi intermediate compartment (ERGIC) and the disappearance of the Golgi compartment (Derré and Isberg, 2005). However, the disruptions seen with over-expression are likely not to be

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physiologically relevant and may be viewed as an over-exaggeration of LidA function since the ERGIC and the Golgi are normally intact during L. pneumophila replication. More recently, co-localization studies of Rab1 in infections using L. pneumophila sidM and lidA mutants suggested that LidA cooperates in the SidM-mediated recruitment of Rab1 (Machner and Isberg, 2006). Machner and Isberg (2006) demonstrated that the two translocated substrates, SidM and LidA, synergistically recruit the small GTPase Rab1 to the LCV in order to manipulate ER-to-Golgi traffic, thus recruiting early secretory vesicles necessary for LCV biogenesis. SidM possesses GEF activity that can stimulate Rab1 and exhibits higher activity than that observed for Arf1-stimulating GEF RalF (Machner and Isberg, 2006). Since Rab1 was shown to be crucial for LCV biogenesis (Derré and Isberg, 2004), the lack of an intracellular growth defect in macrophages infected with sidM mutants was surprising (Machner and Isberg, 2006). However, formation of mature replicative vacuoles was reduced with lidA mutants (Conover et al., 2003), perhaps due to LidA’s interaction with other small GTPases including Rab6 and Rab8 (Machner and Isberg, 2006).

4.4. LepA and LepB LepA and LepB were identified by sequence analysis the L. pneumophila genome for likely T4SS substrates and their export was confirmed to be Dot/Icm dependent (Chen et al., 2004). Typical of other Dot/Icm-substrates, inactivation of lepA, lepB, or both genes did not significantly diminish the intracellular replication of L. pneumophila within macrophages, although a lower survival rate was observed in a protozoan host (Chen et al., 2004). This phenotype appeared to be due to the non-lytic release of vesicles containing L. pneumophila from the amoeba. The release of L. pneumophila in vesicles has been described elsewhere (Berk et al., 1998; Rowbotham, 1986); however, factors involved in the process had not been described. Although the exact mechanism is unknown, host-specific effectors like LepA and LepB might play a valuable role in the environmental dissemination of L. pneumophila during the replicative cycle within its natural protozoan host.

4.5. Ylf and the Vips Using a Saccharomyces cerevisiae model, Campodonico et al. (2005) demonstrated that over-production of L. pneumophila yeast lethal factor (YlfA) resulted in vesicular trafficking defects, an effect attributed to its N-terminal coiled-coil regions. By sequence analysis, a paralog to YflA, YflB, was identified. Both YlfA and YlfB were shown to be secreted into host cells by a Dot/Icm-dependent process, although ylfA ylfB double mutants showed no intracellular growth defects in A. castellanii or murine macrophages. Unlike other Dot/Icm-secreted proteins (RalF, LidA, SdeC, and SidC) that co-localize on the LCV within the first 30 minutes of infection (Bardill et al., 2005, Conover et al., 2003; Luo and Isberg, 2004; Nagai et al., 2002), YlfA localizes to the LCV late in the infection process (Campodonico et al., 2005). It was suggested that YlfA may perform a

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more distant role in the cytoplasm after translocation rather than the direct modulation of the LCV itself (Campodonico et al., 2005). Also using a yeast model, Shohdy et al. (2005) identified L. pneumophila proteins that produced defects in the vacuole protein sorting (VPS) pathway in S. cerevisiae. Three vacuole protein sorting inhibitor proteins (Vips) that were able to alter vesicular trafficking were identified (Shohdy et al., 2005). The Vips shared no common structure and the vesicular trafficking defects differed, but they were all translocated via the Dot/Icm system (Shohdy et al., 2005). VipF had an acetyltransferase domain of unknown function and VipA contained a coiledcoil domain that could be involved in SNARE-mediated interactions (Shohdy et al., 2005). VipD was investigated in more detail since it had previously been identified as a factor that affected growth in phagocytic cells (VanRheenen et al., 2006). VipD contains a phospholipase domain homologous to ExoU, a T3SS effector in Pseudomonas aeruginosa that exhibits lipolytic activity (Rabin et al., 2006). Though VipD could be translocated into macrophages, no phospholipase activity could be detected (VanRheenen et al., 2006). Moreover, Shohdy et al. (2005) had previously shown that a fragment of VipD lacking the phospholipase domain interfered with the secretory pathway to a greater extent than the fulllength protein (Shohdy et al., 2005), that the C-terminal fragment of ExoU was sufficient to target to the host plasma membrane (Rabin et al., 2006), and therefore VipD may affect vesicular trafficking via its C-terminal coiled-coil domain instead of via its enzymatic domain. In summary, L. pneumophila exports a large number of substrates via the Dot/Icm type IV secretion system. These multiple effectors may act in concert to provide a vast repertoire of activities that perturbs or modifies host cell functions, thus allowing the LCV to avoid the endocytic pathway and mature into a replicate phagosome. Although the Dot/Icm system plays a key role in the modulation of host cell functions, mutants lacking single effector proteins do not have strong intracellular growth defects (Luo and Isberg, 2004; Ninio et al., 2005; Campodinico et al., 2005). This is likely due to functional redundancy and has necessitated the development of alterative approaches to determine their specific molecular function.

5. Temporal Modulation of the LCV Surface The identification of host pathways involved in LCV biogenesis may reveal which Dot/Icm substrates are crucial for intracellular growth. Based on this assumption, Dorer et al. (2006) used RNA interference in Drosophila Kc167 cells to inhibit multiple host trafficking pathways that were important for intracellular growth of L. pneumophila. This approach identified Cdc48/p97, a component of endoplasmic reticulum-associated degradation (ERAD), as playing an important role in the intracellular growth of L. pneumophila. Cdc48/p97 is an AAA-ATPase that forms a chaperone complex required for ERAD functions, where misfolded proteins from the ER are ubiquitinated, removed by

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Cdc48/p97, and delivered to the proteasome for degradation (Bar-Nun, 2005). Cdc48/p97 and poly-ubiquitinated proteins were found to localize to the LCV in a Dot/Icm-dependent process in both macrophages and Drosophila cells (Dorer et al., 2006). Specifically, the Cdc48/p97 chaperone complex appeared to be required for the timely removal of the Dot/Icm substrates LidA and SidC from the LCV (Dorer et al., 2006). Consistent with its involvement, inhibition of proteasome function led to decreased intracellular replication of L. pneumophila (Dorer et al., 2006). Thus, L. pneumophila seems to exploit the ubiquitination system to temporally modulate the surface composition of the LCV and promote a vacuole suitable for replication.

6. Conclusion The Dot/Icm secretion system plays a crucial role in the generation of a replication-permissive niche for L. pneumophila in a wide range of protozoan and mammalian hosts. Although a limited amount of work has been done to determine how this T4SS assembles and functions, much labor remains. Recent estimates indicate that the Dot/Icm system may export between 50 and 150 proteins making it one of the most robust bacterial secretion systems ever identified. Although the apparent functional redundancy of these substrates makes elucidation of their molecular function more challenging, it also provides fertile ground for future discoveries that will likely provide key information about host cell processes and pathogenesis.

References Amor, J. C., J. Swails, X. Zhu, C. R. Roy, H. Nagai, A. Ingmundson, X. Cheng, and R. A. Kahn. 2005. The structure of RalF, an ADP-ribosylation factor guanine nucleotide exchange factor from Legionella pneumophila, reveals the presence of a cap over the active site. J Biol Chem 280:1392–400. Backert, S. and T. F. Meyer. 2006. Type IV secretion systems and their effectors in bacterial pathogenesis. Curr Opin Microbiol 9:207–17. Bar-Nun, S. 2005. The role of p97/Cdc48p in endoplasmic reticulum-associated degradation: from the immune system to yeast. Curr Top Microbiol Immunol 300:95–125. Bardill, J. P., J. L. Miller, and J. P. Vogel. 2005. IcmS-dependent translocation of SdeA into macrophages by the Legionella pneumophila type IV secretion system. Mol Microbiol 56:90–103. Berger, K. H. and R. R. Isberg. 1993. Two distinct defects in intracellular growth complemented by a single genetic locus in Legionella pneumophila. Mol Microbiol 7:7–19. Berk, S. G., R. S. Ting, G. W. Turner, and R. J. Ashburn. 1998. Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ Microbiol 64:279–86. Boschiroli, M. L., Ouahrani-Bettache, S., Foulongne, V., Michaux-Charachon, S., Bourg, G., Allardet-Servent, A., Cazevieille, C., Lavigne, J. P., Liautard, J. P., Ramuz, M., and D. O’Callaghan. 2002. Type IV secretion and Brucella virulence. Vet Microbiol 90:341–8.

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Bruggemann, H., A. Hagman, M. Jules, O. Sismeiro, M. A. Dillies, C. Gouyette, F. Kunst, M. Steinert, K. Heuner, J. Y. Coppee, and C. Buchrieser. 2006. Virulence strategies for infecting phagocytes deduced from the in vivo transcriptional program of Legionella pneumophila. Cell Microbiol 8:1228–40. Buscher, B. A., G. M. Conover, J. L. Miller, S. A. Vogel, S. N. Meyers, R. R. Isberg, and J. P. Vogel. 2005. The DotL protein, a member of the TraG-coupling protein family, is essential for viability of Legionella pneumophila strain Lp02. J Bacteriol 187:2927–38. Campodonico, E. M., L. Chesnel, and C. R. Roy. 2005. A yeast genetic system for the identification and characterization of substrate proteins transferred into host cells by the Legionella pneumophila Dot/Icm system. Mol Microbiol 56:918–33. Celli, J. and J. P. Gorvel. 2004. Organelle robbery: Brucella interactions with the endoplasmic reticulum. Curr Opin Microbiol 7:93–7. Chen, J., K. S. de Felipe, M. Clarke, H. Lu, O. R. Anderson, G. Segal, and H. A. Shuman. 2004. Legionella effectors that promote nonlytic release from protozoa. Science 303:1358–61. Christie, P. J. and J. P. Vogel. 2000. Bacterial type IV secretion: conjugation systems adapted to deliver effector molecules to host cells. Trends Microbiol 8:354–60. Christie, P. J., K. Atmakuri, V. Krishnamoorthy, S. Jakubowski, and E. Cascales. 2005. Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol 59:451–85. Coers, J., C. Monahan, and C. R. Roy. 1999. Modulation of phagosome biogenesis by Legionella pneumophila creates an organelle permissive for intracellular growth. Nat Cell Biol 1:451–3. Coers, J., J. C. Kagan, M. Matthews, H. Nagai, D. M. Zuckman, and C. R. Roy. 2000. Identification of Icm protein complexes that play distinct roles in the biogenesis of an organelle permissive for Legionella pneumophila intracellular growth. Mol Microbiol 38:719–36. Conover, G. M., I. Derré, J. P. Vogel, and R. R. Isberg. 2003. The Legionella pneumophila LidA protein: a translocated substrate of the Dot/Icm system associated with maintenance of bacterial integrity. Mol Microbiol 48:305–21. Derré, I. and R. R. Isberg. 2004. Legionella pneumophila replication vacuole formation involves rapid recruitment of proteins of the early secretory system. Infect Immun 72:3048–53. Derré, I. and R. R. Isberg. 2005. LidA, a translocated substrate of the Legionella pneumophila type IV secretion system, interferes with the early secretory pathway. Infect Immun 73:4370–80. Dorer, M. S., D. Kirton, J. S. Bader, and R. R. Isberg. 2006. RNA interference analysis of Legionella in Drosophila cells: exploitation of early secretory apparatus dynamics. PLoS Pathog 2:e34. Duménil, G. and R. R. Isberg. 2001. The Legionella pneumophila IcmR protein exhibits chaperone activity for IcmQ by preventing its participation in high-molecular-weight complexes. Mol Microbiol 40:1113–27. Duménil, G., T. P. Montminy, M. Tang, and R. R. Isberg. 2004. IcmR-regulated membrane insertion and efflux by the Legionella pneumophila IcmQ protein. J Biol Chem 279:4686–95. Gal-Mor, O. and G. Segal. 2003a. Identification of CpxR as a positive regulator of icm and dot virulence genes of Legionella pneumophila. J Bacteriol 185:4908–19. Gal-Mor, O. and G. Segal. 2003b. The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii. Microb Pathog 34:187–94.

3. The Dot/Icm Type IVB Secretion System

61

Gal-Mor, O., T. Zusman, and G. Segal. 2002. Analysis of DNA regulatory elements required for expression of the Legionella pneumophila icm and dot virulence genes. J Bacteriol 184:3823–33. Horwitz, M. A. and F. R. Maxfield. 1984. Legionella pneumophila inhibits acidification of its phagosome in human monocytes. J Cell Biol 99:1936–43. Hueck, C. J. 1998. Type III protein secretion systems in bacterial pathogens of animals and plants. Microbiol Mol Biol Rev 62:379–433. Kagan, J. C. and C. R. Roy. 2002. Legionella phagosomes intercept vesicular traffic from endoplasmic reticulum exit sites. Nat Cell Biol 4:945–54. Kagan, J. C., M. P. Stein, M. Pypaert, and C. R. Roy. 2004. Legionella subvert the functions of Rab1 and Sec22b to create a replicative organelle. J Exp Med 199:1201–11. Kirby, J. E., J. P. Vogel, H. L. Andrews, and R. R. Isberg. 1998. Evidence for pore-forming ability by Legionella pneumophila. Mol Microbiol 27:323–36. Lu, H. and M. Clarke. 2005. Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7:995–1007. Luo, Z. Q. and R. R. Isberg. 2004. Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer. Proc Natl Acad Sci USA 101:841–6. Machner, M. P. and R. R. Isberg. 2006. Targeting of host rab GTPase function by the intravacuolar pathogen Legionella pneumophila. Dev Cell 11:47–56. Marra, A., S. J. Blander, M. A. Horwitz, and H. A. Shuman. 1992. Identification of a Legionella pneumophila locus required for intracellular multiplication in human macrophages. Proc Natl Acad Sci USA 89:9607–11. Matthews, M. and C. R. Roy. (2000). Identification and subcellular localization of the Legionella pneumophila IcmX Protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells. Infect Immun 68:3971–3982. Molofsky, A. B. and M. S. Swanson. (2004). Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol. Microbiol. 53:29–40. Nagai, H. and C. R. Roy. 2003. Show me the substrates: modulation of host cell function by type IV secretion systems. Cell Microbiol 5:373–83. Nagai, H., J. C. Kagan, X. Zhu, R. A. Kahn, and C. R. Roy. 2002. A bacterial guanine nucleotide exchange factor activates ARF on Legionella phagosomes. Science 295:679–82. Ninio, S., D. M. Zuckman-Cholon, E. D. Cambronne, and C. R. Roy. 2005. The Legionella IcmS-IcmW protein complex is important for Dot/Icm-mediated protein translocation. Mol Microbiol 55:912–26. Rabin, S. D., J. L. Veesenmeyer, K. T. Bieging, and A. R. Hauser. 2006. A C-terminal domain targets the Pseudomonas aeruginosa cytotoxin ExoU to the plasma membrane of host cells. Infect Immun 74:2552–61. Rowbotham, T. J. 1986. Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci 22:678–89. Roy, C. R. and R. R. Isberg. 1997. Topology of Legionella pneumophila DotA: an inner membrane protein required for replication in macrophages. Infect Immun 65:571–8. Roy, C. R., K. H. Berger, and R. R. Isberg. 1998. Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol Microbiol 28:663–74. Sauer, J. D., J. G. Shannon, D. Howe, S. F. Hayes, M. S. Swanson, and R. A. Heinzen. 2005. Specificity of Legionella pneumophila and Coxiella burnetii vacuoles and versatility of Legionella pneumophila revealed by coinfection. Infect Immun 73:4494–504.

62

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Segal, G. and H. A. Shuman. 1998. Intracellular multiplication and human macrophage killing by Legionella pneumophila are inhibited by conjugal components of IncQ plasmid RSF1010. Mol Microbiol 30:197–208. Segal, G., J. J. Russo, and H. A. Shuman. 1999. Relationships between a new type IV secretion system and the icm/dot virulence system of Legionella pneumophila. Mol Microbiol 34:799–809. Segal, G., M. Feldman, and T. Zusman. 2005. The Icm/Dot type-IV secretion systems of Legionella pneumophila and Coxiella burnetii. FEMS Microbiol Rev 29:65–81. Sexton, J. A. and J. P. Vogel. 2002. Type IVB secretion by intracellular pathogens. Traffic 3:178–85. Sexton, J. A., J. L. Miller, A. Yoneda, T. E. Kehl-Fie, and J. P. Vogel. 2004a. Legionella pneumophila DotU and IcmF are required for stability of the Dot/Icm complex. Infect Immun 72:5983–92. Sexton, J. A., J. S. Pinkner, R. Roth, J. E. Heuser, S. J. Hultgren, and J. P. Vogel. 2004b. The Legionella pneumophila PilT homologue DotB exhibits ATPase activity that is critical for intracellular growth. J Bacteriol 186:1658–66. Sexton, J. A., H. J. Yeo, and J. P. Vogel. 2005. Genetic analysis of the Legionella pneumophila DotB ATPase reveals a role in type IV secretion system protein export. Mol Microbiol 57:70–84. Shi, C., V. Forsbach-Birk, R. Marre, and T. L. McNealy. 2006. The Legionella pneumophila global regulatory protein LetA affects DotA and Mip. Int J Med Microbiol 296:15–24. Shohdy, N., J. A. Efe, S. D. Emr, and H. A. Shuman. 2005. Pathogen effector protein screening in yeast identifies Legionella factors that interfere with membrane trafficking. Proc Natl Acad Sci USA 102:4866–71. Swanson MS and B.K. Hammer. 2000 Legionella Pneumophila pathogenesis: a fateful journey from amoebae to macrophages. Annu Rev Microbiol 54:567–613. Uchiya, K., M. A. Barbieri, K. Funato, A. H. Shah, P. D. Stahl, and E. A. Groisman. 1999. A Salmonella virulence protein that inhibits cellular trafficking. EMBO J 18:3924–33. VanRheenen, S. M., G. Duménil, and R. R. Isberg. 2004. IcmF and DotU are required for optimal effector translocation and trafficking of the Legionella pneumophila vacuole. Infect Immun 72:5972–82. VanRheenen, S. M., Z. Q. Luo, T. O’Connor, and R. R. Isberg. 2006. Members of a Legionella pneumophila family of proteins with ExoU (phospholipase A) active sites are translocated to target cells. Infect Immun 74:3597–606. Vincent, C. D. and J. P. Vogel. 2006. The Legionella pneumophila IcmS-LvgA protein complex is important for Dot/Icm-dependent intracellular growth. Mol Microbiol 61:596–613. Vincent, C. D., Friedman, J. R., Jeong, K. C., Buford, E. C., Miller, J. L., and J. P. Vogel. 2006. Identification of the core transmembrane complex of the Legionella Dot/Icm type IV secretion system. Mol Microbiol [Epub ahead of print]. Vogel, J. P., C. Roy, and R. R. Isberg. 1996. Use of salt to isolate Legionella pneumophila mutants unable to replicate in macrophages. Ann NY Acad Sci 797:271–2. Vogel, J. P., H. L. Andrews, S. K. Wong, and R. R. Isberg. 1998. Conjugative transfer by the virulence system of Legionella pneumophila. Science 279:873–6. Watarai, M., H. L. Andrews, and R. R. Isberg. 2001. Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth. Mol Microbiol 39:313–29.

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Weber, S. S., C. Ragaz, K. Reus, Y. Nyfeler, and H. Hilbi. 2006. Legionella pneumophila exploits PI(4)P to anchor secreted effector proteins to the replicative vacuole. PLoS Pathog 2:e46. Wiater, L. A., K. Dunn, F. R. Maxfield, and H. A. Shuman. 1998. Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila. Infect Immun 66:4450–60. Zuckman, D. M., J. B. Hung, and C. R. Roy. 1999. Pore-forming activity is not sufficient for Legionella pneumophila phagosome trafficking and intracellular growth. Mol Microbiol 32:990–1001. Zusman, T., O. Gal-Mor, and G. Segal. 2002. Characterization of a Legionella pneumophila relA insertion mutant and toles of RelA and RpoS in virulence gene expression. J Bacteriol 184:67–75.

4 Life Cycle, Growth Cycles and Developmental Cycle of Legionella pneumophila Rafael A. Garduño

1. Introduction The Gram-negative bacterium L. pneumophila is an inhabitant of freshwater. Although free legionellae may be readily detected in freshwater, L. pneumophila is not a free-living organism but an intracellular parasite of amoebae (Fields 1996) that recently became an opportunistic human pathogen. Only accidentally, when water aerosol–containing L. pneumophila is inhaled or contaminated water is aspirated, does L. pneumophila enter the human lung to infect alveolar macrophages. In susceptible individuals (mainly immuno-compromised patients or the elderly), this initial infection may lead to an atypical pneumonia known as Legionnaires’ disease (Winn 1988, Weeratna et al. 1993). Legionnaires’ disease is always transmitted from the environment to humans, and thus constitutes an environmental disease that cannot be transmitted from person to person. A careful evaluation of the ecology of L. pneumophila would unequivocally lead to the conclusion that the accidental infection of humans is not advantageous to L. pneumophila, but rather a “lateral shuffle” (Spriggs 1987) leading to a dead end. Infection of the human host is a diversion from the natural life cycle of L. pneumophila in amoebae. In other words, it would be very difficult to argue that humans have played a defining role in the evolution of L. pneumophila. Despite years of accumulated clinical experience and intense epidemiological research, a satisfactory explanation for the lack of person-to-person transmission of Legionnaires’ disease does not exist. However, some recent studies that will be discussed below are beginning to provide explanations. Central to these studies has been the understanding of the life cycle and the developmental cycle of L. pneumophila. Given the recent advances in our understanding of L. pneumophila’s developmental cycle, I believe that the systematic study of the ecology and natural history of L. pneumophila will shed new light on the pathogenesis of L. pneumophila and the mechanism(s) by which Legionnaires’ disease is transmitted. The purpose of this chapter is dual. It intends to integrate and summarize what we know about the natural history of L. pneumophila. An ever-increasing body of literature continues to describe both the dramatic changes that L. pneumophila 65

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experiences as it grows in different environments, as well as its complex interactions with host cells. Regrettably, the terminology used in the literature to describe these changes and complex interactions is not consistent, and clarification of some concepts has become necessary. Therefore, this chapter also intends to clarify the concepts and definitions associated with the life cycle, the many growth cycles, and the developmental cycle of L. pneumophila.

2. The Life Cycle of L. pneumophila as Currently Understood Life cycles tell us a great deal about the natural history of a living organism. They are a synthesis of years of evolution, adaptation, specialization, and survival in nature. From an infectious disease standpoint, life cycles may provide clues to understand the mechanisms of pathogenesis of a particular parasite. The origin of pathogens and the evolution of virulence lie within the dominion of microbial ecology, and the life cycle of L. pneumophila surely is no exception. Amoebae are the primary natural hosts of L. pneumophila and constitute the central axis around which the life cycle of L. pneumophila revolves (Fig. 4.1). Rowbotham (1980) first recognized L. pneumophila to be an intracellular parasite of amoebae, shortly after the isolation and identification of the “Legionnaires’ disease bacterium” by McDade et al. (1977). Currently, ~40 species of Legionella have been identified (Benson and Fields 1998), all of which are able to parasitize amoebae. In addition, a large number of Legionella-like amoebal pathogens (Adeleke et al. 1996) that cannot be cultured in vitro demonstrate that Legionella has developed unique obligate intracellular relationships with specific amoebae. Finally, Legionella-related endosymbionts (known as X-bacteria) have reached a very specific mutual dependence with Amoeba proteus (Ahn et al. 1994, Jeon 2004). Collectively, these observations suggest that the genus Legionella is ancient, and very well adapted to amoebal hosts. Most likely, members of the genus Legionella (L. pneumophila included) first “learned” how to become intracellular parasites through frequent encounters with amoebae (Molmeret et al. 2005), and have since evolved to establish a wide spectrum of relationships with these hosts, from facultative intracellular and pathogenic to endosymbiotic. The amoebae–L. pneumophila relationship seems to occupy a place on the pathogenic side of the spectrum, as L. pneumophila often kills its host cells. The life cycle of L. pneumophila, first described by Rowbotham (1986) at the light microscope level, starts with the invasion of an amoebal host, and proceeds through a series of intracellular events that lead to the active replication of L. pneumophila and then the release of the bacterial progeny that infect new hosts (Fig. 4.1). As expected, the cycle begins and ends at the same point (i.e., infection of a new host). The following is a brief description of the specific events that constitute the life cycle of L. pneumophila and its progression through the amoebal host.

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FIGURE 4.1. The life cycle of L. pneumophila. The cycle begins with the attachment to and the invasion of an amoebal host. Post-internalization events include inhibition of phagosome–lysosome fusion and alteration of organelle traffic. L. pneumophila replicates in a ribosome-decorated vacuole that associates with the endoplasmic reticulum (ER) and acquires ER markers. After replication is finished the progeny exits the wasted amoeba to reinitiate the cycle. The life cycle of L. pneumophila is associated with bacterial differentiation. The two main morphological forms are the mature intracellular form, or MIF, and the replicative form, or RF, which differentiate into each other via intermediates. MIFs play a central role in the infection of amoebae and are the potential agents that spread Legionnaires’ disease to humans, either free or packaged into vesicles. MIF-ladden vesicles may be directly released by amoebae or indirectly by ciliates via an additional packaging step.

2.1. Attachment and Invasion Attachment of L. pneumophila to and the subsequent invasion of amoebae seem to involve several molecular players. A 170-kD galactose/N-acetylgalactosamine–inhibitable amoebal lectin (Gal/GalNAc lectin) seems to serve as a receptor for L. pneumophila, and be necessary for the invasion of Hartmanella vermiformis (Venkataraman et al. 1997). This receptor is de-phosphorylated upon the interaction of L. pneumophila with H. vermiformis, a process that requires contact with viable bacteria, suggesting that L. pneumophila plays an active role in the invasion process. Because invasion often depends on effective attachment, it is possible for the Gal/GalNAc lectin to serve as a primary binding site required to bring legionellae close to a secondary receptor that mediates invasion. However, the bacterial ligand for the Gal/GalNAc lectin has not been as yet identified. Although one may assume amoebae would actively seek bacteria to phagocytose them as a source of food, it has been determined that the association of

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legionellae to H. vermiformis is very poor, and that virulent L. pneumophila may actually mediate its own internalization (Fields et al. 1993). In support of the multifactorial nature of the attachment and invasion processes, several legionellae factors have been shown to be involved: RtxA (Cirillo et al. 2002), a type IV pilus known as CAP (for competence- and adherence-associated pili) (Stone and Abu Kwaik 1998), and a flagella-related factor (Bosshardt et al. 1997). The receptor for these legionellae ligands, and/or the mechanism by which they act, has not yet been determined. Similarly, it is still not clear what molecular mechanism amoebae deploy to internalize L. pneumophila (or what mechanism does L. pneumophila activate to mediate its internalization by amoebae) since inhibition of actin polymerization by cytochalasin D or disruption of microtubules by colchicine do not inhibit uptake of L. pneumophila (Harb et al. 1998, King et al. 1991). However, cytoskeletal proteins like paxillin and vinculin homologs are selectively dephosphorylated upon interaction of H. vermiformis with L. pneumophila (Venkataraman et al. 1998), suggesting that these proteins may be involved in the internalization of L. pneumophila. Morphologically, L. pneumophila has been seen entering amoebae by coiling phagocytosis (Bozue and Johnson 1996, Venkataraman et al. 1998). However, other forms of internalization (perhaps resembling receptor-mediated endocytosis or macropinocytosis) do occur and seem to be even more common than coiling phagocytosis (Abu Kwaik 1996, Fields 1993, Venkataraman et al. 1998). As implied by Harb et al. (1998) one should not assume that all amoebae use the same mechanisms to internalize L. pneumophila.

2.2. Post-Internalization Events and Intracellular Growth The morphological events that follow internalization of L. pneumophila by amoebae are similar to those thoroughly characterized in mammalian cells. These include the association of the legionellae-containing vacuole with vesicles and the rough endoplasmic reticulum, deformation of the vacuolar membrane to closely follow the contour of replicating legionellae, etc. In addition, biochemical methods and/or immunolabeling have shown that amoebal vacuoles containing L. pneumophila do not fuse with lysosomes, and later acquire endoplasmic reticulum (ER) markers (Bozue and Johnson 1996, Abu Kwaik 1996). It is within this specialized vacuole surrounded by the ER that L. pneumophila replicates. The same morphological and biochemical intracellular events described in Hartmanella and Acanthamoebae also happen in the social amoeba Dictyostelium discoideum (Hägele et al. 2000, Solomon et al. 2000). This social amoeba has emerged as an excellent model to study the L. pneumophila–host interaction, mainly because it is a haploid organism with a known genome sequence, and for which several genetic tools have been developed. Other chapters in this book provide extensive details about the intracellular events associated with L. pneumophila replication, and the effector proteins that mediate these events. Therefore, discussion of these topics is not needed here, except to say that the morphological similarities observed

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between intracellular events in mammalian cells and amoebae are substantiated by the fact that many L. pneumophila genes are required for infection of both amoebae and mammalian cells (Cianciotto and Fields 1992, Cirillo et al. 2002, Gao et al. 1997, Liles et al. 1999, Miyake et al. 2005, Polesky et al. 2001, Pruckler et al. 1995, Segal and Shuman 1999). However, L. pneumophila seems to exclusively use subsets of genes to establish intracellular infections in either mammalian cells (Gao et al. 1998) or amoebae (Brieland et al. 1997b).

2.3. End of Replication and Exit Once the host cell has been wasted after L. pneumophila replication, the bacterial progeny is released to the environment where new host cells are to be found. The life cycle is then completed. The killing of the host cell and the release of the bacterial progeny seem to be two related processes that are driven by L. pneumophila’s ability to form pores in membranes (Gao and Abu Kwaik 2000). A class of mini Tn10 insertion mutants defective in their pore-forming ability were unable to both kill infected Acanthamoeba polyphaga and exit the infected amoebae. These mutants remained inside live amoebae for up to 4 days, but did not continue to replicate. The demise of the amoebal host does not occur via apoptosis (Gao and Abu Kwaik 2000). An alternative way of exiting infected Acanthamoeba has been reported by Berk et al. (1998). Live L. pneumophila can be released within vesicles, likely as a consequence of the amoebae trying to get rid of their food vacuoles before encystation. In this process, vesicles containing hundreds of legionellae are produced, in contrast to the free legionellae released by the lytic process. Because these vesicles are of respirable-size, it has been argued that they could act as complex infectious units in the transmission of Legionnaires’ disease. Furthermore, legionellae inside vesicles seem more resistant to some environmental challenges (Berk et al. 1998), suggesting that this mode of exit is associated with additional fitness advantages. Interestingly, numerous legionellae-laden vesicles are also produced by the ciliate Tetrahymena (McNealy et al. 2002), as discussed below.

2.4. What Role Do Ciliates Play in the Life Cycle of L. pneumophila? In contrast to the clear role that amoebae play as natural hosts for the intracellular replication of L. pneumophila, the permissiveness of ciliates to L. pneumophila replication is dubious. Although a few studies have experimentally shown that the freshwater ciliate Tetrahymena pyriformis supports the multiplication of L. pneumophila at 30–35°C (Barbaree 1986, Fields et al. 1984, 1986, Steele and McLennan 1996), at 25°C T. pyriformis is not permissive (Fields et al. 1984). In addition, L. pneumophila could not grow in the ciliate Cyclidium in axenic culture (Barbaree 1986). Except for Legionella longbeachae, several other Legionella spp. (L. pneumophila included) were either unable to grow in T. pyriformis or showed inconsistent growth in this ciliate (Steele and McLennan 1996). Finally,

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Tetrahymena vorax did not support the intracellular growth of L. pneumophila at 20–22°C (Smith-Somerville 1991). In my lab, we have been unable to grow different L. pneumophila strains in Tetrahymena sp. incubated at ⱕ30°C. Therefore, ciliates do not seem to constitute the preferred host within which L. pneumophila multiplies in nature. Still, ciliates may play an important role in the life cycle of L. pneumophila, either supporting the growth of L. pneumophila in thermally altered waters, as reservoirs of L. pneumophila, or as producers of legionellaeladen vesicles (McNealy et al. 2002). In a tripartite co-culture including A. polyphaga, Tetrahymena sp, and a low concentration of L. pneumophila (⬍1 legionellae/amoebae), numerous legionellae-laden vesicles were produced. In contrast, no vesicles were observed in experiments with amoebae and legionellae, or in experiments with ciliates and legionellae, when the low concentration of L. pneumophila was used (McNealy et al. 2002). However, mixing L. pneumophila and ciliates at a 100:1 ratio, or higher, yields numerous legionellae-laden vesicles (S. G. Berk personal communication). It seems that amoebae can amplify a thinned population of legionellae, so that the thicker bacterial population can be then ingested by Tetrahymena and packaged into legionellae-laden vesicles that could act as potential infectious particles for humans (Rowbotham 1980) with associated fitness advantages as discussed above.

3. The Growth Cycles of L. pneumophila Legionella pneumophila is able to grow on agar plates as well as in nutrient-rich liquid media. The artificial growth of L. pneumophila in the lab underlines the point that L. pneumophila may follow a number of growth cycles that are not part of its natural life cycle described above. Yet, the multiple growth cycles of L. pneumophila are part of its biology and constitute helpful tools to better understand this bacterium. For instance, it is of great advantage that L. pneumophila can be coerced to grow in the lab, as this allows the application of routine genetic, biochemical, and physiological methods to its study. However, we should keep in mind that growth in the lab is irrelevant to the life cycle and natural history of L. pneumophila. The same principle applies to the growth of L. pneumophila in mammalian cells (e.g., human macrophages). Legionella pneumophila grows extracellularly in lab co-cultures with green unicellular algae and/or cyanobacteria (Hume and Hann 1984, Pope et al. 1982, Tison et al. 1980) or other bacterial species (Wadosky and Yee 1983). L. pneumophila respires and/or incorporates carbon compounds present in photosynthetic exudates, as determined with 14C-tracing experiments (Tison 1987). Extracellular growth of legionellae in association with other microorganisms may thus happen in nature, particularly in biofilms. However, the significance of this extracellular growth to the life cycle of L. pneumophila remains to be established, particularly because recent studies suggest that the only manner in which L. pneumophila generates progeny in biofilm systems is through intracellular growth in amoebae (Kuiper et al. 2004, Murga et al. 2001). Following this line of thought, some

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investigators have proposed that in its natural habitat the intracellular environment of protozoa is essential for L. pneumophila replication (Abu Kwaik et al. 1998, Fields 1993). In Fig. 4.2 the extracellular growth cycle of L. pneumophila is portrayed. For the reasons discussed above, this growth cycle is not a primary representation of the growth of L. pneumophila in environmental biofilms, but a representation of the growth of L. pneumophila in nutrient-rich broth or agar. As described by Byrne and Swanson (1998), the extracellular growth cycle alternates between exponential phase legionellae that actively replicate, and post-exponential phase legionellae that eventually stop replicating until placed in fresh medium where growth is reinitiated. There are clear developmental changes associated with the transition between exponential phase and post-exponential phase legionellae (e.g., changes in the ability to infect host cells), which will be discussed in Section 4 of this chapter. When it comes to growing intracellularly, L. pneumophila displays many growth cycles. Numerous mammalian cells in culture support the growth of

Broth or agar

1 2 Germination Extracellular cycle MIF Intermediate Replicative form or exponential phase form Virulent form or postexponential phase form

FIGURE 4.2. The extracellular growth cycle of L. pneumophila. L. pneumophila enters the extracellular growth cycle via differentiation of MIFs into replicative forms in a nutrientrich medium. The presence of amino acids seems to be the trigger for germination. This step occurs through morphological intermediates. The replicative form is non-infectious to cells and cannot initiate intracellular growth cycles. As the nutrients are exhausted, cell division stops and the replicative form differentiates into a virulent form that stays in a stationary growth phase until passed into fresh nutrient-rich media. The virulent form can initiate intracellular growth cycles.

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L. pneumophila, some of which have been listed by Fields (1996). Technically, each of these cultured mammalian cells is associated with a unique L. pneumophila growth cycle that shows similarities and differences with both the L. pneumophila life cycle that primarily evolved in amoebae, and with growth cycles in other host cells. Many of the differences occur in the early stages of infection, e.g., attachment and invasion, whereas many of the similarities are found in the post-internalization and replication stages. The similarities and differences between growth in mammalian cells and amoebae have been highlighted in previous reviews (e.g., Fields 1996, Hoffman and Garduño 1999). Whole mammals (e.g., mice, guinea pigs) and embryonated chicken eggs also support the growth of L. pneumophila. These animals constitute very valuable models for understanding the human infection, but as previously stated in relation to the infection of humans, infection of these animals is irrelevant for the life cycle and the natural history of L. pneumophila. If L. pneumophila displays as many growth cycles as there are hosts, an intriguing point is whether L. pneumophila possesses a plethora of molecular factors that allow it to gain access to and replicate in these many hosts, or whether L. pneumophila cleverly exploits a very fundamental, ancient mechanism that is common to many eukaryotic cells. The current experimental evidence available, as presented above, could be interpreted one way or another, leaving the issue undecided. However, the many differences and variations that characterize the attachment to and entry of L. pneumophila into cells (Cirillo et al. 2000, Garduño et al. 1998a,b, Gibson et al. 1994, Harb et al. 1998, Payne and Horwitz 1987) seem to favor the notion that L. pneumophila has developed many different invasion factors. How did these factors evolve in the context of frequent interactions with amoebae (Molmeret et al. 2005) is not clear, but a subject of interest to understand the evolution of intracellular parasitism.

4. The Developmental Cycle of L. pneumophila The life cycle of L. pneumophila is accompanied by changes in the bacterium, some of which are very dramatic. First noticed by Rowbotham (1986) were the morphological changes in the progeny that emerges from wasted amoeba trophozoites. Besides being shorter than legionellae grown in vitro, the rods released by infected amoebae were also transiently motile. Through the years, it has become clear that growth of L. pneumophila in amoebae is also associated with changes in biochemistry, physiology, and virulence potential. L. pneumophila pre-grown in amoebae has the following characteristics: • • • • • • •

Enhanced resistance to chemicals (biocides) (Barker et al. 1992) Altered fatty acid profile, and possibly altered LPS (Barker et al. 1993) Altered protein profile (Barker et al. 1993, Cirillo et al. 1994) Bright red Giménez staining (Cirillo et al. 1994) Shorter size (Barker et al. 1995, Cirillo et al. 1994, Rowbotham 1986) Increased infectivity to mammalian cells and amoebae (Cirillo et al. 1994) Enhanced resistance to antibiotics (Barker et al. 1995)

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• Increased environmental fitness (Abu Kwaik et al. 1997) • Increased uptake by coiling phagocytosis (Cirillo et al. 1994, 1999) • Increased virulence to mice (Cirillo et al. 1999, Brieland et al. 1997a) Such profound and numerous changes are compatible with bacterial differentiation. At the time when some of the above phenotypes were characterized, it was also determined that they were lost upon subculture of L. pneumophila in lab media (and of course regained upon growth in amoebae), suggesting that L. pneumophila had gone through a reversible transition compatible with a cyclic change. It was in the context of the growth cycle in HeLa cells that the cyclical ultrastructural changes that L. pneumophila experiences during its intracellular growth were observed and studied (Garduño et al. 1998b, 2002a). These studies benefited from working with a highly pure population of HeLa cell-grown L. pneumophila, isolated from legionellae-laden vacuoles and HeLa cell debris by differential centrifugation in continuous density gradients (Garduño et al. 1998b). Actively replicating legionellae inside ribosome-decorated vacuoles were seen undergoing cell division, and displayed a typical Gram-negative envelope ultrastructure. In contrast, purified HeLa cell-grown L. pneumophila were never seen undergoing cell division, and presented a distinct thickening of the inner leaflet of the outer membrane and long invaginations of the cytoplasmic membrane (Garduño et al. 1988b, 2002b). HeLa cell-grown legionellae also showed several of the features listed above for amoebae-grown L. pneumophila including enhanced resistance to chemicals (detergents), an altered protein profile, a bright red Giménez staining, shorter size, increased infectivity to mammalian cells, enhanced resistance to antibiotics, and increased environmental fitness (Garduño et al. 1988b, 2002a, 2002b). Collectively, our HeLa cell results and those obtained in amoebae [mainly by Rowbotham (1986)] indicated a clear alternation between different forms of L. pneumophila that strongly suggested the existence of a developmental cycle. A detailed ultrastructural study (Faulkner and Garduño 2002) further established the cyclical morphological development that L. pneumophila follows when it differentiates along the HeLa cell growth cycle (modeled in Fig. 4.3). Recently, the existence of different L. pneumophila developmental forms was confirmed at the ultrastructural level in H. vermiformis (Greub and Raoult 2003), but even in past literature these forms are easily distinguished in several electron micrographs of infected amoebae, as discussed by Faulkner & Garduño (2002). Therefore, the life cycle of L. pneumophila, as some intracellular growth cycles in cultured cells, is associated with defined morphological, biochemical, and physiological changes that define a developmental cycle along which L. pneumophila differentiates.

4.1. Legionella pneumophila Development Along the Extracellular Growth Cycle The extracellular growth cycle of L. pneumophila is also associated with a differentiation process, initially described by Byrne and Swanson (1998). The post-exponential form (or stationary phase form) of L. pneumophila (Fig. 4.2)

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Mitochondrion Ribosome Vesicle or

HeLa MIF

ER

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Intermediate Replicative form Virulent form or postexponential phase form

FIGURE 4.3. The growth cycle of L. pneumophila in HeLa cells. The cycle begins with the attachment to and the invasion of a HeLa cell, a process mediated (at least partially) by the L. pneumophila chaperonin. Cell infection is initiated by stationary phase forms, or by MIFs, because replicative forms do not proceed to establish an intracellular infection (patterned arrow with the “do not” sign). Post-internalization events are very similar to those observed in amoebae, and L. pneumophila also replicates in a ribosome-decorated vacuole that associates with the endoplasmic reticulum (ER) and acquires ER markers. After replication is finished, the progeny exits the wasted HeLa cell either as free MIFs or as large MIF-ladden vesicles. The intracellular growth cycle in HeLa cells is associated with bacterial differentiation, whereas the intracellular growth cycle in macrophages does not lead to the efficient production of MIFs.

is sodium sensitive, cytotoxic to mammalian cells, osmotically resistant, motile, infectious to cultured mouse macrophages, and capable of inhibiting phagosome–lysosome fusion. All these characteristics were absent in the exponential phase form (Byrne and Swanson 1998), which has been further confirmed to be non-infectious to cells in culture (Joshi et al. 2001). This clear distinction between the two forms of the extracellular growth cycle led to the proposed names by Byrne and Swanson (1998) of “replicative form” for the exponential phase form, and “virulent form” for the post-exponential phase form. The regulatory system that controls L. pneumophila differentiation in the extracellular growth cycle has been studied at the molecular level, mainly in the Swanson lab. As comprehensibly reviewed by Molofsky and Swanson (2004), entry into stationary phase is triggered by the activation of the stringent response regulator RelA and accumulation of the molecular alarmone ppGpp (Hammer and Swanson 1999). To coordinate the expression of virulence traits and differentiation, ppGpp and RelA act in concert with the following molecular players:

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• The two-component regulatory system LetA/S (responsible for signal transduction with help from LetE) (Bachman and Swanson 2004, Hammer et al. 2002) • The sigma factors RpoS, RpoN, and FliA (Bachman and Swanson 2001, Hales and Shuman 1999, Hammer et al. 2002, Heuner et al. 2002, Zussman et al. 2002) • CsrA (a repressor of mRNA translation) (Forsbach-Birk et al. 2004, Molofsky and Swanson 2003). However, the role of RelA may be redundant and/or not essential because L. pneumophila relA mutants still induce cytotoxicity and keep RpoS-dependent functions (Abu-Zant et al. 2006), also suggesting a lack of control of ppGpp levels and the stringent response over RpoS function (Abu-Zant et al. 2006, Zussman et al. 2002). A key question would be: Is the developmental program that gives rise to the post-exponential virulent form in vitro the same that L. pneumophila uses to differentiate in amoebae and HeLa cells? In a head-to-head experimental comparison, HeLa cell-grown legionellae and post-exponential forms from the same L. pneumophila strain grown in vitro had different phenotypes (Garduño et al. 2002b). Post-exponential forms were less infectious to mammalian cells in culture, less resistant to detergents or antibiotics, and had a higher respiration rate than HeLa cell-grown legionellae. Indirect comparison with published data also indicated that post-exponential virulent forms are different from amoebae-grown legionellae (e.g., Cirillo et al. 1994, and Byrne and Swanson 1998 vs. Barker et al. 1992, 1995). However, post-exponential phase and HeLa cell-grown forms share a number of traits, like thermal tolerance, resistance to osmotic shock and protease treatments, and production of cytoplasmic inclusions. Moreover, mutants with a defective LetA/S two component regulatory system, or with a deleted rpoS gene, which are defective in developing the post-exponential virulent phenotype, are also unable to develop the wild type characteristics of HeLa cell-grown legionellae (unpublished results). Finally, post-exponential virulent forms develop the unique morphological features of HeLa cell- or amoebaegrown legionellae upon interaction with the ciliate Tetrahymena, and in the absence of intracellular multiplication (unpublished results). Collectively, these results suggest that (i) L. pneumophila contains a developmental program, (ii) a known trigger of this developmental program (among others) is amino acid starvation, (iii) the developmental program of L. pneumophila involves a regulatory network of which LetA/S and RpoS are essential members, and (iv) the post-exponential virulent form developed in vitro constitutes a partially differentiated form that in the right conditions can further differentiate. By reasons not yet understood, full differentiation into the highly infectious and resilient developmental form reported by Barker et al. (1992, 1993, 1995), Cirillo et al. (1994, 1999), Abu Kwaik et al. (1997), and Garduño et al. (2002b) seems to only take place in the intracellular environment of L. pneumophila hosts. Therefore, in addition to amino acid starvation, other signals seem to be required for full differentiation of L. pneumophila. In spite of representing an immature L. pneumophila form that does not express a full virulence

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phenotype, post-exponential phase forms have served as a practical tool to study L. pneumophila differentiation at the molecular level, and to elucidate the complex regulatory pathway that controls the expression of virulence traits (Molofsky and Swanson 2004).

4.2. MIF: The Result of L. pneumophila Intracellular Differentiation Intracellularly, L. pneumophila possesses two main morphological forms, the replicative form (RF) and the mature intracellular form (MIF), which alternate within the developmental cycle of L. pneumophila via several morphological intermediates (Faulkner and Garduño 2002, Greub and Raoult 2003). The RF actively undergoes cell division, has the typical ultrastructure of Gram-negative bacteria (defined by two membranes and a periplasmic space between them), and does not persist in the extracellular environment. The RF seems equivalent to the replicative form, or exponential phase form characterized by Byrne and Swanson (1998), which is non-infectious (Joshi et al. 2001). In contrast, the MIF does not undergo cell division, has a complex laminated envelope defined by several membranes and layers, displays prominent intracellular inclusions, and is highly infectious to cells in culture and mice (Cirillo et al. 1994, 1999, Garduño et al. 2002b). The MIF is the intracellular equivalent of the postreplicative form, which exits infected cells and persists in the environment. If we accept the notion that in its natural habitat L. pneumophila only replicates in the intracellular environment (see Section 3 above), then we are compelled to accept the notion that the MIF is the L. pneumophila free form that predominates in the environment. That would explain why MIFs are environmentally resilient and highly infectious, loaded with inclusions of storage polymers, and exhibit a very low respiration rate. The MIF is the specialized form of L. pneumophila designed to survive in the environment for several months (James et al. 1999, Lee and West 1991, Schofield 1985, Skaliy and McEachern 1979), and assail potential hosts. Once in the intracellular environment, MIFs differentiate (or “germinate,” to use the same terminology introduced in Fig. 4.2) into RFs, likely in response to an elevated concentration of amino acids putatively transported from the host cell cytoplasm into the replicative vacuole (Sauer et al. 2005). After L. pneumophila replication, and in response to amino acid starvation and other signals, RFs differentiate into MIFs in the intracellular environment. MIFs then exit the wasted host cell and disperse in the environment until a new host is found. Clearly, the MIF-to-RF and RF-to-MIF transitions naturally occur in the intracellular environment, and MIFs naturally constitute the resting cyst-like forms that survive free in the water environment. Whether MIFs are capable to further differentiate (e.g., to produce the viable but non-culturable form of L. pneumophila) remains to be established. One remarkable observation is the fact that MIFs seem inconspicuous in cultured human macrophages (Garduño et al. 2002b). Even in a retrospective analysis of past literature, MIFs could not be clearly identified in micrographs of infected

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macrophages. It is very possible that upon inducing early apoptosis in macrophages (Gao and Abu Kwaik 1999, Hägele et al. 1998), L. pneumophila cuts short its time window available to achieve full differentiation. Alternatively, the signals that lead to full differentiation of L. pneumophila into MIFs may be present in amoebae or in non-lymphoid mammalian cell lines, but absent in macrophages. Assuming that the MIF is the transmissible form of L. pneumophila in nature, the fact that MIFs scantily form in human macrophages (Garduño et al. 2002b) may provide the first clue to explain the environmental quality of Legionnaires’ disease, and its lack of transmission from person to person.

4.3. The Central Role of MIFs in the Study of L. pneumophila Virulence The MIF seems to hold the key to understand the transmission and infectiousness of L. pneumophila. First, in contrast to the non-infectious RF, the MIF is infectious to amoebae, a number of cells cultured in vitro, and mice. The previously reported virulence-enhancing role of amoebae (Brieland et al. 1996, 1997a, 1997b, Cirillo et al. 1994, 1999) is most likely due to the developmental differentiation of L. pneumophila into MIFs inside amoebae. Thus, MIFs seem to play a central role in the infection of both amoebae and mammalian hosts. In the L. pneumophila life cycle, MIFs in water infect amoebae, become established in the intracellular environment, and differentiate into RFs. However, when released from wasted amoebae, L. pneumophila emerges again as MIFs either free (as a result of necrotic lysis of the host) or packaged into vesicles (as described by Berk et al. 1998). Most likely, MIFs released from amoebae are the ones accidentally diverted into the human host to initiate Legionnaires’ disease in susceptible individuals. Finally, free MIFs emerging from infected amoebae are the ones packaged by ciliates into respirable-size vesicles (McNealy et al. 2002) that also may accidentally initiate human infections. MIFs must possess all the factors required to survive in water and infect new hosts. This prediction is based on the facts that MIF’s metabolism is low (Garduño et al. 2002b), and that invasion of new hosts and the intracellular establishment of L. pneumophila takes but minutes after the initial contact (Lu and Clarke 2005, Roy et al. 1998). It is our belief that within this narrow frame there is not enough time for the metabolically challenged MIF to mount a de novo response to the host cell, start the developmental program of differentiation into the RF, initiate a rapid synthesis of virulence effectors, assemble a functional Dot/Icm secretion system, and deliver effectors to the host cell. Instead, it is appealing to view MIFs as preloaded units carrying all the goods required to assure a successful infection of new hosts. In this respect, Roy et al. (1998) demonstrated, by placing the dotA gene under the control of an inducible promoter, that DotA was required for invasion and intracellular establishment, but was dispensable during intracellular growth (the RF phase). When dotA expression was “turned off” toward the end of the intracellular growth cycle, the resulting progeny could not re-infect cells, elegantly suggesting that

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the bacteria emerging from a wasted host cell (i.e., MIFs) must already have a functional Dot/Icm system to assure infection of new hosts. The expression of other virulence-related proteins (e.g., DotO, DotH, and Hsp60) in the late stages of intracellular growth (corresponding to differentiation of RFs into MIFs) but not in the post-exponential phase of the extracellular growth cycle (Garduño et al. 2002b, Watarai et al. 2001) points to the unique array of virulence factors that equip MIFs to best perform their duty: the efficient assailment of new hosts.

5. Concluding Remarks It seems clear now that L. pneumophila has a defined developmental cycle embedded in its life cycle. Developmental cycles are central to the pathogenesis and ecology of obligate intracellular bacterial pathogens with an extracellular phase. Therefore, differentiation into a highly infectious and environmentally resilient form that survives extended periods of starvation in freshwater (just to have the opportunity to gain access into new hosts) constitutes a trait evolved by L. pneumophila that has become an integral part of its natural history. The term “life cycle” should only be used in reference to the growth cycle and differentiation of L. pneumophila in amoebae and other protozoa. Similarly, the term “MIF” should only be used to refer to the differentiated L. pneumophila form that emerges from protozoa, or from mammalian cells that support the full differentiation of L. pneumophila (e.g., HeLa cells). MIFs should be regarded as the transmissive forms of L. pneumophila. Post-exponential virulent forms grown in vitro, unlikely to exist in nature, should not be regarded as the transmissive form of L. pneumophila in nature. In vitro grown legionellae should be held in the highest regard as experimental models to study aspects of L. pneumophila development and its general biology. In many respects, the L. pneumophila life cycle resembles the life cycles of Chlamydia and Coxiella, which are obligate intracellular pathogens that spread via cyst-like forms. In particular, Coxiella burnetti is a bioterrorism agent (CDC list of bioterrorism agents: http://www.bt.cdc.gov/agent/agentlist.asp) that shows striking similarities with Lp: • It has a dimorphic developmental cycle (Coleman et al. 2004). • It is highly infectious by aerosol (Glazunova et al. 2005). • It exploits autophagy (as L. pneumophila does) to establish intracellular infections (Amer and Swanson 2005, Gutierrez et al. 2005). • It possesses similar virulence factors including the Dot/Icm system (Vogel 2004). The complete DNA sequence of its genome has confirmed Coxiella burnetti to be the closest relative of L. pneumophila (Samuel et al. 2003, Vogel 2004). The facts that L. pneumophila grows in vitro (either in broth or on agar plates) and only requires level-2 biohazard containment (in contrast to Coxiella which is an

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obligate intracellular pathogen that cannot grow in vitro, and requires a level-3 containment) are significant advantages for its use as an experimental surrogate for Coxiella. Thus, MIFs have the potential of becoming the preferred experimental model to study intracellular bacteria transmitted by cyst-like forms.

References Abu Kwaik Y (1996) The phagosome containing Legionella pneumophila within the protozoan Hartmannella vermiformis is surrounded by the rough endoplasmic reticulum. Appl Environ Microbiol 62:2022–2028. Abu Kwaik Y, Gao L-Y, Harb OS, and Stone BJ (1997) Transcriptional regulation of the macrophage-induced gene (gspA) of Legionella pneumophila and phenotypic characterization of a null mutant. Mol Microbiol 24:629–642. Abu Kwaik Y, Gao L-Y, Stone BJ, Venkataraman C, and Harb OS (1998) Invasion of protozoa by Legionella pneumophila and its role in bacterial ecology and pathogenesis. Appl Environ Microbiol 64:3127–3133. Abu-Zant A, Asare R, Graham JE, and Abu Kwaik Y (2006) Role of RpoS but not RelA of Legionella pneumophila in modulation of phagosome biogenesis and adaptation to the phagosomal microenvironment. Infect Immun 74:3021—3026. Adeleke A, Pruckler J, Benson R, Rowbotham T, Halablab M, and Fields B (1996) Legionella-like amoebal pathogens—Phylogenetic status and possible role in respiratory disease. Emerg Infect Dis 2:225–230. Ahn TI, Lim ST, Leeu HK, Lee JE, and Jeon KW (1994) A novel strong promoter of the groEx operon of symbiotic bacteria in Amoeba proteus. Gene 148:43–49. Amer AO, and Swanson MS (2005) Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol 7:765–778. Bachman MA, and Swanson MS (2001) RpoS co-operates with other factors to induce Legionella pneumophila virulence in the stationary phase. Mol Microbiol 40:1201–1214. Bachman MA, and Swanson MS (2004) The LetE protein enhances expression of multiple LetA/LetS-dependent transmission traits by Legionella pneumophila. Infect Immun 72:3284–3293. Barbaree JM, Fields BS, Feeley JC, Gorman GW, and Martin WT (1986) Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl Environ Microbiol 51:422–424. Barker J, Brown MRW, Collier PJ, Farrell I, and Gilbert P (1992) Relationship between Legionella pneumophila and Acanthamoeba polyphaga: physiological status and susceptibility to chemical inactivation. Appl Environ Microbiol 58:2420–2425. Barker J, Lambert PA, and Brown MR (1993) Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila. Infect Immun 61:3503–3510. Barker J, Scaife H, and Brown MR (1995) Intraphagocytic growth induces an antibioticresistant phenotype of Legionella pneumophila. Antimicrob Agents Chemother 39:2684–2688. Benson RF, and Fields BS (1998) Classification of the genus Legionella. Semin Respir Infect 13:90–99. Berk SG, Ting RS, Turner GW, and Ashburn RJ (1998) Production of respirable vesicles containing live Legionella pneumophila cells by two Acanthamoeba spp. Appl Environ Microbiol 64:279–286.

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Bosshardt SC, Benson RF, and Fields BS (1997) Flagella are a positive predictor for virulence in Legionella. Microb Pathogen 23:107–112. Bozue, JA, and Johnson W (1996) Interaction of Legionella pneumophila with Acanthamoeba castellani: Uptake by coiling phagocytosis and inhibition of phagosomelysosome fusion. Infect Immun 64:668–673. Brieland J, McClain M, Heath L, Chrisp C, Huffnagle G, LeGendre M, Hurley M, Fantone J, and Engleberg C (1996) Coinoculation with Hartmanella vermiformis enhances replicative Legionella pneumophila lung infection in a murine model of Legionnaires’ disease. Infect Immun 64:2449–2456. Brieland JK, Fantone JC, Remick DG, LeGendre M, McClain M, and Engleberg NC (1997a) The role of Legionella pneumophila-infected Hartmanella vermiformis as an infectious particle in a murine model of Legionnaires’ disease. Infect Immun 65:5330–5333. Brieland J, McClain M, LeGendre M, and Engleberg C (1997b) Intrapulmonary Hartmannella vermiformis: a potential niche for Legionella pneumophila replication in a murine model of legionellosis. Infect Immun 65:4892–4896. Byrne B, and Swanson MS (1998) Expression of Legionella pneumophila virulence traits in response to growth conditions. Infect Immun 66:3029–3034. Cianciotto NP, and Fields BS (1992) Legionella pneumophila mip gene potentiates intracellular infection of protozoa and human macrophages. Proc Natl Acad Sci USA 89:5188–5191. Cirillo JD, Falkow S, and Tompkins LS (1994) Growth of Legionella pneumophila in Acanthamoeba castellani enhances invasion. Infect Immun 62:3254–3261. Cirillo JD, Cirillo SLG, Yan L, Bermudez LE, Falkow S, and Tompkins LS (1999) Intracellular growth in Acanthamoeba castellani affects monocyte entry mechanisms and enhances virulence of Legionella pneumophila. Infect Immun 67:4427–4434. Cirillo SLG, Lum J, and Cirillo JD (2000) Identification of novel loci involved in entry by Legionella pneumophila. Microbiology 146:1345–1359. Cirillo SLG, Yang L, Littman M, Samrakandi MM, and Cirillo JD (2002) Role of the Legionella pneumophila rtxA gene in amoebae. Microbiology 148:1667–1677. Coleman SA, Fischer ER, Howe D, Mead DJ, and Heinzen RA (2004) Temporal analysis of Coxiella burnetii morphological differentiation. J Bacteriol 186:7344–7352. Faulkner G, and Garduño RA (2002) Ultrastructural analysis of differentiation in Legionella pneumophila. J Bacteriol 184:7025–7041. Fields BS (1993) Legionella and protozoa: interaction of a pathogen and its natural host. In: Barbaree JM, Breiman RF, Dufour AP (eds.) Legionella: Current status and emerging perspectives. American Society of Microbiology, Washington, pp 129–136. Fields BS (1996) Molecular ecology of legionellae. Trends Microbiol 4:286–290. Fields BS, Shotts EB Jr, Feeley JC, Gorman GW, and Martin WT (1984) Proliferation of Legionella pneumophila as an intracellular parasite of the ciliated protozoan Tetrahymena pyriformis. Appl Environ Microbiol 47:467–471. Fields BS, Barbaree JM, Shotts EB Jr, Feeley JC, Morrill WE, Sanden GN, and Dykstra MJ (1986) Comparison of guinea pig and protozoan models for determining virulence of Legionella species. Infect Immun 53:553–559. Fields BS, Fields SRU, Loy JNC, White EH, Steffens WL, and Shotts EB (1993) Attachment and entry of Legionella pneumophila in Hartmanella vermiformis. J Infect Dis 167:1146–1150. Forsbach-Birk V, McNealy T, Shi C, Lynch D, and Marre R (2004) Reduced expression of the global regulator protein CsrA in Legionella pneumophila affects

4. Life Cycle, Growth Cycles and Developmental Cycle

81

virulence-associated regulators and growth in Acanthamoeba castellanii. Int J Med Microbiol 294:15–25. Gao LY, and Abu Kwaik Y (1999) Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity. Infect Immun 67:862–870. Gao L-Y, and Abu Kwaik Y (2000) The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila. Environ Microbiol 2:79–90. Gao L-Y, Harb OS, and Abu Kwaik Y (1997) Utilization of similar mechanisms by Legionella pneumophila to parasitize two evolutionarily distant hosts, mammalian and protozoan cells. Infect Immun 65:4738–4746. Gao L-Y, Harb OS, and Abu Kwaik Y (1998) Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa. Infect Immun 66:883–892. Garduño RA, Garduño E, and Hoffman PS (1998a) Surface-associated Hsp60 chaperonin of Legionella pneumophila mediates invasion in a HeLa cell model. Infect Immun 66:4602–4610. Garduño RA, Quinn FD, and Hoffman PS (1998b) HeLa cells as a model to study the invasiveness and biology of Legionella pneumophila. Can J Microbiol 44:430–440. Garduño RA, Garduño E, Hiltz M, Allan D, and Hoffman PS (2002a) Morphological and physiological evidence for a developmental cycle in Legionella pneumophila. In: Marre R, AbuKwaik Y, Bartlett C, Cianciotto NP, Fields BS, Frosch M, Hacker J, and Lück PC (eds.) Legionella. ASM Press, Washington, pp. 82–85. Garduño RA, Garduño E, Hiltz M, and Hoffman PS (2002b) Intracellular growth of Legionella pneumophila gives rise to a differentiated form dissimilar to stationary phase forms. Infect. Immun. 70:6273–6283. Gibson FC III, Tzianabos AO, and Rodgers FG (1994) Adherence of Legionella pneumophila to U-937 cells, guinea pig alveolar macrophages, and MRC-5 cells by a novel complement-independent binding mechanism. Can J Microbiol 40:865–872. Glazunova O, Roux V, Freylikman O, Sekeyova Z, Fournous G, Tyczka J, Tokarevich N, Kovacava E, Marrie TJ, and Raoult D (2005) Coxiella burnetii genotyping. Emerg Infect Dis 11:1211–1217. Greub G, and Raoult D (2003) Morphology of Legionella pneumophila according to their location within Hartmanella vermiformis. Res Microbiol 154:619–621. Gutierrez MG, Vázquez CL, Munafó DB, Zoppino FCM, Berón W, Rabinovitch M, and Colombo MI (2005) Autophagy induction favours the generation and maturation of the Coxiella-replicative vacuoles. Cell Microbiol 7:981–993. Hägele S, Hacker J, and Brand B (1998) Legionella pneumophila kills human phagocytes, but not protozoan host cells by inducing apoptotic cell death. FEMS Microbiol Lett 169:51–58. Hägele S, Kohler R, Merkert H, Schleicher M, Hacker J, and Steinert M (2000) Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol 2:165–171. Hales LM and Shuman HA (1999) The Legionella pneumophila rpoS gene is required for growth within Acanthamoeba castellanii. J Bacteriol 181:4879–4889. Hammer BK, and Swanson MS (1999) Co-ordination of Legionella pneumophila virulence with entry into stationary phase by ppGpp. Mol Microbiol 33:721–731. Hammer BK, Tateda E, and Swanson MS (2002) A two-component regulator induces the transmission phenotype of stationary phase Legionella pneumophila. Mol Microbiol 44:107–118.

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Harb OS, Venkataraman C, Haack BJ, Gao L-Y, and Abu Kwaik Y (1998) Heterogeneity in the attachment and uptake mechanisms of the Legionnaires’ disease bacterium, Legionella pneumophila, by protozoan hosts. Appl Environ Microbiol 64: 126–132. Heuner K, Dietrich C, Skriwan C, Steinert M, and Hacker J (2002) Influence of the alternative sigma-28 factor on virulence and flagellum expression of Legionella pneumophila. Infect Immun 70:1604–1608. Hoffman PS, and Garduño RA (1999) Pathogenesis of Legionella pneumophila infection. In: Paradise LJ, Friedman H, and Bendinelli M (eds) Opportunistic Intracellular Bacteria and Immunity. Plenum Press, New York, pp. 131–147. Hume RD, and Hann WD (1984) Growth relationships of Legionella pneumophila with green algae (Chlorophyta). In: Thornsberry C, Balows A, Feeley JC, and Jakubowski W (eds) Legionella, Proceedings of the 2nd International Conference. American Society of Microbiology, Washington, pp. 323–324. James BW, Mauchline WS, Dennis PJ, Keevil CW, and Wait R (1999) Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments. Appl Environ Microbiol 65:822–827. Jeon KW (2004) Genetic and physiological interactions in the amoeba-bacteria symbiosis. J Eukaryot Microbiol 51:502–508. Joshi AD, Sturgill-Koszycki S, and Swanson MS (2001) Evidence that Dot-dependent and -independent factors isolate the Legionella pneumophila phagosome from the endocytic network in mouse macrophages. Cell Microbiol 3:99–114. King CH, Fields BS, Shotts EB Jr, and White EH (1991) Effects of cytochalasin D and methylamine on intracellular growth of Legionella pneumophila in amoebae and human monocyte-like cells. Infect Immun 59:758–763. Kuiper MW, Wullings BA, Akkermans ADL, Beumer RR, and van der Kooij D (2004) Intracellular proliferation of Legionella pneumophila in Hartmanella vermiformis in aquatic biofilms grown on plasticized polyvinyl chloride. Appl Environ Microbiol 70:6826–6833. Lee JV, and West AA (1991) Survival and growth of Legionella species in the environment. J Appl Bacteriol (Symp Suppl) 70:121S–129S. Liles MR, Edelstein PH, and Cianciotto NP (1999) The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila. Mol Microbiol 31:959–970. Lu H, and Clarke M (2005) Dynamic properties of Legionella-containing phagosomes in Dictyostelium amoebae. Cell Microbiol 7:995–1007. McDade JE, Shepard CC, Fraser DW, Tsai TR, Redus MA, Dowdle WR, et al. (1977) Isolation of a bacterium and demonstration of its role in other respiratory disease. N Engl J Med 297:1197–1203. McNealy T, Newsome AL, Johnson RA, and Berk SG (2002) Impact of amoebae, bacteria and Tetrahymena on Legionella pneumophila multiplication and distribution in an aquatic environment. In: Marre R, AbuKwaik Y, Bartlett C, Cianciotto NP, Fields BS, Frosch M, Hacker J, and Lück PC (eds.) Legionella. ASM Press, Washington, pp. 170–175. Miyake M, Watanabe T, Koike H, Molmeret M, Imai Y, and Abu Kwaik Y (2005) Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages. Infect Immun. 73:6272–6282. Molmeret M, Horn M, Wagner M, Santic M, and Abu Kwaik Y (2005) Amoebae as training grounds for intracellular bacterial pathogens. Appl Environ Microbiol 71:20–28.

4. Life Cycle, Growth Cycles and Developmental Cycle

83

Molofsky AB, and Swanson MS (2003) Legionella pneumophila CsrA is a pivotal repressor of transmission traits and activator of replication. Mol Microbiol 50:445–461. Molofsky AB, and Swanson MS (2004) Differentiate to thrive: lessons from the Legionella pneumophila life cycle. Mol Microbiol 53:29–40. Murga R, Forster TS, Brown E, Pruckler JM, Fields BS, and Donlan RM (2001) Role of biofilms in the survival of Legionella pneumophila in a model potable-water system. Microbiology 147:3121–3126. Payne NR, and Horwitz MA (1987) Phagocytosis of Legionella pneumophila is mediated by human monocyte complement receptors. J Exp Med 166:1377–1389. Polesky AH, Ross JTD, Falkow S, and Tompkins LS (2001) Identification of Legionella pneumophila genes important for infection of amoebas by signature-tagged mutagenesis. Infect Immun 69:977–987. Pope DH, Soracco RJ, Gill HK, and Fliermans CB (1982) Growth of Legionella pneumophila in two-membered cultures with green algae and cyanobacteria. Curr Microbiol 7:319–321. Pruckler JM, Benson RF, Moyenuddin M, Martin WT, and Fields BS (1995) Association of flagellum expression and intracellular growth of Legionella pneumophila. Infect Immun 63:4928–4932. Rowbotham TJ (1980) Preliminary report on the pathogenicity of Legionella pneumophila for freshwater and soil amoebae. J Clin Pathol 33:1179–1183. Rowbotham TJ (1986) Current views on the relationships between amoebae, legionellae and man. Isr J Med Sci 22:678–689. Roy CR, Berger KH, and Isberg RR (1998) Legionella pneumophila DotA protein is required for early phagosome trafficking decisions that occur within minutes of bacterial uptake. Mol Microbiol 28:663–674. Samuel JE, Kiss K, and Varghees S (2003) Molecular pathogenesis of Coxiella burnetii in a genomics era. Ann NY Acad Sci 990:653–663. Sauer J-D, Bachman MA, and Swanson MS (2005) The phagosomal transporter A couples threonine acquisition to differentiation and replication of Legionella pneumophila in macrophages. Proc Natl Acad Sci USA 102:9924–9929. Schofield GM (1985) A note on the survival of Legionella pneumophila in stagnant tap water. J Appl Bacteriol 59:333–335. Segal G, and Shuman HA (1999) Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages. Infect Immun 67:2117–2124. Skaliy D, and McEachern HV (1979) Survival of the Legionnaires’ disease bacterium in water. Ann Intern Med 90:662–663. Smith-Somerville H., Huryn VB, Walker C, and Winters AL (1991) Survival of Legionella pneumophila in the cold-water ciliate Tetrahymena vorax. Appl Environ Microbiol 57:2742–2749. Solomon JM, Rupper A, Cardell JA, and Isberg RR (2000) Intracellular growth of Legionella pneumophila in Dictyostelium discoideum, a system for genetic analysis of host-pathogen interactions. Infect Immun 68:2939–2947. Spriggs DR (1987) Legionella, microbial ecology, and inconspicuous consumption. J Infect Dis 155:1086–1087. Steele TW, and McLennan AM (1996) Infection of Tetrahymena pyriformis by Legionella longbeachae and other Legionella species found in potting mixes. Appl Environ Microbiol 62:1081–1083.

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Stone BJ, and Kwaik YA (1998) Natural competence for DNA transformation by Legionella pneumophila and its association with expressin of type IV pilli. J Bacteriol 181:1395–1402. Tison DL (1987) Microbial ecology of Legionella. J Infect Dis 156:852. Tison DL, Pope DH, Cherry WB, and Fliermans CB (1980) Growth of Legionella pneumophila in association with blue-green algae (cyanobacteria). Appl Environ Microbiol 39:456–459. Venkataraman C, Haack BJ, Bondada S, and Abu Kwaik Y (1997) Identification of a Gal/GalNAc lectin in the protozoan Hartmannella vermiformis as a potential receptor for attachment and invasion by the Legionnaires’ disease bacterium. J Exp Med 186:537–547. Venkataraman C, Gao LY, Bondada S, and Abu Kwaik Y (1998) Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires’ disease bacterium, Legionella pneumophila. J Exp Med 188:505–514. Vogel, JP (2004) Turning a tiger into a house cat: Using Legionella pneumophila to study Coxiella burnetii. Trends Microbiol 12:103–105. Wadosky RM, and Yee RB (1983) Satellite growth of Legionella pneumophila with an environmental isolate of Flavobacterium breve. Appl Environ Microbiol 46:1447–1449. Wataraj M, Andrews HL, and Isberg RR (2001) Formation of a fibrous structure on the surface of Legionella pneumophila associated with exposure of DotH and DotO proteins after intracellular growth. Mol Microbiol 39:313–329. Weeratna R, Logan SM, Hoskin D, Hoffman PS, Marrie TJ, Yates L, Burbridge S, Haldane D, and Bezanson G (1993) Legionnaires’ disease in cardiac transplant patients—A cell mediated immune response develops despite cyclosporin A therapy. J Infect Dis 168:521–522. Winn WC Jr. (1988) Legionnaires disease: Historical perspective. Clin Microbiol Rev 150:60–81. Zussman T, Gal-Mor O, and Segal G (2002) Characterization of a Legionella pneumophila relA insertion mutant and roles of RelA and RpoS in virulence gene expression. J Bacteriol 184:67–75.

5 Legionella in the Environment Barry S. Fields

1. Introduction Legionellae survive in environments with their primary hosts, free-living protozoa. These bacteria probably survive in many of the same environments inhabited protozoa although the full range of these environments is unknown. Legionellae bacteria are well suited to aquatic environments and freshwater is the primary reservoir for legionellae worldwide (Fliermans et al.1981). Although legionellae can be found in waters ranging from cold to very hot, their multiplication is limited to temperatures between 25 and 42°C with an optimal growth at 35°C. Therefore the bacteria colonize and persist in moist environments within this temperature range whereas their presence in other environments is probably transitory. Some outbreaks of legionellosis have been associated with construction, and it was originally believed that the bacteria could survive and be transmitted to humans via soil. However, legionellae do not survive in dry environments, and these outbreaks are more likely the result of massive descalement of plumbing systems due to changes in water pressure during construction (Katz and Hammel 1987; Mermel et al. 1995).

2. The Distribution of Legionella as a Function of Various Detection Methods Descriptions of the prevalence and distribution of legionella depends upon the detection method used. Results from culture-independent methods suggest a greater distribution and concentration of legionellae in the environment. These methods offer the potential of greatly increased sensitivity. However, culture remains the method of choice for detecting legionellae, primarily because cultureindependent methods cannot provide information regarding the viability of the bacteria. These non-culture methods include detection of the organisms with specific antisera by DFA staining and procedures to detect nucleic acids of legionellae using PCR.

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The use of DFA to detect legionellae is limited by the number of specific antisera that can be used. Since there are no antisera which specifically react with all Legionella species, a different antiserum must be used for each species or serogroup. Reports on the sensitivity and specificity of DFA testing of environmental specimens vary greatly, with most studies indicating that the test is relatively insensitive and non-specific (Joly 1993). The use of PCR for detecting nucleic acids of legionellae in the environment has proved to be valuable in some investigations of outbreaks of legionellosis (Miller et al. 1993). A number of Legionella genes, including 5S rRNA, 16S rRNA, dotA, and mip genes, have been used as targets for PCR (Mahbubani et al. 1990; Starnbach et al. 1989; Yanez et al. 2005). Use of PCR to detect legionellae in the environment has indicated that up to 80% of freshwaters are positive while only 20–60% are positive by culture (Arnow et al. 1985; Fields et al. 2002; Flannery et al. 2006). Culture-independent techniques, particularly PCR, have indicated that some sites contain unusually high concentrations of legionellae when compared with culture results (Fliermans et al. 1981; Tison et al. 1983; Ortiz-Roque and Hazen 1987; Sheehan et al. 2005). PCR assays have also detected legionellae nucleic acids in environments considered unfavorable for the survival of these bacteria (Palmer et al. 1993). There are several potential explanations for the discrepancy between culturebased and culture-independent environmental analysis. First, a viable but nonculturable state (VNBC) has been described for L. pneumophila (Steinert et al. 1997). VNBC Legionella probably represent stressed bacteria. Bacteria stressed in water and wastewaters frequently become incapable of growth and colony formation under standard conditions because of structural or metabolic injury (Anonymous 1985). These bacteria can usually be cultivated through recovery enhancement procedures which provide essential nutrients or remove toxic or inhibitory compounds. For Legionella spp. the addition of host protozoa can reestablish the ability to culture the organism. Most environmental testing for legionellae is performed to assess the potential for disease transmission. If the differences in PCR and culture data are due to the presence of VNBC Legionella, the clinical relevance of these organisms must be established. Another potential explanation for the difference in culture and cultureindependent results is the presence of Legionella species which do not grow on laboratory media or are difficult to culture. These bacteria were originally described as Legionella-like amoebal pathogens or LLAPs and were detected by examining water samples for bacteria-infecting protozoa present in these samples (Rowbotham 1993; Adeleke et al. 1996). The number of new species of Legionella continues to grow and there may be a number of species of this organism which still cannot be cultured by conventional means. If these organisms account for the numerous PCR positive/culture negative samples, then their clinical relevance must be established as well. Finally, the differences in culture and culture-independent results may be the result of a non-specific reaction with unrelated organisms. As previously stated, a number of PCR and fluorescent antibody procedures for detecting legionellae in

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the environment have been described. Some of these assays were subject to limited or no validation procedures and may have resulted in unfounded conclusions regarding the distribution of legionellae in the environment. Until some of these issues are resolved, studies should not rely solely on culture-independent assays but should use these in conjunction with culture.

3. Natural Environments A limited number of studies have looked at the distribution of legionellae in natural waters. Most studies have focused on man-made aquatic systems associated with disease transmission. Several early studies of natural environments relied heavily on direct fluorescent antibody staining for the detection of the bacteria in these samples. One study examined hundreds of samples from numerous lakes and rivers in the US (Fleirmans et al. 1981). This study detected legionellae in the majority of samples using DFA but only 15% by isolation in guinea pigs. Natural freshwater environments are rarely associated with outbreaks of legionellosis. Most cases of legionellosis can be traced to man-made aquatic environments where the water temperature is higher than ambient temperature. At lower temperature, L. pneumophila multiplies more slowly in its protozoan host. This probably results in a natural balance between the bacteria and their protozoan host at concentrations below the threshold for human disease. Thermally altering aquatic environments can shift the balance between protozoa and bacteria, resulting in rapid multiplication of legionellae, which can translate into human disease. Legionella pneumophila multiplies at temperatures between 25 and 42°C with an optimal growth temperature of 35°C (Figure 5.1). The previously mentioned study of US lakes and streams detected a seasonal effect in one lake with higher rates of isolate in May, June, and July. Several studies have detected legionellae in naturally occurring thermal springs. A study of lakes and streams in Mount St. Helens blast zone in 1983 detected several species of legionellae with higher concentrations in water warmed by thermal vents (Tison et al. 1983). Studies in continental Portugal and the Azores identified multiple species of legionellae from waters ranging from Temperature Range of Legionellae Celsius

25

35

42

45

55

Farenheit

77

95

108

113

131

Dormant

Growth

Death

FIGURE 5.1. Temperature Range of Legionellae.

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22 to 67.5°C (Verissimo et al. 1991). A PCR-based study of acidic biofilm communites in Yellowstone National Park detected several species of these bacteria in a pH 2.7 geothermal stream with temperatures ranging from 30to 38°C (Sheehan et al. 2005). These authors suggested that natural thermal gradients influence the Legionella species distribution. Legionellae have been detected in hot springs and associated resorts in multiple sites in Japan (Ohno et al. 2003). Many of these hot springs are modified for guest use and have incorporated the water treatment programs and recirculation and filtration systems. It is not clear if these legionellae populations naturally inhabit these enviroments with such distribution and concentration or if this is enhanced by the human manipulation of these waters. Although legionellae are realtively easy to detect in surface waters, the bacteria are believe to exist at low concentrations in ground water (Costas et al. 2005). The primary reason for this is believed to be the relatively colder temperatures of most ground water. However, a recent culture and PCR-based survey study found that 28–29% of ground waters tested were positive for legionellae with some of these positive ground waters as cold as 9°C (Brooks et al. 2004). Nevertheless, temperature appears to be the the primary environmental parameter associated with the prevalence of legioellae (Lee and West 1991; Flannery et al. 2006). Other factors such as osmolarity and pH appear to be less critical. High concentrations of sodium are known to be toxic to L. pneumophila and it is generally accepted that the bacteria cannot grow in sea water (Barbaree et al. 1983). Legionellae have not been cultured from sea water although one study did detect the organisms using PCR (Palmer et al. 1993). Conclusive studies are still needed to confirm the inhibition effect of sea water on the growth of Legionella. Although Legionella spp. are primarily associated with freshwater environments, the ecology of L. longbeachae appears to be a notable exception. This species is the leading cause of legionellosis in Australia which occurs in gardeners and those exposed to commercial potting soil (Steele 1989; Ruehlemann and Crawford 1996). The first US cases of L. longbeachae infection associated with potting soil were in 2000 (Centers for Disease Control and Prevention 2000). The factors that allow L. longbeachae to predominate in potting soil and thereby cause human disease are not yet understood.

4. Man-Made Environments Most of the information regarding the distribution of legionellae in man-made enviroments has been obtained during investigations of outbreaks of legionellosis. An overwhelming majority of cases of legionellosis can be traced to manmade aquatic environments where the water temperature is higher than ambient temperature. As stated in the 1992 Institute of Medicine report (Lederberg et al. 1992), “ Emerging Infections: Microbial Threats to Health in the United States,” “technology and industry can . . . cause, or at least contribute to, the emergence of infectious disease.” This is precisely the case with Legionnaires’ disease which may be regarded as a consequence of altering the environment for human benefit. A number of devices have been implicated as sources of aerosol transmission of

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TABLE 5.1. Building water systems known to transmit legionellae via aerosol. Type of water

Transmitting device

Potable

Showers, tap water faucets, respiratory therapy equipment Cooling towers and evaporative condensers, whirlpool spas, decorative fountains, ultrasonic mist machines, humidifiers

Non-potable

legionellae (Table 5.1). These sources are of two general types: those producing aerosols from potable water such as showers and faucets, and those from nonpotable water, such as cooling towers and whirlpool spas. Legionellae are common in many building water systems and only rarely cause disease. Prospective studies have found as many as 60% of buildings are colonized by these bacteria. A survey conducted in the UK found 60% of large buildings and 44% of cooling towers contained legionellae (Lee and West 1991). A study of buildings in San Francisco demonstrated that Legionella colonized 60% of hot water systems of 53 buildings tested over a two-year period (Flannery et al. 2006). Despite efforts to detect cases during the two-year study period, no case of Legionnaires’ disease were identified. Increased prevalence of Legionella colonization was associated with water heater temperatures below 50°C, buildings taller than 10 stories, and interruptions in water service. Approximately 30% of these buildings were consistently colonized over the two-year period. In most cases one or two strains or species of Legionella colonized a single building. In some cases a single strain or species was unique to a certain building. A similar study conducted in Pinellas County Florida detected Legionella in approximately 20% of the buildings tested (Moore et al. 2006). This demonstrates that the prevalence of Legionella varies in different geographic areas. The reason for this variablity is probably the result of many factors such as climate and water chemistry. The type of primary disinfectant used for municipal water supplies will effect the prevalence of legionellae in building water supplies. Monochloramine disinfection of water supplies is associated with decreased risk of Legionnaires’ disease (Kool et al. 1999; Heffelfinger et al. 2000). It is believed that monochloramine persists and penetrates biofilms better than free chlorine. Two prospective studies (mentioned above) looked at conversion from free chlorine to monochloramine as the primary disinfectant for the municipal water supply. In Pinellas County, Florida, the percent of buildings colonized with legionellae decreased from 19.8 to 6.2 % one month after conversion to monochloramine. In San Francisco County, Legionella colonization decreased from 60 to 4% after conversion and remained at this low level for the following year. Based on these studies the primary disinfectant used by a municipality should be a major factor in the prevalence of legionellae in man-made evironments. It is unclear if such treatment would have any effect of legionellae in non-potable systems such as cooling towers or whirlpool spas.

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5. Interaction with Other Organisms Legionellae require a distinct combination of nutrients in order to amplify in an environment. This unusual nutritional requirement of legionellae would appear to contradict the pervasiveness of the bacteria in aquatic environments. The levels of nutrients that legionellae require would rarely be found in freshwater and, if present, would serve only to amplify faster-growing bacteria that would compete with the legionellae. However, these nutrients represent an intracellular environment, not soluble nutrients commonly found in fresh water. Legionellae survive in aquatic and in some moist soil environments as intracellular parasites of freeliving protozoa (Rowbotham 1980; Fields 1996). Legionellae have been labeled “protozoonotic,” a term which appears to suit their natural history (Fields 1996). Legionellae have been reported to multiply in 14 species of amoebae, 2 species of ciliated protozoa, and 1 species of slime mold, while growth of legionellae in the absence of protozoa has been documented only on laboratory media (Fields 1996; Hägele et al. 2000; Solomon and Isberg 2000). Protozoa naturally present in environments implicated as sources of Legionnaires’ disease can support intracellular growth of legionellae in vitro (Barbaree et al. 1986). Legionella can infect and multiply intracellularly in both protozoa and human phagocytic cells (Horwitz and Silverstein 1980; Fields 1996). It appears that protozoa are the natural hosts of legionellae, whereas human phagocytic cells occasionally become ill-fated surrogates. Understanding the crucial role of protozoa in the ecology of legionellae is important for the development of successful prevention strategies.

6. Association with Biofilms Interaction of legionellae and protozoa is impacted by other microorganisms that comprise biofilms in building water systems. Legionellae are known to colonize these biofilms and can persist within these microbial communities for years. They are more easily detected from swab samples of biofilm than from flowing water, suggesting that the majority of the legionellae are biofilm associated (Rogers et al. 1994). A limited number of studies have attempted to characterize the bacteria’s interaction within these complex ecosystems. These studies have evaluated the effect of temperature and surface materials on the growth of L. pneumophila as well as the effect of biocides on sessile legionellae. The type of material used in the construction of plumbing systems has been shown to impact the ability of L. pneumophila to colonize these surfaces. L. pneumophila appears to be more abundant on elastomeric surfaces than copper or stainless steel (Rogers et al. 1994; Van der Kooij et al. 2005). However, the inhibitory effect of the metals is lost over time (Van der Kooij et al. 2005). The use of biofilm models to evaluate biocide efficacy against L. pneumophila represents a vast improvement over previous studies, which primarily evaluated the susceptibility of agar-grown bacteria in sterile water (Kusnetsov et al. 1994; Storey et al. 2004). These studies have

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also documented that amoebae provide protection from disinfectants in model biofilms (Donlan et al. 2005). The majority of Legionella/biofilm studies that have been conducted employ naturally occurring microbial communities. Such studies have the advantage of representing a true and natural microbial community, but all the organisms present have not been identified and their contribution to the survival and multiplication of legionellae remains unknown. Biofilm matrices are known to provide shelter and a gradient of nutrients. The complex nutrients available with biofilms have led some researchers to propose that the biofilms support the survival and multiplication of legionellae outside a host cell (Rogers and Keevil 1992). This concept is certainly plausible; most facultative intracellular bacteria are known to multiply extracellularly in some environments. If legionellae can multiply extracellularly within biofilms, the characterization of this phenomenon would have impact on control strategies for the prevention of legionellosis. Investigators have attempted to detect extracellular growth of L. pneumophila using a biofilm reactor and a defined bacterial biofilm grown on non-supplemented potable water (Murga et al. 2001). The base biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae, and the Flavobacterium-like organism isolated from a water sample containing legionellae. The addition of the amoeba Hartmannella vermiformis to the reactor resulted in a reproducible equilibrium between the amoeba and heterotrophic bacteria. L. pneumophila associated with, and persisted in, these biofilms with and without H. vermiformis. L. pneumophila cells did not appear to develop microcolonies, and growth measurement studies indicate that L. pneumophila did not multiply within this biofilm in the absence of amoebae. L. pneumophila did multiply in the biofilm and planktonic phase in the presence of H. vermiformis, and the majority of these bacteria appeared to be shed into the planktonic phase. These studies suggest that L. pneumophila may persist in biofilms in the absence of amoebae, but in the model, the amoebae were required for multiplication of the bacteria. This model biofilm was constructed of five preselected organisms and does not represent the many potentially diverse biofilms that may support the growth of legionellae in the environment. Additional studies are needed to determine if legionellae possess a means to multiply independent of a host cell within biofilms. The control of biofilm-associated legionellae may lead to the most effective control measures to prevent legionellosis. Institutions that have experienced outbreaks of legionellosis are all too aware of how tenacious legionellae can be within building water system biofilms.

References Adeleke A, Pruckler J, Benson R, Rowbotham T, Halablab M, Fields B (1996) Legionellalike amebal pathogens—phylogenetic status and possible role in respiratory disease. Emerg Infect Dis 2:225–230. Anonymous (1985) Stress organisms. In: Franson MAH, Clesceri LS, Greenberg AE, Trussell RR, (eds.) Standard methods for the examination of water and wastewater, 16th edition. American Public Health Association. Washington, DC. pp. 1036–1038.

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Arnow PM, Weil D, Para MF (1985) Prevalence and significance of Legionella pneumophila contamination of residential hot-tap water systems. J Infect Dis 152:145–151. Barbaree JM, Sanchez A, Sanden GN (1983) Tolerance of Legionella species to sodium chloride. Curr Microbiol 9:1–5. Barbaree JM, Fields BS, Feeley JC, Gorman GW, Martin WT (1986) Isolation of protozoa from water associated with a legionellosis outbreak and demonstration of intracellular multiplication of Legionella pneumophila. Appl Environ Microbiol 51:422–424. Brooks T, Osicki RA, Springthrorpe VS, Sattar SA, Filion L, Abrial D, Riffard S (2004) Detection and identification of Legionella species from ground waters. J Toxicol Environ Health, Part A 67:1845–1859. Centers for Disease Control and Prevention. (2000) Legionnaires’ Disease associated with potting soil—California, Oregon, and Washington, May–June 2000. MMWR 49(34):777–778. Costas J, Tiago I, da Costa MS, Verissiom A. (2005) Presence and persistence of Legionella spp in groundwater. Appl Environ Microbiol 71:663–671. Donlan RM, Forster T, Murga R, Brown E, Lucas C, Carpenter J, Fields B (2005) Legionella pneumophila associated with the protozoan Hartmannella vermiformis in a model multispecies biofilm has reduced susceptibility to disinfectants. Biofouling 21: 1–7. Fields BS (1996) The molecular ecology of Legionellae. Trends Microbiol 4:286–290. Fields BS, Benson RF, Besser RE (2002) Legionella and Legionnaires’ Disease: 25 years of investigation. Clin Microbiol Rev 15:506–526. Flannery B, Gelling LB, Vugia DJ, Weintraub JM, Salerno JJ, Conroy MJ, Stevens VA, Rose CE, Moore MR, Fields BS, and Besser RE (2006) Reduced Legionella after monochloramine disinfection. Emerg Infect Dis. 12:588–596. Fliermans CB, Cherry WB, Orrison LH, Smith SJ, Tison DL, Pope DH (1981) Ecological distribution of Legionella pneumophila. Appl Environ Microbiol 41:9–16. Green PN, Pirrie RS (1993) A laboratory apparatus for the generation and biocide efficacy testing of Legionella biofilms. J Appl Bacteriol 74:388–393. Hägele S, Kohler R, Merkert H, Schleicher M, Hacker J, Steinert M (2000) Dictyostelium discoideum: a new host model system for intracellular pathogens of the genus Legionella. Cell Microbiol 2:165–171. Heffelfinger JD, Kool JL, Fridkin SK, Fraser VJ, Hageman J, Carpenter, J, Whitney CG; Society for Healthcare Epidemiology of America (2000) Risk of hospital-acquired legionnaires’ disease in cities using monochloramine versus other water disinfectants. Infect Control Hops Epidemiol 24:569–574. Horwitz MA, Silverstein SC (1980) The legionnaires’ disease bacterium (Legionella pneumophila) multiplies intracellularly in human monocytes. J Clin Inves 66: 441–450. Joly JR (1993) Monitoring for the presence of Legionella: where, when, and how? In: Barbaree JM, Breiman RF, Dufour AP (eds.). Legionella: Current status and emerging perspectives. American Society for Microbiology, Washington, DC, pp. 211–216. Katz SM, Hammel, JM (1987) The effect of drying, heat, and pH on the survival of Legionella pneumophila. Ann Clin Lab Sci 17:150–156. Kool JL, Carpenter JC, Fields BS (1999) Effect of monochloramine disinfection of municipal drinking water on risk of nosocomial Legionnaires’ disease. Lancet 353:272–277. Kusnetsov JM, Keskitalo PJ, Ahonen HE, Tulkki AI, Miettinen IT, Martikanen PJ (1994) Growth of Legionella and other heterotrophic bacteria in a circulating cooling water system exposed to ultraviolet irradiation. J Appl Bacteriol 77:461–466. Lederberg J, Shoppe RE,Oaks SCJr.(eds.). (1992) Emerging Infections: Microbial Threats to health in the United States. National Academy Press, Washington, DC

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Lee JV, West AA (1991) Survival and growth of Legionella species in the environment. J Appl Bacteriol Symp Supp 70:121–129. Mahbubani MH, Bej AK, Miller R, Haff L, DiCesare J, Atlas RM (1990) Detection of Legionella pneumophila with polymerase chain reaction and gene probe methods. Mol Cell Probes 4:175–187. Mermel LA, Joesephson SL, Giorgio CH, Dempsey J, Parenteau S (1995) Association of Legionnaires’ disease with construction: contamination of potable water? Infect Contr Hosp Epidemiol 16:76–80. Miller LA, Beebe JL, Butler JC, Martin WT, Benson R, Hoffman RE, Fields BS (1993) Use of polymerase chain reaction in an epidemic investigation of Pontiac fever. J Infect Dis 168:769–772. Moore MR, Pryor M, Fields B, Lucas C, Phelan M, Besser Re (2006) Introduction of monochloramine into a municipal water system: impact on colonization of buildings by Legionella. Appl Environ Microbiol. 72:373–383. Murga R, Forster TS, Brown E, Pruckler JM, Fields BS, Donlan RM (2001) The role of biofilms in the