USMLE Step 1 Review - Home Study Program - Vol I - General Principles 1

Contents Section I: Biochemistry Chapter 1. Acid-Base Equilibrium and Buffering ........................... 3 Chapter 2. Proteins, Enzymes, and Coenzymes ........................... 7 Chapter 3. Bioenergetics ............................................ 23 Chapter 4. Carbohydrates ........................................... 37 Chapter 5. Lipids .................................................. 63 Chapter 6. Amino Acid Metabolism ................................... 89 Chapter 7. Summary of Metabolism .................................. 103

Section II: Molecular Biology Chapter 1. Nucleic Acids ........................................... 115 Chapter 2. The Nucleus, the Cell Cycle, and Meiosis ..................... 123 Chapter3. DNA Replication and Repair ............................... 129 Chapter 4. Transcription ............................................ 135 Chapter 5. Protein Synthesis ........................................ 143 Chapter 6. Regulation of Gene Expression ............................. 151 Chapter 7. Genetic Engineering ...................................... 159

I KAPLAlf med lea

vii

Section III: Cell Biology Chapter 1. Overview of Cell Types .................................... 177 Chapter 2. Nucleus and Nucleolus ................................... 183 Chapter 3. Subcellular Organelles .................................... 187 Chapter 4. Cytoskeleton ........................................... 199 Chapter 5. Cellular Adhesion and Extracellular Matrix ..................... 205

Section IV: Genetics Chapter 1. Mendelian Inheritance .................................... 213 Chapter 2. Chromosomal Abnormalities ............................... 223 Chapter 3. Linkage ............................................... 237 Chapter 4. Population Genetics ...................................... 243

Section V: Microbiology Chapter 1. Introduction ............................................ 251 Chapter 2. Bacteriology Overview .................................... 255 Chapter 3. Bacteriology: Gram-Positive Cocci .......................... 267 Chapter 4. Bacteriology: Gram-Positive Bacilli .......................... 277 Chapter 5. Bacteriology: Gram-Negative Organisms ..................... 283 Chapter 6. Bacteriology: Anaerobes .................................. 299 Chapter 7. Bacteriology: Mycobacteria and Actinomycetes ................ 303 Chapter 8. Bacteriology: Rickettsiae and Chlamydiae .................... 309 Chapter 9. Bacteriology: Spirochetes ................................. 315 Chapter 10. Bacteriology: Mycoplasma and Ureaplasma ................. 321

viii

meClical

----------- ----- - - - - - - -

Chapter 11. Virology .............................................. 323 Chapter 12. Mycology ., ........................................... 343 Chapter 13. Protozoa .............................................. 351 Chapter 14. Helminths ............................................ 359 Chapter 15. Antimicrobial Agents .................................... 365

Section VI: Immunology Chapter 1. Basic Immunology ....................................... 405 Chapter 2. Clinical Immunology ..................................... 449 Index ............. , .. , .......... " ...... , .. , .. " .......................... 483

meClical

ix

SECTION I

Biochemistry

Acid-Base Equilibrium and Buffering Homeostasis requires that the pH of biologic fluids be maintained at a constant and characteristic value. The control of pH in body fluids and subcellular compartments is achieved by a number of biologic buffers. The most important buffers in physiologic systems are bicarbonate, phosphate, and proteins. Buffers consist of a weak acid and its conjugate base that are in equilibrium and are able to donate and accept protons from the surrounding medium. The relationship between pH, pKa, and the concentration of the conjugate acid and base of a buffering system is described by the HendersonHasselbalch equation. Disturbances in the acid-base balance can lead to acidosis (a plasma pH less than 7.35) or alkalosis (a plasma pH greater than 7.45). This chapter reviews the concepts underlying acid-base equilibrium, the pH of various body fluids, and the properties of the buffer systems that resist fluctuations in pH.

ACID-BASE EQUILIBRIUM The most appropriate biochemical definition for acids and bases is that acids are proton donors and bases are proton acceptors. Many important compounds found in biologic fluids, including amino acids, lactic acid, and acetoacetic acid, are weak acids. A. Properties of weak acids. Weak acids, in contrast to strong acids such as Hel or H 2S04, are not completely dissociated under the conditions that exist within biologic systems. The undissociated form (HA) that retains the proton is known as the conjugate acid, whereas the corresponding deprotonated form (A-) is a conjugate base that can accept a proton to recreate the acid. The reaction that describes the dissociation of a weak acid is shown in Figure I-l-l.

HA

~,============~'

conjugate acid

H+

+ conjugate base

Figure 1-1-1. Dissociation of a weak acid.

KAPLlIf . ._ I me 1C8

3

Biochemistry

B. The Henderson-Hasselbalch equation shown in Figure 1-1-2 describes the relationship

between pH and the concentration of a weak acid and its conjugate base. In this equation, pH is defined as the negative log of [H+], and pKa is defined as the negative log of the . . constant Ka' were h Ka = [H+] [A-] . For any weak'd K'IS a constant t h at · d IssoClatlOn [HA] aCl, th epa

Note

is characteristic of that particular ionizing group. The Henderson-Hassselbach equation is

A corollary of the equation: When the pH is greater than the pKa, the base (A-) form is dominant; when the pH is less than the PKa, the acid (HA) form is dominant.

useful because it provides a meaningful definition of pKa and permits calculation of the amounts of the undissociated acid and its conjugated base at any pH.

Figure 1-1-2. Henderson-Hasselbalch equation.

1. A functional definition of pKa can be deduced from the Henderson-Hasselbalch equation. The pKa of any ionizing group can be defined as the pH where the concentrations of the acid (HA) and its conjugate base (A-) are equal. When the pH is equal to the pKa of the acid, the concentrations of HA and A-are equal. Inspection of the equation indicates that when [HA] = [A-J, the last term of the equation becomes zero, and the equation reduces to pH = pKa. 2. The logarithmic nature of the Henderson-Hasselbalch equation indicates that small changes in pH will bring about large changes in the relative concentrations of HA and A-. These effects are summarized in Table I-I-I, where lactic acid, having a pKa of 3.8, is considered as an example of a weak acid. The same principles apply for any weak acid, i.e., a change of 1 pH unit results in a lO-fold change in the ratio of conjugate acid to conjugate base. Table 1-1-1. Effect of pH on the relative amounts of lactic acid and lactate. pH 1.8 12.8 pKa 3.8 4.8 5.8

[Lactic Acid] I [Lactate-] 100 101 1 0.1 0.01

TITRATION AND BUFFERING PROPERTIES OF WEAK ACIDS Titration can be defmed as the addition of a strong base to a weak acid (or a strong acid to a weak base) for the purpose of buffering the solution, i.e., maintaining a constant pH. A. Titration curve. The addition of a strong base such as sodium hydroxide to a weak acid may be written as:

4

KAPLA~. I meulca

Acid-Base Equilibrium and Buffering

As NaOH is added, the protons released by the weak acid combine with hydroxide to form water. The continued addition of sodium hydroxide progressively decreases the [H+J and increases the pH. A graph of the titration of a weak acid with a strong base is shown in Figure 1-1-3.

---------- ~--z-~--~h7'77T

Buffering range

HA

o

0.5

1.0

NaOH (equivalents) Figure 1-1-3. Titration curve for a weak acid.

At the midpoint in the titration, half of the acid has been neutralized and, therefore, [HAJ = [A-J. The Henderson-Hasselbalch equation shows that when [HAJ = [A-J, the pH of the solution is equal to the pKa of the weak acid. B. Buffering capacity. Buffers are mixtures of weak acids and their conjugate bases. The capac-

ity of a buffer, to resist change in pH is dependent on two factors: the concentration of the buffer, and the pH at which it is used. A buffer is most effective when it is used in a pH range near the pKa. It is clear from Figure 1-1-3 that in the pH range defined by 1.0 pH unit above and below the pKa' the addition of either strong acid or strong base will result in minimal change in the pH.

Note The best buffering occurs when pKa = pH, resulting in 50% of acid and 50% of base. This system can then handle either an addition of base or an addition of acid.

PHYSIOLOGICALLY IMPORTANT BUFFERING SYSTEMS The three most important buffering systems in biologic fluids are proteins, bicarbonate, and phosphate. The conjugate acid-base pairs and the pKa values for these buffers are summarized in Table 1-1-2. The major buffer in plasma and interstitial fluid is bicarbonate, whereas protein and organic phosphate esters are the major buffers of intracellular fluid. A. Protein buffering systems. The cytosol of cells contains high concentrations of proteins with amino acid side chains that are weak acids and bases. These side chains impart great buffering capacity to proteins. The acidic amino acids (glutamic and aspartic acids) and the basic amino acids (lysine, histidine, and arginine) all contain ionizable side chains (discussed in the next chapter). However, histidine is the only amino acid with good buffering capacity at physiologic pH. The imidazole side chain of histidine has a pKa that ranges from 5.6-7.0, depending on its microenvironment within the protein.

Bridge to Physiology Histidine is frequently found in the catalytic site of enzymes where the imidazole ring can donate or accept protons in the formation and breaking of bonds.

meCtical

5

Biochemistry

Table 1-1-2. Properties of major physiologic buffers. Buffering System Protein systems: Histidine side chains

5.6-7.0

Bicarbonate system: HC03-ICOz

6.1

Phosphate systems: HP042-/HzP04-

6.8

Organic phosphate esters

6.5-7.5

The buffering reaction for proteins is shown in Figure 1-1-4, in which the imidazole side chain of histidine can reversibly donate and accept protons. The presence of multiple histidine side chains within each protein molecule contributes to the buffering capacity of the protein. Histidine plays a key role in making hemoglobin an excellent buffer in red blood cells.

O rotein

Bridge to Physiology Acidosis, a plasma pH below 7.35, can result from the accumulation of acids such as lactic acid, acetoacetic acid, and B-hydroxybutyric acid (resulting in a metabolic acidosis), or from hypoventilation that results in the accumulation of CO 2 (respiratory acidosis). Alkalosis, a plasma pH above 7.45, can result from a loss of stomach acids due to excessive vomiting (metabolic alkalosis) or from hyperventilation (respiratory alkalosis). Acidosis and alkalosis are discussed in greater detail in the Respiratory Physiology and Renal Physiology sections.

6

KAPLAN"d_

me lea

I

CH

I

NH

=

CH

I

+ NH

H

+

+

orotein CH = I NH

"~

"

CH

CH

CH

I

N

~

Figure 1-1-4. Protein buffering system. B. Bicarbonate buffering system. The bicarbonate-C0 2 system is the most important buffer in

maintaining the pH of blood plasma and interstitial fluid at its normal value of 7.4. Figure I -1-5 summarizes the properties of this buffering system. Carbonic acid (H 2 C03 ) is the proton donor, bicarbonate anion (HC0 3-) is the proton acceptor, and the pKa for this reaction is 6.1. The strength of this buffering system lies in the ability of carbonic acid to be converted to carbon dioxide.

pKa = 6.1

Figure 1-1-5. The bicarbonate/C0 2 buffering system.

C. Phosphate buffering system. Intracellular fluids contain high concentrations of inorganic

phosphate and many organic phosphate esters that contribute significantly to the buffering power of the cytosol. The phosphate buffering system is of little importance in plasma and interstitial fluid because of the low concentrations of phosphates in extracellular fluids. The phosphate buffering system consists of H 2 P0 4- as the proton donor and HPoi- as the proton acceptor. The pKa of 6.8 is sufficiently close to the normal intracellular pH to make it an ideal buffer in those fluids that contain high concentrations of phosphates, such as red blood cells and kidney tubules.

- - - - - - - - - -

-------

- - - -

Proteins, Enzymes, and Coenzymes Proteins are linear polymers of amino acids, each having distinct chemical and structural properties. They are the largest class of molecules found in living systems and are responsible for most of the diverse functions of the cell. They act as transport proteins, storage proteins, hormone receptors, structural proteins, regulatory proteins, and enzymes. Each protein has a unique amino acid sequence that determines its precise three-dimensional conformation and specifies its function. The most versatile class of proteins is enzymes, which act as biologic catalysts, enhancing the rate of chemical reactions occurring in cells. Most enzymes catalyze only one reaction, and their catalytic activities may be stringently regulated, allowing the cell to control and coordinate its metabolic pathways. The kinetic parameters, Km and Vmax, are constants that describe the catalytic properties of enzymes. The function of many enzymes requires a cofactor-a metal or a small organic coenzyme derived from a vitamin precursor. This chapter reviews the structure and properties of amino acids that are found in proteins, the kinetic and regulatory properties of enzymes, and the relationship between coenzymes and their vitamin precursors.

AMINO ACIDS: THE BUILDING BLOCKS OF PROTEINS A. Amino acid structure. The building blocks for proteins are the 20 common amino acids that are encoded in the DNA of the cell. 1. General structure. Nineteen of the twenty common amino acids can be represented by the structure shown in Figure 1-2-1. They all have a central carbon atom attached to a carboxyl group, an amino group, and a hydrogen atom. The amino acids differ from one another only in the chemical nature of the side chain (R). The only amino acid that is not described by this structure is proline, an imino acid in which the side chain forms a cyclic structure with the amino group (Figure 1-2-2). NH2

I R-Ca-COOH I H

In a Nutshell Amino Acids with Hydrophobic Side Chains

Figure 1-2-1. General structure of a-amino acids.

2. Classification. The amino acids can be classified as either hydrophobic or hydrophilic, depending on the ease with which their side chains interact with water. This particular method of classification is especially useful when considering amino acids as the building blocks of proteins. The properties of amino acid side chains influence how the protein folds into more compact structures. In general, proteins fold so that the hydrophobic side

Aliphatic Ala Val Leu lie Pro

Aromatic Phe Tyr Trp

meClical

7

Biochemistry

In a Nutshell

chains are in the interior of the molecule, where they are protected from water, and the hydrophilic side chains are on the surface.

Amino Acids with Hydrophilic Side Chains Positive Negative Lys Asp Arg Glu His

a. Hydrophobic amino acids have side chains with aliphatic groups or aromatic ring structures (Figure 1-2-2).

Neutral Ser

b. Hydrophilic amino acids have side chains that contain 0, N, or S atoms. Some of the hydrophilic side chains are charged at physiologic pH. The acidic amino acids (aspartic and glutamic acids) have carboxyl groups that are negatively charged, whereas the basic amino acids (lysine, arginine, and histidine) have nitrogen atoms that are positively charged. Other hydrophilic amino acids with uncharged side chains contain hydroxyl groups (serine and threonine), a sulfur atom (cysteine), or amide groups (asparagine and glutamine). The structures of the R groups for the hydrophilic amino acids are shown in Figure 1-2-3. Methionine, tyrosine, cysteine, and glycine may be found either in the interior or on the surface of globular proteins.

Thr Cys Met Asn Gin

Nonpolar, Aliphatic Side Chains

coo· + I H3N -C-H

GJ Glycine

coo· I

H3N+-C --ti

coo· I

H3N+-C-H

~ Alanine

CH2

Leucine

+

H3N~-H

CH CH3/

'CH3

Valine

H3N -C -H

COO· +

I

H3N -C-H

oQ OH

Phenylalanine

I

I

CH3 Isoleucine

Proline

Figure 1-2-2. The hydrophobic amino acids.

8

I

H3~-C-H

CH

"CH 3

coo·

I

I

H-C-CH3

CH 3/

coo·

coo·

CH2

I

Aromatic Side Chains

liP LIN"iIme leaI

- - - - - - - - -

Tyrosine

Tryptophan

Proteins, Enzymes, and Coenzymes

Positively charged R groups

Polar, uncharged R groups

coo +

coo

I

+

H3 N - C -H

I

H3N -C-H

I

I

CH 2

H-C-OH

CH 2

CH 3

I

I

I

CH 2

I

Serine

Threonine

Cysteine

Methionine

Asparagine

Glutamine

NH

I

+

C=NH 2

I

NH2 Lysine

Arginine

Histidine

Negatively charged R groups

coo +

I

H3 N - C-H

I CH 2

I

COO

Aspartate

Glutamate

Figure 1-2-3. The hydrophilic amino acids.

3. Modified amino acids. In addition to the 20 common amino acids that are encoded by DNA, proteins often contain other amino acids that are generated by modification of the common amino acids after the protein has been synthesized. This process is called posttranslational modification. a. Cystine is formed in proteins by the reaction of two cysteine side chains to form a disulfide linkage. It is found most frequently in extracellular proteins where the disulfide bond stabilizes the three-dimensional structure of the protein. b. Hydroxyproline is formed in an oxygen-dependent hydroxylation reaction that occurs in t1broblasts. It is found in collagen, where it stabilizes the triple helical structure. c. Phosphotyrosine, phosphoserine, and phosphothreonine are formed by transferring

phosphate from ATP to the hydroxyl group of serine, tyrosine, or threonine. These amino acids are found in many enzymes and proteins, where they serve as regulatory signals.

In a Nutshell • S-S bridges:

Cystine

• Collagen triple helix:

Hydroxyproline

Phosphotyrosine • Signal transduction: Phosphoserine Phosphothreonine

meCtical

9

Biochemistry

B. Properties of amino acids. The properties of proteins are influenced by the properties of

Note There are other amino acids, like ornithine and citrulline, that play important roles in the cell but that are not building blocks for protein synthesis.

their constituent amino acids. In particular, the stereochemistry and ionic properties of amino acids have an impact on the structure and/or properties of the protein. 1. Stereochemistry. The a-carbon atom of all amino acids except glycine is linked to four

different chemical groups, making the a-carbon atom an asymmetric center. As shown in Figure 1-2-4, an asymmetric center has two stereoisomers (enantiomers) that are mirror images of each other and are designated as D- and L-amino acids. Only L-amino acids are incorporated into proteins.

coo-

cooI

I

H-C- NH3+

I

+H N-C-H

I

3

CH3

CH 3 D-alanine

L-alanine

Figure 1-2-4. Stereoisomers of an a-amino acid.

2. Ionic properties. Depending on the pH, an amino acid may have no net charge, or it may have either a positive or a negative charge. The pH at which a molecule is electrically neutral is defined as the isoelectric point (pI). The equilibrium between the ionic forms of an amino acid is shown in Figure 1-2-5. The pKa values for the a-carboxyl and a-amino groups are approximately 2.1 and 9.8, respectively. At a pH below the pKa' the protonated species predominates, whereas at a pH above the pKa' the deprotonated species predominates. The pI for the amino acid shown in Figure 1-2-5 is the average of the two pKa values. At neutral pH, the species that has both positive charge and negative charge is called a zwitterion.

+NH3

+NH3 NH2 pK a = 2.1 I pK a 9.8 \ ....R -CH -COO ~ ...==~"" R-CH-COOR -CH -COOH ...

I

Net charge = + 1 pH = 1

Net charge = 0 pH = 7

Net charge = -1 pH = 11

Figure 1-2-5. The ionic equilibrium for an a-amino acid.

The acidic and basic amino acids also have ionizing groups in their side chains. Aspartic and glutamic acids have side chain carboxyl groups with a pKa of approximately 4.0 and are negatively charged at physiologic pH. Lysine and arginine have side chains with protonated nitrogen atoms having pKa values of approximately 10 and 12, respectively, and are positively charged at physiologic pH. Histidine has an imidazole side chain with a pKa of approximately 6.0, a value sufficiently close to the physiologic pH to allow some of the histidine side chains to be positively charged, whereas others have no charge. The ratio of positively charged histidine side chains to uncharged side chains at any pH can be calculated from the Henderson-Hasselbalch equation (described in the previous chapter).

10

KAPLAlfdme leaI

Proteins, Enzymes, and Coenzymes

PROTEINS Each protein has a unique three-dimensional structure dictated by its amino acid sequence and is responsible for the highly specific function of the protein. A. Structural features. The amino acids in a protein are linked together by peptide bonds in which the a-carboxyl of one amino acid is linked to the a-amino group of another amino acid (Figure 1-2-6). The peptide (amide) bond has partial double-bond character, making it planar and rigid, thereby imposing limitations on higher orders of structural organization in a protein. 1. Primary structure. The sequence in which the amino acids occur in the polypeptide is

defined as the primary structure. This sequence is encoded by the DNA, and it determines how the protein folds into a more compact three-dimensional structure. 2. Native conformation. The final structure that a particular protein assumes proceeds through several levels of organization. In contrast to the primary structure, which is stabilized by strong covalent bonds, higher orders of structure are stabilized by numerous weak non covalent interactions.

+

H[JR I

H N-C 3

I

R

II

C-NH

0

I

II

CHlEJNH CH-C-COO-

II

I

0

R

Figure 1-2-6. The peptide bond.

a. Secondary structure in proteins is organized around the polypeptide backbone and is stabilized by large numbers of hydrogen bonds formed between the amide hydrogen atom of one peptide bond and the carbonyl oxygen atom of another. Proteins contain two major types of secondary structure: a-helical structure, which is stabilized by intrachain hydrogen bonds, and 13-sheet structure, which is stabilized by interchain hydrogen bonds. In some fibrous proteins, the highest order of structure is secondary. These proteins are elongated asymmetrical proteins that frequently function as structural components of cells and tissues. Simple combinations of a few secondary structural elements are called motifs and are often repeated multiple times in proteins. An example of a motif would be a 13-a-13 loop motif that can be used to connect two parallel13 strands. The a-helix in the middle of the two 13 strands allows for the reversal of direction. b. Tertiary and quaternary structure. Segments of secondary structure in globular proteins associate with one another and fold into a tertiary structure that is stabilized by non covalent interactions between amino acid side chains. The fundamental unit of tertiary structure is the domain, a part of the polypeptide chain that folds into a stable tertiary structure and is encoded by an exon in DNA. Proteins that are composed of more than one polypeptide chain have quaternary structure. Both tertiary and quaternary structures are stabilized by ionic and hydrophobic interactions and by hydrogen bonding between side chains. 3. Denaturation is defined as the loss of native conformation, resulting in a random coil that has little, if any, of the biologic activity of the native protein. Denaturation may be caused by heat, extremes in pH, or detergents.

In a Nutshell 1° structure: • Amino acid sequence

2° structure: • a-helix

• 13-sheet 3° structure: • Overall protein

structure • How a-helix and 13-sheet fold with respect to each other 4° structure: • Mutiple polypeptide chains

• How chains fold with

respect to each other

meClical

11

Biochemistry

B. Function. Proteins perform most of the important functions of the cell. Many of the pro-

teins in skin, bone, muscle, and hair perform structural roles. Other proteins carry out the dynamic functions of the cell. Many plasma proteins transport small molecules such as iron or oxygen; immunoglobulins and clotting factors participate in defense functions; hormones, hormone receptors, and transcription factors play critical regulatory roles in the cell; and enzymes function as biologic catalysts.

ENZYMES Virtually all biologically important reactions are catalyzed by enzymes. Most of these reactions occur under very mild conditions of temperature and pH that are compatible with living organisms. In the absence of enzymes, reactions in the cell would proceed at insignificant or undetectable rates. Enzymes differ from inorganic catalysts in their specificity, catalytic efficiency, and regulatory properties. A. Classification. Enzymes are divided into six different classes on the basis of the types of reactions they catalyze (Table 1-2-1). All of the thousands of different reactions occurring in cells fall into one of these six types of reactions. B. Specificity. The high specificity of enzyme-catalyzed reactions occurs because enzymes have

active sites composed of a small number of amino acid side chains. The side chains come together to form a three-dimensional site on the surface of the enzyme that is complementary to the structure of the substrate. Some of the amino acid side chains in the active site participate in binding the substrate to the enzyme; others act as catalytic groups and enhance the rate of the reaction by acting as acids, bases, or nucleophiles.

Table 1-2-1. The six classes of enzymes.

12

KAPLAlfdme leaI

Class

Type of Reaction

Oxidoreductases

Oxidation-reduction reactions Frequently use coenzymes NAD+, FAD, NADP+' or O 2 as electron acceptors (dehydrogenase, oxidase, reductase)

Transferases

Transfer of a chemical group from a donor to an acceptor Groups transferred include amino, carboxyl, acyl, glycosyl, phosphoryl (transaminase, kinase)

Hydrolases

Cleavage of a bond between carbon and some other atom by the addition of water (protease, phosphatase, amylase)

Lyases

Nonhydrolytic cleavage of carbon-carbon, carbon-sulfur, and some carbon-nitrogen bonds (aldolase, decarboxylase, dehydratase)

Isomerases

Interconversion of isomers (epimerase, mutase)

Ligases

Formation of bonds between carbon and oxygen, nitrogen, or sulfur atoms in reactions that require energy (carboxylase, thiokinase)

Proteins, Enzymes, and Coenzymes

C. Catalytic properties. The energy profile of a chemical reaction, as it proceeds from substrate

to product, is shown in Figure 1-2-7. The highest point on the curve is the energy of the transition state, an intermediate whose properties resemble both the substrate and the product. The energy barrier created by the transition state is also known as the activation energy (Act) of the reaction. To initiate the reaction, energy must be expended to overcome the energy barrier. The rate of a reaction is inversely proportional to the magnitude of the activation energy. As shown by the dotted line in Figure 1-2-7, enzymes increase the rate of a reaction by decreasing the activation energy barrier. They have no effect on the equilibrium constant for the reaction, which is related to ~G, the energy difference between the products and substrates.

Transition state

~-.-__ ~_ ~ minus enzyme

>OJ .....

\3s

Q)

~

~-------

" -------------- - --

.... ;

Q)

c

~G* = G TS - G s

/",-

Q)

"

LL

~G

plus enzyme

= Gp-G s

Reaction progress Figure 1-2-7. Energy profile for a catalyzed and uncatalyzed reaction.

D. Kinetic properties of enzymes. Kinetics is the study of the rate at which chemical reactions occur. 1. Factors affecting the rate of enzyme-catalyzed reactions include temperature, pH, enzyme concentration, and substrate concentration. Typical rate responses to these factors are shown in Figure 1-2-8. a. Temperature. The rate of most reactions increases approximately twofold with a 1O.ODC increase in temperature. For enzyme-catalyzed reactions, however, there is an optimum temperature beyond which the rate rapidly decreases due to denaturation of the enzyme. b. pH. The optimal activity of most enzymes occurs between pH 5 and 9. The shape of the rate-versus-pH curve reflects different ionization states for specific amino acid side chains that are required for substrate binding or for catalysis.

mectical

13

Biochemistry

Mnemonic STEP up the reaction rate: Substrate concentration Temperature Enzyme concentration

c. Enzyme concentration. The rate of an enzyme-catalyzed reaction is directly proportional to the concentration of enzyme provided the substrate is present in concentrations sufficient to saturate the binding sites. d. Substrate concentration. At very low concentrations of substrate, first -order kinetics are observed, with the rate being directly proportional to [S]. When the concentration of substrate is sufficiently high that all of the binding sites are occupied, zero-order kinetics are seen, with the rate being independent of [5]. 2. The Mic:haelis-Menten equation is an expression that quantifies the relationship between the rate of an enzyme-catalyzed reaction and the substrate concentration. In deriving an equation for the curve shown in Figure 1-2-SD, it is assumed that the limiting rate observed at an infinitely high substrate concentration is due to a finite number of sites on the enzyme and that when all of the sites are occupied, the maximum rate is obtained. The Michaelis-Menten expression for velocity is illustrated by the following equation:

pH

In this expression, Vmax and Km are constants that are characteristic of the enzyme. They are defined as follows: a. The maximum velocity, Vmax' is the rate obtained when all of the enzyme is present as an E·S complex, with substrate bound to the active site. The Vmax increases as the concentration of enzyme increases.

In a Nutshell • Vmax: theoretical rate when all enzymes are working

• Km: substrate concentration at which the reaction is running at half of the maximum rate (where V= 1/2 Vmax>

b. The Km is the substrate concentration that is required to achieve half of the maximum velocity and is independent of the enzyme concentration. These relationships are shown in Figure 1-2-9. Rough estimates of the Vmax and Km values can be obtained from this figure. However, values for both Vmax and Km obtained from this type of graph are inherently subject to large errors because of the shape of the curve; the value for Vmax must be extrapolated from an infinitely large value for [S]. Much more accurate values of these two kinetic parameters can be obtained from secondary plots, as described below.

A.

>

~

B.

0

~

Temp C.

pH

D.

zer~ order

> Q)

iii '-

~order Enzyme Concentration

Substrate Concentration

Figure 1-2-8. Factors affecting the rate of enzyme-catalyzed reactions.

14

KAPLA~.

I

meulCa

Proteins, Enzymes, and Coenzymes

Vm~r-------------------~====~

t

>

0.5

Vm~ 1----7/'

[Sl~

Figure 1-2-9. Dependence of rate on substrate concentration.

3. Lineweaver-Burk plot. A more useful graph for obtaining values of Km and Vmax can be obtained by taking the reciprocal of the Michaelis-Menten equation and rearranging it to give the Lineweaver-Burk equation: 1

Km

1

1

V

Vmax

5

Vmax

Note Remember that the equation for a straight line is:

-=--e-+--

As shown in Figure I-2-lO, this equation describes a straight line when IN is plotted against 1/[S]. The slope of the line is equal to KmNmax, the intercept of the line on the y-axis is equal to l/Vmax' and the intercept on the x-axis is equal to -l/Km.

y=mx+b, where m = slope and b = y-i ntercept.

In a Nutshell Lineweaver-Burk Plot x-axIs: y-axIs: slope: x-intercept: y-intercept:

o

1

[8]

Note • Km

Figure 1-2-10. Lineweaver-Burk plot.

4. Significance of Km and Vmax' Each of these kinetic constants reflects an intrinsic property of the enzyme.

l/[S] 1/V = l/rate Km/Vmax -l/Km l/Vmax

oc

l/affinity

. .i Km ~ i

substrate

affinity Km ~ .i substrate affinity

· i

meClical

15

Biochemistry

a. Km is related to the affinity of the enzyme for the substrate. The lower the value for Km , the higher the affinity of the enzyme for the substrate, and vice versa. Frequently, the Km value of an enzyme for its substrate is approximately equal to the substrate concentration found in the cell. b. Vmax is related to the efficiency with which substrate is converted to product when all of the active sites are occupied. Some enzymes, such as catalase, are highly efficient, with the rate of product formation approximating the rate of substrate binding; others, like RNA polymerase, are much less efficient at converting substrate to product. E. Inhibitors of enzyme activity. Much of toxicology and pharmacology is based on the principles of enzyme inhibition. Many metabolic poisons, such as insecticides and nerve gases, are enzyme inhibitors. Similarly, many drugs are inhibitors of key enzymes in metabolic pathways and often have structures similar to the normal enzyme substrate. There are two large classes of enzyme inhibitors: reversible and irreversible. 1. Reversible inhibitors alter the kinetic properties of an enzyme by binding noncova-

In a Nutshell Competitive Inhibition

• Vmax is unchanged • Slope increases • y-intercept is unchanged

lently to the enzyme through multiple interactions with amino acid side chains. The effect of a reversible inhibitor is removed by its dissociation from the enzyme. There are three types of reversible inhibitors: competitive, noncompetitive, and uncompetitive. a. Competitive inhibitors are structural analogs of the substrate and compete with the substrate for binding to the active site. The effect of a competitive inhibitor can be overcome by increasing the concentration of substrate. The Km for the substrate is increased (i.e., because both inhibitor and substrate are competing for the same site, more substrate is needed to reach half-maximum velocity). Vmax remains unchanged. Competitive inhibitors are easily identified by Lineweaver-Burk plots (Figure 1-2-11), in which the slope is increased but the intercept on the 1IV axis is unchanged. The intercept on the x-axis is shifted closer to zero (corresponding to an increase in Km).

• x-intercept shifts to the right

In a Nutshell Noncompetitive Inhibition • Km is unchanged

• -tV max • Slope increases • y-intercept shifts upward • x-intercept is unchanged

16

meclical

o

1

[8]

Figure 1-2-11. Lineweaver-Burk plot of competitive inhibition.

b. Noncompetitive inhibitors bind to some site other than the active site and are not structural analogs of the substrate. The Vmax of the reaction is decreased, but the Km for the substrate remains unchanged. The effect of a noncompetitive inhibitor cannot be overcome by increasing the substrate concentration. In a Lineweaver-Burk plot (Figure 1-2-12), the intercept on the 1IV axis is increased (corresponding to a decrease in Vmax ), and the intercept on the 11[5] axis is unchanged.

Proteins, Enzymes, and Coenzymes

In a Nutshell Uncompetitive Inhibition •

Km and Vmax are changed

• Slope remains the same • x-intercept shifts to the left

o

1

• y-intercept shifts upward

[8] Figure 1-2-12. Lineweaver-Burk plot of noncompetitive inhibition.

c. Uncompetitive inhibitors bind directly to the enzyme substrate complex but not to

free enzyme. This binding causes a conformational change at the active site that renders the enzyme inactive. Both Vmax and Km are changed. Both Vmax and Km decrease, but the slope of the inhibited reaction is parallel with the slope of the uninhibited reaction (Km/Vmax)' Lineweaver-Burk plots at various concentrations of the inhibitor will be a series of parallel lines. 2. Irreversible inhibitors covalently bind to the enzyme, resulting in permanent inactivation of the enzyme. Some irreversible inhibitors are referred to as "mechanism-based inhibitors" or "suicide substrates" because they bind to the active site and mimic the enzyme by beginning to undergo catalysis; however, the catalytic cycle is never completed because they become covalently linked to the enzyme. The effect of irreversible inhibitors on the kinetic parameters of an enzyme is identical to that of a noncompetitive inhibitor. F. Regulation of enzyme activity. Homeostasis requires that the rate of metabolic pathways be carefully regulated and coordinated with one another. This is typically achieved by the regulation of one or two key enzymes in each pathway. The key enzymes that are regulated usually catalyze either the rate-limiting reaction in the pathway or a reaction that is essentially irreversible. There are three major mechanisms for controlling the activity of enzymes. 1. Allosteric regulation. The activity of allosteric enzymes is regulated by the reversible bind-

ing of an effector molecule to a site other than the active site. Substrate saturation curves for allosteric enzymes are usually sigmoidal (Figure 1-2-13). Allosteric effectors can be either positive (activators) or negative (inhibitors) and act by altering either the K m , Vmax' or both. Activators either decrease the Km or increase the Vmax' whereas inhibitors increase the Km or decrease the Vmax' Common types of effectors include end products of pathways (heme, cholesterol, purine, and pyrimidine nucleotides) or molecules that reflect the energy state of the cell (ATP, ADP, AMP, NADH, NAD+, acetyl-CoA).

Clinical Correlate Lead toxicity is caused by lead's inhibitory effect on a number of enzymes having essential sulfhydryl groups in their active sites. The sulfhydryl groups react covalently with lead, resulting in irreversible inactivation of the enzyme. Two enzymes in the pathway of heme synthesis (aminolevulinic acid dehydratase and ferrocheletase) are inactivated by low levels of lead, resulting in decreased synthesis of heme and hemoglobin and increased excretion of aminolevulinic acid. Treatment with chelators such as penicillamine decreases the toxicity by forming nontoxic complexes with lead.

In a Nutshell Allosteric Regulation • Activators: J, Km or i Vmax

i

Km or

J, Vmax

KAPLA~. I meulca

17

• Inhibitors:

Biochemistry

Clinical Correlate Growth factors (i.e., hormones) activate receptors via tyrosine phosphorylation; the newly phosphorylated tyrosine can induce signal transduction in the cell. In many cancers, this pathway is often disregarded and the growth signal is always on, causing unregulated cell proliferation.

v

Figure 1-2-13. Substrate saturation curve for an allosteric enzyme.

Clinical Correlate Patients with chest pain who are suspects for acute myocardial infarction (AMI) are evaluated by EKG and serial measurements of serum creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) isoenzymes. The CPKMB isoenzyme is a marker for cardiac tissue damage. In patients with AMI, the tissue damage results in a transient increase of CPK-MB measurable 6-8 hours after the infarction, peaking between 12 and 24 hours, and returning to normal within 48-72 hours. If the patient is admitted 24 hours after the AMI, LDH isoenzyme analysis is performed. Following an AMI, the total serum LDH is elevated and the relationship between LDH1 and LDH2 is inverted, so that LDH1 > LDH2. The change in LDH isoenzymes occurs within 24-36 hours post-AMI.

18

meClical

2. Covalent modification. The activity of many enzymes is regulated by a phosphorylation/dephosphorylation cycle in which a specific serine, threonine, or tyrosine side chain becomes modified (Figure 1-2-14). The addition and removal of the phosphate group are catalyzed by a family of protein kinases and protein phosphatases that respond to hormonal stimulation of cells. Phosphorylation can increase or decrease the activity of an enzyme, or it can alter the regulatory properties.

Protein kinase

0

00-p~=

0H

Altered activity Protein phosphatase

Figure 1-2-14. Regulation of enzyme activity by covalent modification.

3. Induction and repression of enzyme synthesis. Because enzyme activity is directly proportional to the amount of enzyme present, one way to regulate enzyme-catalyzed reactions is to alter the rate at which enzymes are synthesized. This type of regulation is frequently mediated by steroid or thyroid hormones that act in the nucleus to increase or decrease the rate of transcription, and, secondarily, protein synthesis. G. Isoenzymes. Isoenzymes are different proteins that catalyze the same reaction but have different catalytic and regulatory properties and frequently differ in tissue and/or organelle specificity. The appearance of tissue-specific isoenzymes in plasma is of diagnostic value in identifying sites of tissue damage.

Proteins, Enzymes, and Coenzymes

COENZYMES AND VITAMINS Most enzymes that catalyze transfer reactions or oxidation/reduction reactions require a coenzyme that serves as an intermediate carrier of some specific functional group. Coenzymes are small organic molecules that are more stable than proteins. Coenzymes are derived from vitamins. Vitamins cannot be synthesized de novo by human tissues and must therefore be supplied by the diet. In addition to dietary sources, some vitamins are also synthesized by bacteria normally present in the intestinal flora. Vitamins may be classified as either water soluble or fat soluble. All of the water-soluble vitamins and some of the fat-soluble vitamins serve as precursors for coenzymes. Other fat-soluble vitamins control the rate of transcription of specific genes, thereby influencing the rate of enzyme-catalyzed reactions by altering the amount of enzyme present in the cell. A. Water-soluble vitamins 1. Niacin, also known as vitamin B3 or nicotinic acid, is present in whole grains, meat, and

nuts. Up to 50% of the body's niacin supply can be derived from the amino acid tryptophan. Niacin is converted to nicotinamide, which is then incorporated into the coenzymes NAD+ and NADP+. These coenzymes are very important in both lipid and carbohydrate metabolism, where they act as carriers of hydride ions (two electrons and a proton) in oxidation and reduction reactions. NAD+ is generally involved in oxidative, catabolic pathways and is more concentrated in mitochondria than in cytosol; NADPH is used in reductive, anabolic pathways and is found primarily in the cytosol. 2. Riboflavin, vitamin Bz, is present in organ meat, whole grains, and dairy products. The two coenzymes derived from riboflavin are FMN and FAD, both of which act as carriers of hydrogen atoms in oxidation and reduction reactions. These coenzymes are important in the oxidation of carbohydrates, lipids, and amino acids and are found primarily in the mitochondria. Human riboflavin requirements increase with increased protein use during growth periods, pregnancy, lactation, and wound healing. Patients with a deficiency of riboflavin develop lesions of the lips, mouth, skin, and genitalia. 3. Thiamine, vitamin Bl> is present in meat, beans, peas, and grains. The coenzyme derived

from thiamine is thiamine pyrophosphate, which functions in oxidative decarboxylation of a-ketoacids. The four major enzymes requiring thiamine pyrophosphate are pyruvate dehydrogenase, a-ketoglutarate dehydrogenase, branched-chain a-keto acid dehydrogenase, and transketolase. 4. Pyridoxine, vitamin B6, is present in many foods, including most meats and vegetables. The coenzyme derived from this vitamin is pyridoxal phosphate, which acts as a carrier of amino groups in transamination, decarboxylation, racemization, and dehydration reactions. Pyridoxine deficiency may develop during pregnancy, alcoholism, with prolonged exposure to isoniazid or penicillamide therapy, and in women on oral contraceptives. Both a deficit and excess of pyridoxine may lead to peripheral neuropathy and dermatitis.

Clinical Correlate Side effects of exogenous niacin use include vasodilatation, flushing, and itching. Deficiency results in pellagra, producing the clinical triad of the three Os: diarrhea, dermatitis, and dementia. (If untreated, a fourth possible "0" is death.)

Clinical Correlate Mild thiamine deficiency leads to peripheral neuropathy (dry beriberi). More severe vitamin depletion leads to high-output cardiac failure (wet beriberi) and the dementia, ataxia, and ophthalmoplegia of the W~nicke-Korsakoff syndr~ll}e

frequently seen in chronic alcoholics.

5. Pantothenic acid is widely distributed in foods, particularly in meats and grains. The coenzyme derived from this vitamin is coenzyme A, which acts as a carrier of acyl groups and is particularly important in lipid metabolism. The key feature of this coenzyme is a sulfhydryl group that forms a high-energy thioester linkage with the carboxyl group of fatty acids. Pantothenic acid deficiency is rare. 6. Biotin is present in many foods, including liver, grains, and eggs. It is also synthesized by intestinal bacteria. The conversion to a coenzyme simply requires that it be covalently linked to the appropriate enzymes. Biotin acts as a carrier of "activated carboxyl" groups for three key enzymes that catalyze carboxylation reactions. The enzymes and the pathways they participate in are: pyruvate carboxylase (gluconeogenesis), acetyl-CoA

meClical

19

Biochemistry

Clinical Correlate Folic acid deficiency is most commonly seen in pregnancy and alcoholism. Deficiency states can result in megaloblastic anemia and neural tube defects. Supplementation is recommended before and during pregnancy.

Clinical Correlate Vitamin C deficiency results in scurvy, characterized by poor wound healing and bone formation, gum changes, and petechiae.

carboxylase (fatty acid synthesis), and propionyl-CoA carboxylase (branched-chain amino acid catabolism). Biotin deficiency is rare. Excessive consumption of raw egg impairs biotin absorption because of the presence of a biotin-binding protein, avidin, in egg whites. Antibiotics that alter the intestinal flora can also lead to biotin deficiency. Symptoms of deficiency include alopecia, skin and bowel inflammation, and muscle pain. 7. Folic acid is present in liver, fresh fruit, and leafy green vegetables. The coenzyme form of this vitamin is tetrahydrofolic acid (THF), which acts as a carrier of one-carbon fragments in metabolism. THF carries one-carbon units at all stages of oxidation except carboxyl groups. The coenzyme is important in amino acid catabolism and in the synthesis of purines and pyrimidines. The donors of the one-carbon units are serine, histidine, glycine, and tryptophan; the acceptors are intermediates in the pathways of purine and pyrimidine nucleotide synthesis. 8. Vitamin C, or ascorbic acid, is found in fresh fruits and vegetables. This vitamin exists in both oxidized and reduced forms, with the reduced form being the active form. Vitamin C facilitates iron absorption from the gut and is also required in a number of hydroxyl ation reactions, including proline and lysine hydroxylation for collagen synthesis and dopamine hydroxylation in catecholamine synthesis. Vitamin C also serves as an antioxidant in cells. 9. Vitamin B12, or cobalamin, is synthesized exclusively by microorganisms but is conserved in animal tissues, where it is found in high concentrations in the liver and kidney. It is required as the coenzyme for two reactions in human biochemistry: the methylation of homocysteine to methionine, and the conversion of methylmalonyl-CoA to succinylCoA. The absorption of vitamin B12 requires intrinsic factor, a protein synthesized by parietal cells of the gastric mucosa. A deficiency of vitamin B12 is rare, with the most common cause being a defect in absorption. A deficiency of vitamin B12 leads to megaloblastic anemia, with or without an associated neuropathy due to widespread demyelination. B. Fat-soluble vitamins

Clinical Correlate

1. Vitamin A is supplied by several sources, including yellow and orange vegetables, liver, milk, and eggs. Vitamin A exists in three forms: retinal, retinol, and retinoic acid. Retinal acts as a cofactor for the protein opsin to form a rhodopsin complex, which acts as the light receptor in the visual process. Retinol and retinoic acid are required for growth, differentiation, and maintenance of epithelial cells; they bind to nuclear receptors and regulate the rate of transcription of specific genes, including the gene for keratin. Patients with fat malabsorption or celiac disease may become vitamin A deficient, producing night blindness and dryness of the conjunctiva, cornea, and skin (hyperkeratosis). Excessive amounts of this vitamin are also toxic to humans, producing joint pain, headache, and long-bone thickening.

A deficiency of vitamin D occurs with lack of sunshine or renal failure and leads to rickets in children and osteomalacia in adults. Excessive amounts of vitamin D lead to hypercalcemia and widespread calcification of soft tissues.

2. Vitamin D is found in high concentrations in fish oils, liver, and fortified milk. It can also be synthesized in human skin by ultraviolet irradiation of sterols. The vitamin is metabolically activated by sequential hydroxylation in the liver and kidney to produce the active form of the vitamin 1,25-(OH)z-Vit D. One of the major roles of vitamin D is to increase intestinal calcium and phosphate absorption. It binds to nuclear receptors and increases the rate of transcription of the gene coding for a protein that transports calcium from the lumen into the intestinal mucosal cell. It also acts in concert with parathyroid hormone to mobilize calcium from bone.

Bridge to Pharmacology Isotretinoin, a form of retinoic acid, is used in the treatment of acne. It is teratogenic and is therefore used with caution in women of child-bearing age.

3. Vitamin K is synthesized by intestinal bacteria and is supplied by leafy green vegetables. Vitamin K acts as a coenzyme for glutamate carboxylase, an enzyme that catalyzes the carboxylation of glutamic acid side chains in several of the clotting factors (factors II, VII, IX, and X). A deficiency of vitamin K results in an accumulation of preprothrombin, a

20

meClical

Proteins, Enzymes, and Coenzymes

deficiency in prothrombin, and an increase in clotting time. Newborns are vitamin K deficient because their intestinal tract is still sterile. Antibiotic sterilization of the gut or malabsorption of fat can lead to deficiency and bleeding complications. 4. Vitamin E, also known as tocopherol, is found in leafy vegetables, vegetable oils, and grains. The major function of vitamin E is as an antioxidant, where its first line of defense is against peroxidation of polyunsaturated fatty acids found in cellular membranes. Fat malabsorption may lead to vitamin E deficiency. In newborns, symptoms include hemolytic anemia; in adults, sensory ataxia due to spinocerebellar degeneration may occur. In humans, vitamin E deficiency is seen frequently in premature infants and malabsorption syndromes.

Bri~Je to .~~~rma~()I()fl' Warfarin, an oral anticoagulant, acts as an antagonist to vitamin K and interferes with the synthesis of vitamin K-dependent clotting factors. Vitamin K can be administered to reverse the effects of warfarin. Warfarin is discussed in greater detail in the HematologicfLymphoreticular Pharmacology chapter of Organ Systems Book 1 (Volume III).

KAPLAlf I med1C8

21

Bioenergetics

Cells extract energy from food by the oxidation of carbohydrates, proteins, and fats to CO2 and H20. Catabolic (degradative) pathways involve oxidation reactions that release energy, most of which is captured in the high-energy phosphate bonds of ATP. In contrast, anabolic (synthetic) pathways involve reduction reactions that require the input of energy, with the reducing power and energy being supplied by NADPH and ATP, respectively. The pathways for catabolism of the metabolic fuels converge at acetyl-CoA, a common intermediate in the degradation of carbohydrates, proteins, and fats. Acetyl-CoA is oxidized in the mitochondria via the citric acid cycle. The released energy is conserved as electron pairs that are transferred from acetyl-CoA to NADH and FADH2. The electrons are then transferred sequentially from NADH and FADH2 to O2, resulting in the formation of H20. The oxidation of NADH and FADH2 by molecular oxygen, a process catalyzed by the electron transport chain, is a highly exergonic process. The energy released is used to drive the phosphorylation of ADP to form ATP, an endergonic reaction. Most of the ATP supply in the cell is derived from oxidative phosphorylation. The regulation of oxidative phosphorylation and the citric acid cycle are closely linked-they are both dependent upon the availability of molecular oxygen. This chapter reviews the principles of thermodynamics and oxidation-reduction reactions that form the basis for energy metabolism.

METABOLIC SOURCES OF ENERGY Energy is extracted from food via oxidation, resulting in the end products of carbon dioxide and water. This process occurs in four stages (Figure 1-3-1). In the first stage, metabolic fuels are hydrolyzed to a diverse set of monomeric building blocks, including glucose, amino acids, and fatty acids. In the second stage, the building blocks are degraded by various pathways to a common metabolic intermediate, acetyl-CoA. Most of the energy contained in metabolic fuels is conserved in the chemical bonds (electrons) of acetyl-CoA. In stage three, the citric acid (Krebs, or tricarboxylic acid, TCA) cycle oxidizes acetyl-CoA to CO 2, and the electron pairs present in the carbon-carbon and carbon-hydrogen bonds are transferred to the electron carriers NADH and FADH 2 . The final stage in the extraction of energy from food is oxidative phosphorylation, where the energy in the electron pairs of NADH and FADH2 is released via the electron transport chain (ETC) and is used to synthesize ATP.

I KAPLAlf medlea

23

Biochemistry

Stage

Carbohydrate

Protein

Gluctse

AminJ Acids

~pyruvate

t

Fat Fatt}Acids

________

-------------Acetyl-Co~

II

III

IV

Figure 1-3-1. Extraction of energy from metabolic fuels.

THERMODYNAMIC PRINCIPLES The principles underlying energy metabolism are based on the thermodynamics of chemical reactions. Some of the abbreviations and corresponding definitions involved are: G = free energy (energy available to do work)

= enthalpy (heat content of a compound) S = entropy (randomness of a system) H

T = absolute temperature (measured in OK) R = gas constant (l.987 cal/mol·degree) F = Faraday's constant (23 kcal/volt·mol) A. Free energy change (ilG) of reactions. The free energy change of a system is the portion of the total energy that is available for useful work. For any chemical reaction, the ilG is equal to the difference in energy between the products and the reactants. The free energy change predicts the direction in which a reaction will proceed spontaneously. ilG = ilH - TilS = Gproducts - Greactants O

The standard free energy change (ilG is a constant for any given reaction. (The superscript indicates "standard conditions:' which are pH 7, 25°C, and all reactants and products at 1.0 M concentration.) The ilGo is related to the equilibrium constant, Keq. For the reaction )

A+B - - - - - - i..~ C + D

24

KAPLA~.

meulCa

I

Bioenergetics

the equilibrium constant is defined as:

[C] [D] Keq = [A] [B]

LlGo = -RT In Ke q = -2.3 RT log Keq

Therefore, if the concentrations of reactants and products at equilibrium are known, the Ke q and the LlGo for the reaction can be calculated. B. Exergonic and endergonic reactions are distinguished on the basis of whether the LlG of the

reaction is positive or negative. Exergonic reactions have a negative LlG and will proceed spontaneously in the direction written. In contrast, endergonic reactions have a positive LlG and require the input of energy to proceed in the direction written. If LlG = 0, the reaction is at equilibrium, and the rates of the forward and the reverse reactions are equal. C. Coupled reaction systems. Endergonic reactions in metabolism frequently proceed by being

coupled to an exergonic reaction. The requirement for a coupled reaction system is that the product of the first reaction must be the substrate for the second reaction. Many enzymecatalyzed reactions that use ATP are examples of coupled reactions, where the energy released by the hydrolysis of ATP is used to drive an endergonic reaction. For example, consider the phosphorylation of glucose to glucose-6-phosphate, a reaction that occurs in all cells and is catalyzed by hexokinase.

Glucose + Pi

----il .....

Glucose-6-phosphate

LlGo

= +3.3 kcalimol

Because this reaction is endergonic, it will not proceed spontaneously in the direction of glucose-6-phosphate formation unless energy is supplied. However, if the formation of glucose-6-phosphate is coupled to the hydrolysis of ATP, the sum of the two reactions is exergonic.

ATP+HzO Glucose + Pi Sum:

Glucose + ATP

---I..... ---I..... ---I....

ADP + Pi Glucose-6-phosphate Glucose-6-phosphate + ADP

In a Nutshell • LlG < 0 ---7 spontaneous reaction • LlG > 0 ---7 nonspontaneous reaction • LlG = 0 ---7 equilibrium

Note Cells often couple energetically unfavorable reactions with ATP hydrolysis to force the reaction to proceed.

=-7.3 kcalimol = +3.3 kcalimol LlGo = -4.0 kcalimol LlGo LlGo

The function of hexokinase, therefore, is to couple these two reactions so that the formation of glucose-6-phosphate is thermodynamically favorable. D. Central role of adenine nucleotides in energy transduction. Some of the common

phosphate-containing compounds found in cells and the energy released by hydrolysis of their phosphate bonds under standard conditions are shown in Table 1-3-1.

KAPLAN'iIme leaI

25

Biochemistry

Table 1-3-1. Energy of hydrolysis of phosphate compounds. ~GO

Compound

(kcallmol)

Phosphoenolpyruvate

-14.8

1,3-Bisphosphoglycerate

-11.8

Creatine phosphate

-10.3

Pyrophosphate

-8.0

ATP Glucose-l-phosphate

1-7.31 -5.0

Fructose-6-phosphate

-3.8

AMP

-3.4

Glucose-6-phosphate

-3.3

Glycerol-3-phosphate

-2.2

The positioning of these compounds in the table illustrates important concepts in energy transfer reactions. Note the structure of ATP (Figure 1-3-2).

000 II II II

Adenine-rib0-;Jse --0 -~

-0

0-

ester bond (low energy)

\~O(~ 0-

-0-

0-

anhydride bonds (high energy)

Figure 1-3-2. Structure of ATP.

The energy in the terminal anhydride bond of ATP is greater than the energy found in the compounds listed after it in Table 1-3-1. Therefore, the energy released by hydrolysis can be used to drive the synthesis of these compounds. Conversely, the compounds listed before ATP ATP formation via substrate-level have more energy in their phosphate bonds than does ATp, thus allowing these compounds phosphorylation occurs with: to transfer their phosphate groups to ADP with the formation of ATP. The conversion of ADP • PhosPhOenOlPyrUvate} . glycolysIs to ATP by the use of high-energy phosphate metabolites is known as substrate-level phosphorylation. As shown in Table 1-3-1, there are three phosphorylated intermediates in • l,3-Bisphosphoglycerate cells with sufficient energy to participate in substrate-level phosphorylation. Phospho• Creatine phosphate ~ enolpyruvate and 1,3-bisphosphoglycerate are intermediates in glycolysis, and muscle creatine phosphate serves as a reservoir of high-energy phosphate bonds in muscle.

In a Nutshell

E. Other high-energy carriers. Many chemical groups that are transferred in metabolic reactions require "high-energy carriers" for the reaction to be exergonic. Examples of some groups that are transferred, their high-energy carriers, and the types of reactions and/or pathways in which they participate are summarized in Table 1-3-2.

26

KAPLAlfdI me lea

------------~

.---~~

Bioenergetics

Table 1-3-2. High-energy carriers of chemical groups in metabolism. Group p.

Carrier

Pathways/reactions

ATP

Kinase reactions

Sugars

UDP-sugar

Polysaccharide synthesis

Acetate

Acetyl-CoA

Fatty acid synthesis

Fatty acids

Acyl-CoA

Triacylglycerol synthesis

Amino acids

AMP-amino acid

Protein synthesis

Methyl

SAM*

Methylation reactions

Carboxyl

Carboxy-biotin

Carboxylation reactions

Sulfate

PAPst

Sulfation reactions

1

*S-adenosylmethionine; t phosphoadenosine phosphosulfate

OXIDATION-REDUCTION REACTIONS The principles of oxidation and reduction are an integral part of energy conservation in cells. The energy contained in metabolic fuels is released through a series of oxidation-reduction (redox) reactions that occur mainly in the mitochondria. Approximately 40% of this energy is conserved as ATP. A. General principles. Oxidation-reduction reactions involve the transfer of electrons between a donor and an acceptor. 1. Definitions. Oxidation is defined as the loss of electrons, and reduction is defined as the gain of electrons. Every oxidation is accompanied by a reduction, each of which is considered to be a half-reaction. 2. The standard reduction potential, E", is a constant that describes the tendency of a compound to act as a reducing agent (lose electrons). It is expressed in volts and is measured under standard conditions defined as 2SoC, pH 7, and at concentrations of 1.0M for electron donors and acceptors. Values of standard reduction potentials for some common half-reactions are shown in Table 1-3-3. Stronger electron donors have a more negative reduction potential. Thus, under standard conditions, NADH with an E' of -0.32 volt will reduce pyruvate (or any other compound with a less negative E').

Mnemonic LEO the lion says GER: loss of electrons = oxidation gain of electrons = reduction OIL RIG: oxidation is loss (of electrons) reduction is gain (of electrons)

Table 1-3-3. Standard reduction potentials for common redox pairs. * E'(V)

Half-reaction NAD+ + 2e- + H+

~

NADH

-0.32

Pyruvate + 2e- + 2H+

~

Lactate

-0.19

Oxaloacetate + 2e- + 2H+~

Malate

-0.17

FAD + 2e- + 2H+

~

FADH2

-0.06

CoQ + 2e- + 2H+

~

COQH2

+0.10

Fumarate + 2e- + 2H+

~ ~

Succinate Cyt a (Fe2+)

+0.13

Cyt a (Fe3 +) + e-

+0.29

112 O2 + 2e- + 2H+

~

H 2O

+0.82

*Note that all of the reactions are written as reductions.

KAPLAlf I meillea

27

Biochemistry

Note If the reduction potential is given, reverse the sign to find the oxidation potential:

3. Relationship between ABO and LiGO. The change in the standard reduction potential for an oxidation-reduction reaction is related to the standard free energy change (LiGO) for the reaction by the expression shown below, where n is the number of electrons transferred, F is the Faraday constant, and LiEO = EO electron acceptor - E' electron donor' LiG = -nFLiEo O

E'red = -0.32 ~ E'QX = +0.32

From this relationship, it is clear that for an oxidation-reduction reaction to be exergonic and to proceed spontaneously, LiEO must be a positive value. 4. Example. These thermodynamic principles can be illustrated by considering the transfer of electrons from NADH to molecular oxygen. In this example, NADH is the electron donor (it has the more negative standard reduction potential), and oxygen is the electron acceptor. The overall oxidation-reduction reaction can be written as the sum of two half-reactions. Oxidation of NADH: NADH ----~..~ NAD+ + H+ + 2eReduction of 0: - - - - - - l.. ~ H 20 Overall reaction:

NADH + 1/2 O 2 + H+ ~ NAD+ + H 20

LiEO LiEo

= +0.32 volt = +0.82 volt

LiEo = + 1.14 volt

The positive value of LiEO indicates that the reaction will proceed spontaneously. As defined above, the change in standard free energy can be calculated (n = 2 electrons being transferred, and F = 23 kcal/volt) as follows: AGO = -nFLiEo AGO = -(2)(23 kcallvolt)(1.14 volt) = -52.4 kcal Because AGO is negative, the oxidation of NADH by oxygen is an exergonic reaction. This exergonic reaction is the overall reaction catalyzed by the electron transport chain. The electrons in NADH are transferred sequentially through a series of carriers to oxygen, where each electron carrier is reduced by the preceding carrier. B. Major electron carriers. A wide variety of dehydrogenases participate in the oxidation of

In a Nutshell • Electron acceptors

~

• Electron donors

~

NAD+ FAD NADPH NADH

metabolic fuels. Most of these enzymes use either NAD+ or FAD as electron acceptors. The major carrier of electrons in reductive biosynthetic reactions is NADPH . 1. NAD+ is the electron accceptor in reactions involving oxidation of hydroxylated carbon

atoms (Figure 1-3-3). NAD+ accepts a hydride ion, H- (two electrons and a proton), to form NADH, and the hydroxyl group is oxidized to a carbonyl group.

OH

I

o II

R-C-COOH + NAD+ ~ R-C-COOH + NADH + H+

I

H Figure 1-3-3. The reduction of NAD+ to NADH.

2. FAD is the electron acceptor in reactions involving the oxidation of two adjacent carbons, resulting in the formation of a carbon-carbon double bond (Figure 1-3-4). A hydrogen atom is removed from each carbon atom and is transferred to FAD to form FADH2•

28

KAPLA~. I meulca

Bioenergetics

H I

H I

I

I

R-C-C-R' + FAD

~

R-C =C -R' + FADH

H H

I H

I H

2

Figure 1-3-4. The reduction of FAD to FADH 2 •

3. NADPH is the major source of reducing power for biosynthetic pathways. In contrast to NADH, which is generated and used primarily in the mitochondria, most of the NADPH is formed and used in extramitochondrial reactions (described in Chapter 4 of this section).

CITRIC ACID CYCLE The citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or the Krebs cycle, is localized in the mitochondria. A. Functions. The pathway is amphibolic, playing important roles in both catabolic and anabolic pathways. 1. Catabolic function. The major catabolic function of the citric acid cycle is to transfer

electron pairs (potential energy) from acetyl-CoA to NAD+ and FAD. With every turn of the cycle, two carbon atoms enter as acetyl-CoA, two carbon atoms leave as COl> and the four pairs of electrons in the carbon-hydrogen and the carbon-carbon bonds are transferred to NAD+ and FAD. The overall reaction for the oxidation of acetyl-CoA is shown in Figure 1-3-5.

Acetyl-CoA + 3 NAD+ + FAD + GDP + Pi -----. 2 C02 + 3 NADH + FADH2 + GTP + CoA Figure 1-3-5. Stoichiometry of the citric acid cycle.

Oxidation of acetyl-CoA to CO 2 results in the production of one high-energy phosphate bond (GTP) by substrate level phosphorylation. Assuming adequate oxygen is available, subsequent oxidation of NADH and FADH2 will result in the synthesis of 11 molecules of ATP by oxidative phosphorylation (described below). Therefore, the complete oxidation of one molecule of acetyl-CoA to CO 2 and H 20 results in the synthesis of 12 ATP equivalents. 2. Anabolic functions. Intermediates in the citric acid cycle are used as substrates for vari-

ous biosynthetic pathways. a. Citrate is a substrate for fatty acid synthesis. b. Oxaloacetate is the first intermediate in gluconeogenesis. c. Succinyl-CoA is required for the synthesis of heme.

Note Some amino acids can be converted to a-ketoglutarate and can enter the citric acid cycle at this intermediate.

d. Oxaloacetate and a-ketoglutarate are substrates for amino acid synthesis.

I KAPLAlf med lea

29

Biochemistry

B. Reactions of the citric acid cycle. The reactions by which acetyl-CoA is oxidized to CO 2 are

shown in Figure 1-3-6. AcetylCoA

~Citr"e~

NADHfo.cel.,e

c;o-\.,e

L-M~late

Isocltrate

[7

3

Fumarate

a-Ketoglutarate

FADH,.\ 6 \

~NADH+CO'

fNADH + CO, Sucdnyl-CoA

Succinate

5

GTP

Enzymes 1. Citrate synthase 2. Aconitase 3. Isocitrate dehydrogenase 4. a-Ketoglutarate dehydrogenase 5. Succinyl-CoA thiokinase 6. Succinate dehydrogenase 7. Fumarase 8. Malate dehydrogenase

Figure 1-3-6. The citric acid cycle.

Mnemonic Citric acid is Krebs starting substrate for mitochondrial oxidation: citrate, cis-aconitate, isocitrate, a-ketoglutarate, succinyl-CoA, succinate, fumarate, malate, oxaloacetate

The entry of acetyl-CoA into the cycle involves condensation with oxaloacetate to produce citrate, a C6 tricarboxylic acid. After isomerization to isocitrate, two sequential oxidative decarboxylation reactions occur, resulting in the formation of succinyl-CoA. Each of these reactions releases CO 2 and transfers electrons to NADH. Succinyl-CoA contains a thioester linkage that has sufficient energy to drive the synthesis of a high-energy phosphate bond. Thus, in the next reaction, hydrolysis of succinyl-CoA is coupled with the synthesis of GTP from GDP and Pi. GTP is energetically equivalent to ATP, and the two molecules are interconvertible. The remaining reactions complete the cycle by regenerating oxaloacetate. They include two oxidation reactions: the oxidation of succinate to fumarate, producing FADH2 ; and the oxidation of malate to oxaloacetate, producing NADH. All of the enzymes of the cycle are located in the mitochondrial matrix except for succinate dehydrogenase, which is embedded in the inner mitochondrial membrane. The localization of these enzymes provides for efficient transfer of electrons from the carriers, NADH and FADH2 , to the electron transport chain.

e. Key

enzymes. The four dehydrogenases (isocitrate, a-ketoglutarate, succinate, and malate) all catalyze oxidation reactions. In the isocitrate and a-ketoglutarate dehydrogenase reactions, decarboxylation also occurs. Isocitrate dehydrogenase catalyzes one of the

30

meClical

Bioenergetics

rate-limiting steps in the cycle. This enzyme is allosterically inhibited by ATP and NADH and is activated by ADP. Secondary sites of regulation are a-ketoglutarate dehydrogenase and citrate synthase, which are also inhibited by ATP and NADH. These three enzymes catalyze reactions that are essentially irreversible. Although pyruvate dehydrogenase is not an enzyme in the citric acid cycle, it oxidizes pyruvate to acetyl-CoA, thereby allowing carbohydrate to be oxydized to COz. D. Anaplerotic ("filling-up") reactions. When intermediates of the cycle are removed for synthetic purposes, they must be replenished in order to ensure that acetyl-CoA can continue to be oxidized. The most important reaction for replenishing the cycle with intermediates is the conversion of pyruvate to oxaloacetate. This reaction, catalyzed by pyruvate carboxylase, requires biotin, ATP, and bicarbonate as substrates, and it has an absolute requirement for acetyl-CoA as an allosteric activator (Figure 1-3-7). The accumulation of acetyl-CoA, due to insufficient levels of oxaloacetate, activates the synthesis of this intermediate.

In a Nutshell Enzyme

Inhibitors Activators

Pyruvate ATP dehydrogenase Acetyl-CoA NADH

ADP CoASH NAD+ Ca 2+ Insulin

Citrate synthase

ATP

Iso citrate ATP dehydrogenase NADH

ADP

a-Ketoglutarate Succinyl-CoA dehydrogenase NADH ATP

biotin pyruvate + HC03 - + ATP - - - - - - - - - - - - - - . . . Oxaloacetate + ADP + Pi pyruvate carboxylase

0 acetyl-CoA Figure 1-3-7. The pyruvate carboxylase reaction.

ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION The final step in the aerobic oxidation of metabolic fuels is achieved by the electron transport chain (ETC). The ETC accepts electrons from NADH and FADH z and passes them, through a series of carriers, to molecular oxygen, forming water. The energy released at specific steps in the ETC is used to synthesize ATP via a process called oxidative phosphorylation. All of the enzymes and cofactors required for electron transport and oxidative phosphorylation are localized in the inner mitochondrial membrane. A. The electron transport chain 1. Components. The ETC is composed of a specific sequence of enzymes and their coenzymes, including NAD+ and FAD-linked dehydrogenases, iron-sulfur proteins (FeS), coenzyme Q, and several cytochromes (Figure 1-3-8). The components of the chain can be separated into four protein-lipid complexes (I, II, III, and IV) and two mobile components (CoQ and cyt c) that move freely in the lipid bilayer.

meCtical

31

Biochemistry

Succinate

Complex II FAD FeS

Complex III

Complex I NADH

~

Complex IV ~

FMN

~

FeS

cytc

~

cyt a

t

~cyt

a3

~~

fatty acyl-CoA

a-glycerol-P

Figure 1-3-8. Components of the electron transport chain.

2. Sources ofNADH and FADH. The electrons in NADH arise mainly from the mitochondrial oxidations and to a lesser extent from cytosolic oxidations. Almost all of the FADH arises from mitochondrial oxidations.

a. Mitochondrial oxidations. Several substrates, including isocitrate, a-ketoglutarate, malate, pyruvate, and glutamate, are oxidized in mitochondria with the formation of NADH. The electrons in FADH2 come from the oxidation of succinate, fatty acylCoA, and a-glycerol phosphate. The electrons from NADH and FADH2 enter at different positions along the electron transport chain. Electrons from NADH enter at complex I, whereas electrons from several FAD-linked dehydrogenases enter at CoQ. Note that complex II is succinate dehydrogenase, the same enzyme utilized in the citric acid cycle. b. Shuttles for getting cytoplasmic NADH electrons into mitochondria. Although most oxidations occur in the mitochondria, there are a few oxidative reactions in the cytosol that produce NADH. The mitochondrial membrane, however, is impermeable to NADH; thus, the transfer of electrons from cytosolic NADH into the mitochondria for oxidation by the electron transport chain requires special shuttle systems. Two types of shuttles are found in cells: the malate shuttle and the a-glycerol phosphate shuttle (Figure 1-3-9). In these shuttles, malate and a-glycerol phosphate act as carriers of electrons across the inner mitochondrial membrane. In the malate shuttle, electrons originating in the cytosol are incorporated into mitochondrial NADH, whereas in the a-glycerol phosphate shuttle, cytosolic electrons are incorporated into mitochondrial FADH 2.

32

meClical

Bioenergetics

Inner mitochondrial membrane

Cytosol

Malate Shuttle

NADH

X

NAD+

X

Mitochondrial matrix

Aspartate

Aspartate

t

t

Oxaloacetate

Oxaloacetate

Malate

Malate

X

NAD+

a-Glycerol-P Shuttle NADH NAD+

NADH

Dihydroxyacetone-P

Dihydroxyacetone-P

a-Glycerol-P

a-Glycerol-P

X FAD

Figure 1-3-9. Shuttles for getting cytosolic NADH electrons into the mitochondria.

3. Energetics. A simplified version of the electron transport chain is shown in Figure 1-3-10. The carriers in the electron transport chain are arranged so that the reduction potential progressively increases from negative to positive values. Thus, as electrons move from one carrier to the next in the chain, the ~EO > 0 and the ~GO < 0, ensuring that electrons flow spontaneously to oxygen. FADH2

NADH

~

---J"~ CoQ

-0.32

+0.10

\.."--'y~

__~1

..

cyt b

..

+0.07

\

cyt c

+0.25

Tm

I

+0.42 volt

+0.18

-19.3 kcal/mol

-9.4 kcal/mol

..

..

cyt a

..

cyt a3

+0.55

+0.29

\

1/202 +0.82 volt

I

T +0.53 volt

-24.4 kcal/mol

Figure 1-3-10. Energy released by electron flow through the ETC.

B. Oxidative phosphorylation. The energy required for synthesis of ATP is 7.3 kcal/mol. It is clear from Figure 1-3-10 that transfer of electrons from 1 mol ofNADH through complexes I, III, and IV releases sufficient energy to drive the synthesis of ATP. Thus, it is at these three sites in the electron transport chain that energy can be harnessed for oxidative phosphorylation. Note that electrons from FADH 2 bypass complex I and flow through only two of these energy transduction sites.

I KAPLAlf medlea

33

Biochemistry

In a Nutshell P/O ratio NADH FADH2

3 2

DNP (2A-dinitrophenol, an uncoupler that dissipates the W gradient) has a P/O ratio of o.

1. PIO ratio. By definition, the PIO ratio is the number of ATP molecules produced per 0 atom reduced. Thus, substrates that are oxidized with the generation of NADH (malate, isocitrate, a-ketoglutarate) support the synthesis ofthree molecules of ATP and have PIO ratios of 3. Substrates that are oxidized with the production of FADH2 (succinate, aglycerol phosphate) have PIO ratios of 2. 2. Efficiency. The calculations shown below indicate that the oxidation ofNADH by the ETC releases a total of 53 kcal of energy. LlE' LlG'

= E' oxygen -

E' NADH

= -nFL\E' = -(2)

= +0.82 -

(-0.32)

= 1.14 volts

(23 kcallvolt) (1.14 volt) = -53 kcal

The synthesis of ATP requires 7.3 kcal. Thus, in theory, the oxidation of 1 mole of NADH should release sufficient energy to drive the synthesis of 7 moles of ATP. However, because only 3 moles of ATP are synthesized per mole of NADH oxidized, the efficiency of oxidative phosphorylation is approximately only 40%. The remainder of the energy is released as heat. 3. Mechanism of energy transduction. The chemiosmotic hypothesis is the most widely accepted theory for how electron transport is coupled with ATP synthesis (Figure 1-3-11). This theory is based on the observation that when electrons are flowing through the ETC, complexes I, III, and IV are pumping protons out of the matrix, creating a proton gradient across the inner mitochondrial membrane. Complex II does not pump protons. The chemiosmotic hypothesis asserts that the protomotive force associated with the proton gradient drives the synthesis of ATP. The movement of protons down the gradient as they re-enter the matrix releases energy that is available for ATP synthesis. ATP synthase, also known as complex V, is associated with the inner mitochondrial membrane in close proximity to the electron transport chain (Figure 1-3-11).

Clinical Correlate LHON (Leber hereditary optic neuropathy) is a disease that affects the central nervous system, resulting in the loss of bilateral vision secondary to insufficient ATP to support neuronal activity. The disease has been shown to arise from point mutations in regions of mitochondrial DNA that code for proteins in complex I and complex III of the electron transport chain. Mutations in either of these genes impairs ATP production by oxidative phosphorylation. Patients frequently present with lactic acidosis arising from an inhibition of pyruvate dehydrogenase secondary to NADH accumulation.

34

KAPLAN"dme leaI

ADP+ Pi

Matrix

NADH

l e-

ATP

FADH2

!

e-

Inner mitochondrial membrane

Cytosol

Figure 1-3-11. Structure of mitochondrial ATP synthase.

ATP synthase consists of two types of subunits. The Fo subunits span the membrane, creating a proton channel that allows protons to move back into the matrix. The FI subunit protrudes into the matrix and, in the presence of a proton gradient, catalyzes the condensation of ADP and Pi to form ATP.

Bioenergetics

4. Inhibitors of oxidative phosphorylation. A large number of compounds inhibit oxidative phosphorylation. The sites at which selected inhibitors exert their effects are shown in Figure 1-3-12. Inhibitors of oxidative phosphorylation always decrease ATP production. They are organized into three major classes based on their effects. a. Inhibitors of electron transport bind to specific components of the electron transport chain, blocking electron transfer. This blockage results in a decreased proton gradient and decreased ATP synthesis. For example, cyanide binds to cyt a3 and blocks the transfer of electrons from complex IV to oxygen; antimycin binds to complex III and blocks the transfer of electrons from cyt b to cyt c; rotenone and barbiturates bind to complex I and inhibit the transfer of electrons to CoQ.

NADH

..

------~~

Rotenone, Amytal

cyt b

---

~

cyt c

..

-----I~ cyt a

Antimycin A

~

o

Cyanide

Figure 1-3-12. Inhibitors of electron transport.

b. Inhibitors of ATP synthase bind directly to the enzyme. For example, oligomycin binds to the Fo subunit and prevents re-entry of protons into the mitochondrial matrix. The increased proton gradient across the inner mitochondrial membrane eventually leads to diminished electron transport. c. Uncouplers of oxidation and phosphorylation make the membrane permeable and

abolish the proton gradient. Electron transport is increased by uncouplers, but ATP synthesis cannot occur because the energy cannot be harnessed. The energy resulting from electron transport is dissipated as heat. Examples of uncouplers are dinitrophenol and the protein thermogenin, which is found in the mitochondrial membrane of brown fat. Brown fat surrounds the major blood vessels of the neonate, where it functions to keep the blood warm.

Clinical Correlate Cyanide binds to the heme Fe3+ of cytochrome a3 in the ETC, blocking the transfer of electrons to oxygen and, consequently, the synthesis of ATP. Inhibition of mitochondrial respiration can lead to coma and death unless diagnosed and treated early.

COORDINATE REGULATION OF THE CITRIC ACID CYCLE AND OXIDATIVE PHOSPHORYLATION The rates of oxidative phosphorylation and the citric acid cycle are closely coordinated and are dependent mainly upon the availability of O 2 and ADP. If O 2 is limited, the rate of oxidative phosphorylation decreases, and the concentrations of NADH and FADH2 increase. The accumulation of NADH, in turn, inhibits the citric acid cycle. The coordinated regulation of these pathways is known as "respiratory control." In the presence of adequate 02> the rate of oxidative-phosphorylation is dependent upon the availability of ADP. The concentrations of ADP and ATP are reciprocally related; an accumulation of ADP is accompanied by a decrease in ATP and the amount of energy available to the cell. Therefore, ADP accumulation signals the need for ATP synthesis. ADP allosterically activates isocitrate dehydrogenase, thereby increasing the rate of the citric acid cycle and the production of NADH and FADH 2. The elevated levels of these reduced coenzymes, in turn, increases the rate of electron transport and ATP synthesis.

KAPLAdlf • me leaI

35

Biochemistry

CONGENITAL DEFECTS IN OXIDATIVE PHOSPHORYLATION Mitochondrial DNA contains genes coding for ATP synthase and for some of the proteins in each of the complexes of the electron transport chain. Defects in several of these components have been found. Infants and children present with various combinations of seizures, hypotonia, failure to thrive, and delay of developmental milestones. Focal deficits include abnormalities of eye movements and stroke-like symptoms. Identified entities include mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS). All of the defects arising from mutations in mitochondrial DNA are inherited maternally.

36

KAPLllfilme leaI

Carbohydrates

Carbohydrates participate in many metabolic pathways and serve as structural components of cells and tissues. In human biochemistry, there are three major classes of carbohydrates: monosaccharides, disaccharides, and polysaccharides. The most important monosaccharide in human metabolism is glucose. All cells and tissues require the use of some glucose for energy. The brain relies almost entirely on glucose for its energy, and red blood cells derive all of their energy from glucose. Following a highcarbohydrate meal, glycogenesis in liver and skeletal muscle provides a pathway for storage of excess glucose. Conversely, the mobilization of glucose from glycogen is achieved via glycogenolysis. Gluconeogenesis is the pathway for de novo synthesis of glucose from amino acids, glycerol, propionate, and lactate. This chapter emphasizes the role that these pathways play in homeostasis, the tissues where they occur, the key enzymes involved, and the mechanisms for regulation. Numerous clinical examples are provided that stress the importance of carbohydrates in both health and disease.

CLASSIFICATION Carbohydrates are chains of carbon atoms with attached hydrogen and hydroxyl groups. The length of the carbon chain may vary, and carbohydrates can generally be represented by the formula Cn(H 2 0)n, where n is a minimum of three carbons. This formula emphasizes the idea that this class of molecules are "hydrates of carbon:' In some specialized monosaccharides, one of the hydroxyl groups may be replaced by another chemical group, such as a hydrogen atom (deoxyribose in DNA), an amino group (glucosamine in proteoglycans), or phosphate groups (most intermediates of carbohydrate metabolism pathways). A. Monosaccharides. The simplest carbohydrates are the monosaccharides. Most monosaccharides in human metabolism are trioses (C 3 ), pentoses (Cs ), and hexoses (C 6 ). Monosaccharides containing an aldehyde are known as aldoses, and those containing a keto group are called ketoses. The most important monosaccharide is D-glucose, a hexose and an aldose whose structure is shown in Figure 1-4-1.

meClical

37

Biochemistry

---I."

Aldehyde group (Anomeric carbon)

---I."

Penultimate carbon

D-Glucose Figure 1-4-1. Structure of o-glucose.

1. Nomenclature and definitions. The numbering system for monosaccharides always starts with the carbon atom nearest the carbonyl group (::::C=O). a. Anomeric carbon. The carbonyl carbon is defined as the anomeric carbon. This carbon participates in internal ring structures and glycosidic bonds between monosaccharides (discussed below). b. Penultimate carbon. The penultimate carbon is the next-to-the-last carbon in the chain. For hexoses, it is C-5; for pentoses it is C-4. c. D and L isomers. The "D" designation describes the configuration around the penulti-

mate carbon. If the hydroxyl group is on the right, it is a D-sugar; if it is on the left, it is an L-sugar. Almost all important monosaccharides in human biochemistry have the D configuration. d. Epimers. Two monosaccharides are epimers if they differ in the configuration around a single carbon atom. For example, galactose is a 4-epimer of glucose. Thus, knowing the structure of glucose allows you to automatically recognize the structure of galactose. Enzymes that catalyze the interconversions of these compounds are epimerases. e. Aldose-ketose isomers. Glucose, an aldose, and fructose, a ketose, differ only in the position of the carbonyl group. In glucose, the carbonyl group is at C-1, whereas in fructose, it is at C-2. 2. Cyclic structure of monosaccharides. The straight chain structure of monosaccharides exists in equilibrium with a ring structure, resulting from the reaction of the hydroxyl of the penultimate carbon with the anomeric carbon. The cyclic structure of glucose is a sixmember ring (a pyranose) that can be drawn in one of two ways (Figure 1-4-2).

or

Figure 1-4-2. Cyclic structures for l3-o-glucose.

38

meClical ~~~~~~~~~-

~--

------~---

~

Carbohydrates

In the structure on the right, the ring projects from the plane of the paper and the hydroxyl and hydrogen groups project up and down from the carbons in the ring. By convention, hydroxyl groups on the right point down, and those on the left point up. When a ring structure is formed, the anomeric carbon can exist in two configurations, (J, and ~. In (J,-Dglucose, the hydroxyl group on C-l points down, whereas in ~-D-glucose it points up. The predominant form of glucose in the body is ~-D-glucose. D-fructose, shown in Figure 1-4-3, is an important hexose that forms a five-member ring (a furanose).

6

----J.~ ~:J;OH '~6H OH a-O-Fruclose

Figure 1-4-3. Structure of fructose.

3. Derivatives of glucose. Glucose can be modified to give three important compounds found in metabolism (Figure 1-4-4). a. D-gluconic acid is formed by the oxidation of the aldehyde at C-l, producing an "aldonic" acid. The phosphorylated form of gluconic acid is an important intermediate in the hexose monophosphate shunt. b. D-glucuronic acid is formed by oxidation of the alcohol at C-6, producing a "uronic" acid. Ascorbic acid (vitamin C) is synthesized from glucuronic acid. The activated carrier of glucuronic acid (UDP-glucuronic acid) is used in the synthesis of proteoglycans. c. D-glucosamine is formed by the substitution of an amino group for the hydroxyl group of C-2, resulting in glucosamine. Glucosamine and galactosamine are important structural components of glycoproteins and proteoglycans.

CHO

COOH

I

I

H-C-OH

H-C-OH

I

I

HO-C-H

HO-C-H

I

I

H-C-OH

H-C-OH

I

I

H-C-OH

H-C-OH

I

I

COOH

CHpH

O-Gluconic Acid

O-Glucuronic Acid

In a Nutshell • D-gluconic acid ~ hexose monophosphate shunt • D-glu(uronic acid proteoglycans

~

• D-glucosamine ~ glycoproteins, proteoglycans, and glycoilpids

CHO

I I

H-C-NH 2

HO-C-H

I

H-C-OH

I

H-C-OH

I

CH2 0H

O-Glucosamine

Figure 1-4-4. Derivatives of D-glucose. B. Disaccharides are formed when two monosaccharides are connected by a glycosidic linkage.

The anomeric carbon of one monosaccharide is usually linked to a hydroxyl group on the

,mattical

39

Biochemistry

Clinical Correlate Primary lactose intolerance is caused by a hereditary deficiency of lactase, most commonly found in persons of Asian and African descent. Secondary lactose intolerance can be precipitated at any age by gastrointestinal disturbances such as celiac sprue, colitis, or viral-induced damage to intestinal mucosa. Common symptoms of lactose intolerance include vomiting, bloating, explosive and watery diarrhea, cramps, and dehydration. The symptoms can be attributed to bacterial fermentation of lactose to a mixture of CH 4, H2, and small organic acids. The acids are osmotically active and result in the movement of water into the intestinal lumen. Diagnosis is based on a positive hydrogen breath test following an oral lactose load. Treatment is by dietary restriction of milk and milk products or by lactase pills, which can be added to the diet.

Note Heparan is a proteoglycan found in the extracellular matrix and is associated with the cell surface of skin and fibroblasts; heparin is a proteoglycan found mainly in mast cell granules and functions primarily as an anticoagulant.

40

meClical

second monosaccharide. The glycosidic bond is designated as a or 13, depending on the configuration of the anomeric carbon in the linkage. The two most important disaccharides in human biochemistry are lactose and sucrose (Figure 1-4-5).

Galactose

Glucose Lactose

Glucose

Fructose Sucrose

Figure 1-4-5. Structures of lactose and sucrose.

1. Lactose, also known as milk sugar, is a disaccharide of galactose linked through its

anomeric carbon to the 4-hydroxyl group of glucose. Thus, the monosaccharides are joined by a 13-1,4 glycosidic linkage. Lactose is hydrolyzed to glucose and galactose by lactase, an enzyme found in the brush border membrane of the small intestine. 2. Sucrose, or table sugar, is a disaccharide of glucose and fructose linked together through their anomeric carbon atoms in an a-l,2 linkage. Sucrose is hydrolyzed to glucose and fructose in the small intestine by the enzyme sucrase. C. Oligo saccharides are arbitrarily defined as having between 2 and 10 monosaccharides

linked by glycosidic bonds. They are found in mucoproteins and glycolipids. D. Polysaccharides are arbitrarily defined as having more than 10 monosaccharide units. They

serve as structural components of cells and the extracellular matrix, as storage forms for monosaccharides, and as dietary fiber. The most common polysaccharides are starch, glycogen, cellulose, and proteoglycans. 1. Starch and glycogen are both storage forms for glucose. Starch, the major plant polysac-

charide, is composed of amylose and amylopectin. Amylose is a long, unbranched chain of glucose units linked by a-l,4 bonds. Amylopectin has a similar structure, but it also contains branches, where the monosaccharide at the branch point is linked to a monosaccharide in the straight chain by an a-l,6 glycosidic bond. Glycogen, the major animal polysaccharide, is structurally similar to amylopectin, except it is more highly branched. 2. Cellulose is a linear plant polysaccharide composed of glucose units linked together by 131,4 glycosidic bonds. Cellulose is not digested by humans because there is no intestinal enzyme for hydrolyzing glucose units linked by 13-1,4 glycosidic bonds. Therefore, cellulose acts as a dietary fiber, providing "roughage" in the diet. 3. Proteoglycans, also known as mucopolysaccharides, are major structural components of the extracellular matrix. Examples are hyaluronic acid, chondroitin sulfate, dermatan sulfate, heparan sulfate, and keratan sulfate. These compounds are linear carbohydrate polymers with more than one kind of monosaccharide, linked covalently to a protein core. The carbohydrate portions of proteoglycans are known as glycosaminoglycans (GAGs). GAGs contain repeating disaccharides, usually a hexosamine (glucosamine or galactosamine) and a uronic acid (either glucuronic acid or iduronic acid). The polymers are usually sulfated, and the hexosamines are acylated. The exception is hyaluronic acid; it is not linked to a protein core, it is not sulfated, and it is the only member of this class of compounds found in bacterial sources. The proteoglycans are highly asymmetrical and have a high density of negative charge, allowing them to absorb large quantities of water and form

Carbohydrates

viscous solutions. These physical properties account for their ability to serve as excellent shock absorbers and lubricants. a. Mucopolysaccharidoses are a group of diseases that result from the inability of lysosomes to degrade proteoglycans because of a deficiency of a number of lysosomal hydrolases. The enzyme deficiency leads to excessive storage and urinary excretion of partially degraded mucopolysaccharides. Musculoskeletal deformities and mental retardation are seen in affected individuals. Table 1-4-1 lists the enzyme defect, the urinary metabolite, and the signs and symptoms of the various mucopolysaccharidoses. The enzymatic defects in the mucopolysaccharidoses are inherited in an autosomal recessive manner, except for Hunter disease, which is X-linked recessive.

Mnemonic A Hunter will aim for the X: Hunter disease is the only mucopolysaccharidosis that is X-linked recessive.

Table 1-4-1. Mucopolysaccharidoses. Urinary Metabolites'

Signs and Symptomst

a-L-iduronidase Iduronosulfate sulfatase 4 variants, incl. heparan N-sulfatase (sulfamidase) N-acetylglucosaminidase N-acetylgalactosamine-6sulfatase a-L-iduronidase N-acetylgalactosamine-4sulfatase

HS,DS HS,DS HS

C, MS, CNS, CV MS,CNS MS,CNS

KS, C-6-S

C,MS,CV

HS,DS DS

C,MS,CV C,MS,CV

~-glucuronidase

HS,DS

C(+/-), MS, CNS( +/-), CV

Type

Enzyme Defect

III

(Hurler) (Hunter) (Sanfilippo)

IV

(Morquio)

V VI

(Scheie) (MaroteauxLamy) (Sly)

II

VII

*HS = heparan sulfate; DS = dermatan sulfate; KS = keratan sulfate; C-6-S = chondroitin-6-sulfate. corneal clouding; MS = musculoskeletal abnormalities (dwarfism); CNS = neurologic deterioration; CV = cardiovascular abnormalities.

Clinical Correlate

tc =

4. Glycoproteins and mucoproteins also contain carbohydrate covalently linked to protein, but there are no repeating disaccharides, and the carbohydrate portion usually contains fewer than 20 monosaccharides. Glycoproteins are found in connective tissue (collagen), in plasma (plasma proteins and some peptide hormones), on cell surfaces as antigens (i.e., blood groups), and as components of mucus. There are three types of linkages between protein and carbohydrate. The amino acid side chains involved in the protein-carbohydrate linkage are: a. Asparagine (N-linkage) is found in plasma and cell surface proteins. b. Serine (O-1inkage) is found in mucous and connective tissue proteins. c. 5-Hydroxylysine (O-linkage) is found in collagen.

Glycoprotein synthesis starts in the endoplasmic reticulum and is completed in the Golgi. One of the last steps in glycoprotein synthesis is putting "zip codes" on proteins that are targeted for a particular destination. For example, glycoproteins that are targeted for lysosomes have mannose-6-phosphate in their carbohydrate chains. Receptors on lysosomes recognize this signal and vesicles containing these proteins pinch off and fuse with lysosomes.

I-cell disease is characterized by coarse facial features, musculoskeletal deformities, and recurrent respiratory problems; death usually occurs within the first decade. The distinguishing feature of I-cell disease is a deficiency of mUltiple lysosomal enzymes and the presence of high concentrations of these enzymes in the plasma. The enzymes are being synthesized but are being sent to the wrong destination due to a deficiency in N-acetylglucosaminylphosphotransferase, resulting in the absence of mannose-6-phosphate.

meClical

41

Biochemistry

GLUCOSE ENTRY AND TRAPPING IN CELLS Circulating glucose supplies all cells with a source of energy. The entry of glucose into cells is mediated by a group of carrier proteins (GLUT proteins) that span the plasma membrane. Net transport across the membrane is ensured by coupling glucose transport with phosphorylation, a process that keeps the intracellular glucose concentration low and continues to shift the equilibrium toward glucose uptake by cells (Figure 1-4-6). The membrane is highly impermeable to the phosphorylated compounds. Therefore, phosphorylation renders glucose transport irreversible.

Hexokinase (all tissues)

[Glucose]

[Glucose]

mM

J..LM

--------il..~

Glu-6-P

Glucokinase (liver)

GLUT Carrier protein (tissue specific)

Figure 1-4-6. Glucose transport and trapping in cells.

A. Glucose carriers (GLUT proteins). A family of homologous proteins, known as GLUT proteins, are responsible for transporting glucose into the cell. These proteins differ in tissue specificity, their affinity for glucose, and the maximum rate at which they can transport glucose across the plasma membrane. 1. Skeletal muscle and adipose tissue respond to insulin by increasing their uptake of glucose. These tissues contain GLUT-4, a glucose carrier with spare copies in the Golgi membrane. Stimulation by insulin results in translocation of spare carriers into the plasma membrane. 2. Liver has a high density of GLUT-2, a transporter that has a low affinity (high Km) for glucose and is not saturated by the increased concentration of glucose in the portal circulation following a high-carbohydrate meal.

Mnemonic Hexokinase places a hex on glucose, with lKm and lv max. Hexokinase is inhibited by i[G6P]. Glucokinase is a glutton for glucose, with iKm and ivmax ; it does not seem to be affected by the amount of glucose-6-phosphate.

3. The brush-border membrane of intestinal and kidney cells contains S-GLUT, a carrier that requires sodium for glucose transport.

4. Brain and red blood cells are rich in GLUT-I. This transporter has a high affinity (low Km) for glucose and is normally saturated, ensuring a constant supply of glucose to these tissues. B. Glucose phosphorylation and trapping. The phosphorylation of glucose in most cells is catalyzed by hexokinase. Liver contains glucokinase, an isozyme that has a much lower affinity (higher Km) for glucose and is never saturated under physiologic conditions. Therefore, the properties of glucokinase, in concert with GLUT-2, allow the liver to reduce the concentration of glucose in the portal circulation following a meal.

GLYCOLYSIS Glycolysis, also known as the Embden-Myerhof pathway, is the central pathway of glucose metabolism. It occurs in the cytosol of all cells. Glycolysis is defined as the pathway that converts glucose to pyruvate. For each mol of glucose converted to pyruvate, 2 mols of ATP are consumed and 4 mols are generated, with a net production of 2 mols of ATP per mol of glucose (discussed below). In the presence of well oxygenated mitochondria, pyruvate can be completely oxidized to CO 2 and H 20, resulting in a total of 36-38 mols ATP per mol of glucose. Under anaerobic conditions, however, pyruvate is converted to lactate. The formation of lactate provides a mechanism

42

KAPLA~. I me....ca

Carbohydrates

for regenerating NAD+ from NADH, a condition that is essential for glycolysis to continue. Anaerobic glycolysis plays an important role in ATP production under conditions where O2 is limited, e.g., in exercising skeletal muscle or in cells that lack mitochondria, such as red blood cells. A. Functions. Glycolysis generates ATP and provides intermediates that can be used in other pathways. For example, both glucose-6-phosphate and pyruvate act as branch points in metabolism because they are substrates for enzymes in other pathways. B. Pathway and enzymes. The pathway of glycolysis is shown in Figure 1-4-7. It can be divided

into two stages. The first stage requires the expenditure of ATP, whereas the second stage results in the net production of ATP.

*2(3-Phosphoglycerate)

a-D-glucose

A~+1 Glucokinase Hexokinase

COOH I H-C-OH

Mg ADP

I Glucose-6-Phosphate

1

1

~I Phosphoglucoisomerase

CH -o-® 11 Phosphoglycerate ~ Mutase

I

Fructose-6-®

M;;+1 ADP

1

*2(2-Phosphoglycerate)

H-1~:~

Phosphofructokinase- 1 1

I

CH20H

~

Fructose-1 ,6-bisphosphate

1:

HjJ

Glyceraldehyde-3-®

Dihydroxyacetone- ®

H-?=O H-C-OH

9H2- 0- ® C=O

I

CH2-0-®

R

NAD+

~

NADH + H+

Enolase 1

1

[AfdOfase]

I C~OH

~ *2(2-Phosphoglycerate)

Triosephosphate Isomerase

IGlyceraldehyde-3P-Dehydrogenase I

*2( Phosphoenolpyruvate) COOH I c-cr® II CH 2

2ADP~

I

I

MgH pyruvate Kinase . . 2ATP *2(Enolpyruvate) 00H

9

C-OH II CH 2

*2(1,3)-Bisphosphoglycerate

tSpontaneous

O=?-O-® H-C-OH

2(Pyruvate)

I

2A~;~~ ::hogl~emte K'oose I 1

*2ATP

2(3-Phosphoglycerate) - - - - - - - - - - - - '

EH NADH+~ NAD

+

.-'L-a-,ct-at;-e----, Dehydrogenase

*2 (Lactate) COOH I

HO-C-H I

*2 moles per mole glucose

CH3

Figure 1-4-7. Glycolysis.

meClical

43

Biochemistry

1. Stage 1 of glycolysis. Glucose is converted to fructose-1,6-P 2 through three sequential reac-

tions. ATP is required for the addition of phosphate groups to both ends of the monosaccharide. The two key enzymes in stage 1 are hexokinase and phosphofructokinase-1 (PFK-1). a. Hexokinase (or glucokinase in liver) uses ATP to convert glucose to glucose-6phosphate. In the next step, glucose-6-phosphate is isomerized to fructose-6-phosphate. b. PFK-l uses ATP to add phosphate to the C-1 of fructose-6-phosphate, with the formation of fructose-1,6-bisphosphate. This reaction is the rate-limiting step in glycolysis. PFK-1 is therefore the primary site of regulation in glycolysis. c. Phosphofructokinase-l and phosphofructokinase-2 both use fructose-6-phosphate as substrate. However, these enzymes have distinctly different functions. The product of the PFK-1 reaction is fructose-1 ,6-P 2, an intermediate in glycolysis. The product of the PFK-2 reaction is fructose-2,6-P 2 • The only known function offructose-2,6-P 2 is to act as an allosteric effector that activates glycolysis and inhibits the opposing pathway of gluconeogenesis. The relationship between PFK-1 and PFK-2 is shown in Figure 1-4-8.

GLUCOSE ~-------

!i

Fructose-6-phosphate

Pi Fructose-1,6-bisphosphatase

f

\ : Fructose-2,6-bisphosphate

..... . . . . .

/

',,+, . . . . . .v_/ /

!i

Fructose-1,6-bisphosphate

---- ----------

' - ....... (activator)

.........

/ /

PYRUVATE ..-/

/

/

e

(inhibitor)

/ /"

Figure 1-4-8. Relationship between PFK-1 and PFK-2.

2. Stage 2 of glycolysis. The function of the second stage is to produce ATP. It begins with the cleavage of fructose-1,6-bisphosphate by aldolase into two phosphorylated trioses (dihydroxyacetone phosphate and glyceraldehyde-3-phosphate). These two compounds are interconvertible through the action of triosephosphate isomerase. This reaction allows both trioses to proceed by a common pathway. Thus, beginning from this step in glycolysis, each mol of glucose can be considered to produce two mols of glyceraldehyde-3-phosphate and all subsequent intermediates. The remaining steps in the pathway are concerned with generating intermediates having high-energy phosphate groups that can be transferred to ADP with the formation of ATP. Two intermediates, 1,3-bisphosphoglycerate and phosphoenolpyruvate, have enough energy to drive the synthesis of ATP. There are three important enzymes in stage 2 of glycolysis: glyceraldehyde-3-phosphate dehydrogenase (G-3-P DH), 3-phosphoglycerate kinase, and pyruvate kinase. Under anaerobic conditions, lactate dehydrogenase is an also important enzyme in glycolysis. a. Glyceraldehyde-3-phosphate dehydrogenase catalyzes a reversible reaction that occurs in two steps. First, the aldehyde group is oxidized to a carboxylic acid, with NAD+ being reduced to NADH. (Note that this is the only oxidation reaction in glycolysis.) Second,

44

KAPLA~. I meulca

---------------

Carbohydrates

inorganic phosphate is covalently linked to the carboxyl group, forming 1,3bisphosphoglycerate. The phosphate bond created in this reaction is a high-energy bond, with a L1GO of -ll.S kcal/mol (only 7.3 kcal/mol are needed to convert ADP to ATP). b. 3-Phosphoglycerate kinase transfers the high-energy phosphate group from 1, 3-bisphosphoglycerate to ADP, producing ATP and 3-phosphoglycerate. Thus, 3-phosphoglycerate kinase is one of the ATP-producing enzymes in glycolysis. At this point, the energy consumed in stage 1 has been replaced. In the next two reactions, the phosphate group of 3-phosphoglycerate is rearranged to form another high-energy bond. Phosphoglycerate mutase moves the phosphate group from carbon-3 to carbon2, forming 2-phosphoglycerate. Enolase then dehydrates 2-phosphoglycerate to form phosphoenolpyruvate, a high-energy compound with a L1GO of -14.S kcal/mol.

In a Nutshell Intermediate Enzyme

Product

1,3-bisphospho- Phosphoglycerate glycerate kinase

ATP

Phosphoenolpyruvate

Pyruvate ATP kinase (irreversible)

Glyceraldehyde- G-3P 3-phosphate dehydrogenase

NADH

c. Pyruvate kinase catalyzes the last reaction in glycolysis. The phosphate group from phosphoenolpyruvate is transferred to ADP with the formation of ATP and pyruvate. Thus, the pathway results in a net of mols of ATP per mol of glucose. This reaction is irreversible, and pyruvate kinase is a secondary site for regulation of glycolysis.

d. Lactate dehydrogenase participates in glycolysis only under anaerobic conditions. Lactate dehydrogenase reduces pyruvate to lactate in a reaction that uses NADH and regenerates NAD+. In cells with well oxygenated mitochondria, lactate does not form in significant amounts. However, when oxygenation is poor, as in heavily exercising muscle, shock, or cardiopulmonary arrest, both the citric acid cycle and oxidative phosphorylation become relatively inactive, and most of the cellular ATP is generated by glycolysis. Because glycolysis requires NAD+ (step "a" above), the formation of lactate serves as a mechanism for regenerating NAD+ so that glycolysis can continue. In states of prolonged anoxia, large amounts oflactate accumulate. The lactate is transported to the liver where it can be used to resynthesize glucose. C. Regulation of glycolysis. There are three steps in glycolysis that are regulated: The primary

site of regulation is PFK-1, the enzyme that catalyzes the slowest step in the pathway. Secondary sites of regulation are hexokinase/glucokinase and pyruvate kinase. The regulatory properties of these enzymes are summarized in Table 1-4-2. PFK-1 is inhibited by ATP and citrate, molecules that indicate a high-energy status. When AMP accumulates, signaling the need for ATP, PFK-1 is activated. Pyruvate kinase is also inhibited by molecules that indicate a high-energy state (ATP and acetyl-CoA). When fructose-1,6-bisphosphate accumulates, it feeds forward and activates pyruvate kinase. In liver, the most important activator of glycolysis is fructose-2,6-bisphosphate, a compound whose concentration is increased by insulin and decreased by glucagon.

Table 1-4-2. Regulation of glycolysis. Enzyme

Mode of Regulation

Effect

Hexokinase

Allosteric

Inhibition: glucose-6-P*

Glucokinase t

Enzyme synthesis

Induced by insulin, not inhibited by glucose-6-P

PFK-1

Allosteric

Activation: AMP, fructose-2,6-P 2 t Inhibition: ATP, citrate

Pyruvate kinase

Allosteric

Activation: fructose-1 ,6-P2 Inhibition: ATP, acetyl-CoA, alanine t

Covalent t

Inhibited by phosphorylation t

Clinical Correlate Hemolytic anemia is caused by excessive destruction of red blood cells. A deficiency in pyruvate kinase is a common enzyme deficiency that results in premature lysis of the red blood cell. The red blood cell has no mitochondria and is totally dependent on glycolysis for ATP. A deficiency in pyruvate kinase, therefore, markedly decreases the ability to synthesize ATP. Maintenance of the biconcave shape of the red blood cell is dependent on ATP. Loss of shape signals uptake and turnover by the spleen. In addition, decreased ion pumping by Na+/K+ ATPase results in loss of ion balance and causes osmotic fragility, leading to swelling and lysis.

'Accumulates when ATP is high. t Effects are liver specific.

meCtical

45

Biochemistry

D. Fate(s) of pyruvate. Pyruvate, the end product of glycolysis, plays a central role in metabolism, as shown in Figure 1-4-9. Pyruvate can be reversibly converted to lactate and to alanine. Both of these reactions occur in the cytosol. Lactate is generated under anaerobic conditions by lactate dehydrogenase. Alanine is formed mainly in skeletal muscle, where it serves as a vehicle for transferring amino groups from muscle to liver, where they are then incorporated into urea (discussed in Chapter 6 of this section). Pyruvate also undergoes two irreversible reactions, both occurring in the mitochondria. It can be carboxylated to oxalo-acetate, which can replenish TCA cycle intermediates or be used for gluconeogenesis. The oxidative decarboxylation of pyruvate results in acetyl-CoA, which can be used as a building block for fatty acids, or it can be oxidized to C02 and H 20 by the TCA cycle and oxidative phosphorylation to generate ATP.

Pyruvate

2

Lactate

4

Alanine

3

Acetyl-CoA

Oxaloacetate

/~

/~ Fatty acids

TeA cycle

Gluconeogenesis

Enzymes: 1. Lactate dehydrogenase; 2. transaminase; 3. pyruvate carboxylase; 4. pyruvate dehydrogenase

Figure 1-4-9. Metabolic fates of pyruvate.

1. Pyruvate dehydrogenase (PDR). PDH is a multienzyme complex (Figure 1-4-10) that

converts pyruvate to acetyl-CoA by oxidative decarboxylation. This reaction is irreversible, and there is no enzyme in humans that will reverse the reaction. The overall reaction catalyzed by PDH is: Pyruvate + CoA + NAD+

46

KAPLAlfdme leaI

-7

Acetyl-CoA + CO 2 + NADH

The enzyme complex is located in the mitochondria and consists of three distinct enzyme activities (decarboxylase, dihydrolipoyl transacetylase, and dihydrolipoyl dehydrogenase). Five coenzymes are required (thiamine pyrophosphate, lipoic acid, Coenzyme A, FAD, and NAD+). The reaction occurs in several steps as shown in Figure 14-10. In the first step, the decarboxylase (E 1) and its cofactor, thiamine pyrophosphate (TPP), release CO 2. The C2 fragment that remains is oxidized by lipoic acid, transferred to CoA, and finally released as acetyl-CoA. These reactions are catalyzed by dihydrolipoyl transacetylase (E z). The last step, catalyzed dihydrolipoyl dehydrogenase (E 3 ), requires FAD and NAD+ to regenerate the initial oxidized form of lipoic acid. The cofactors TPP, lipoic acid, and FAD never leave the enzyme complex.

carbohydrates

CHTCH-TPP

I OH

Figure 1-4-10. The pyruvate dehydrogenase complex.

2. Regulation of pyruvate dehydrogenase. The activity of PDH is dependent on the energy state of the cell as reflected by the levels of acetyl-CoA, ATP, and NADH. It is activated by ADP, CoA, and NAD+. Acetyl-CoA and NADH are powerful inhibitors of pyruvate dehydrogenase activity. As shown in Figure 1-4-11, PDH undergoes phosphorylation/dephosphorylation. A specific protein kinase and protein phosphatase are associated with the multienzyme complex. The kinase is activated by acetyl-CoA and NADH. The phosphatase is activated by insulin. The regulation of pyruvate dehydrogenase is important to fuel conservation. For example, the oxidation of carbohydrate, fat, and proteins all produce acetyl-CoA and NADH (as seen in Figure 1-3-1 in the Bioenergetics chapter). Thus, when fatty acids are being oxidized, carbohydrate (pyruvate) and protein are conserved.

NADH ~~ Acetyl-CoA

.lG> Kinase

Insulin

~ Phosphatase

Figure 1-4-11. Regulation of PDH activity.

KAPLA~.

I

meulC8

47

Biochemistry

3. Homology of pyruvate dehydrogenase with other enzymes. Two other multienzyme complexes in human metabolism have similar structures to pyruvate dehydrogenase. These are a-ketoglutarate dehydrogenase (in the TCA cycle) and branched-chain keto acid dehydrogenase (in amino acid catabolism). All three of these multienzyme complexes catalyze the oxidative decarboxylation of a-ketoacids. They all have E1> E2> and E3 enzyme activities that require the same five coenzymes.

Clinical Correlate Lactic acidosis arising from a deficiency in pyruvate dehydrogenase (PDH) may be an acquired or an inherited condition. The inherited form frequently presents in the neonatal period with vomiting, hypotonia, neurologic deficits, and persistent acidosis. The accumulation of pyruvate behind the metabolic block pushes the equilibrium of the reversible lactate dehydrogenase and alanine transaminase reactions, thus accumulating lactate and alanine. Acquired PDH deficiency is frequently seen in chronic alcoholics who have a thiamine deficiency. The elevated fat content of the diet given as treatment results in an excess of acetyl-eoA, which is converted to ketones. The ketones can be used as fuel by the brain, thereby bypassing the PDH reaction.

E. Energetics of glycolysis. The amount of ATP derived from glucose oxidation is dependent on the availability of oxygen. 1. Anaerobic glycolysis. When oxygen is limited, glycolysis can be summarized by the equation given below. For each mol of glucose consumed, 2 mols oflactate and 2 mols of ATP are produced. There is no net accumulation of NADH. Glucose + 2 ADP + 2 Pi ~ 2 lactate + 2 ATP 2. Aerobic glycolysis. When oxygen is plentiful, glycolysis can be summarized as follows: Glucose + 2 NAD+ + 2 ADP + 2 Pi ~ 2 pyruvate + 2 ATP + 2 NADH For each mol of glucose consumed, 2 mols each of pyruvate, ATP, and NADH are produced. The NADH can undergo oxidative phosphorylation, producing either 2 or 3 mols of ATP, depending on how electrons in the cytosolic NADH are transported into the mitochondria. Use of the malate shuttle results in 3 mols of ATP per NADH, whereas use of the a-glycerol-phosphate shuttle results in 2 mols of ATP per NADH. Additionally, each mol of pyruvate can be transported into the mitochondria and oxidized to CO 2 and H 2 0 by the TCA cycle and oxidative phosphorylation, resulting in 15 mols of high-energy phosphate (ATP or GTP) per mol of pyruvate. The enzymes involved in generating ATP (or GTP) from glucose are summarized in Table 1-4-3.

F. Poisons of glycolysis. Many compounds shut down the glycolytic pathway by inhibiting one or more of the enzymes. 1. 2-Deoxyglucose is converted by hexokinase to 2-deoxyglucose-6-phosphate. This compound is not a substrate for phosphoglucoisomerase. It accumulates and inhibits hexokinase, preventing the phosphorylation of glucose to glucose-6-phosphate. 2. Iodoacetate and other reagents that react with sulfhydryl groups inactivate glyceraldehyde3-phosphate dehydrogenase, which has an essential sulfhydryl group in its active site. 3. Fluoride inhibits enolase by complexing with 2-phosphoglycerate and Mg2+, making the substrate unavailable. 4. Arsenate inhibits ATP production by acting as a substrate for glyceraldehyde-3-phosphate dehydrogenase, producing a compound that breaks down spontaneously to 3-phosphoglycerate and arsenate. Thus, no ATP is formed in the reaction.

48

meclical

Carbohydrates

Table 1-4-3. Energetics of aerobic oxidation of glucose. Pathway

Enzyme

Product

Method of Energy Generation

1. Glyceraldehyde-3-P dehydrogenase

2 NADH

Oxidative phosphorylation

2 Phosphoglycerate kinase

2 ATP

Substrate-level phosphorylation

2

3. Pyruvate kinase

2 ATP

Substrate-level phosphorylation

2

ATP/Glucose

(Cytosol) Glycolysis 4-6*

ATP produced/glucose ATP consumed/glucose (hexokinase and phosphofructokinase)

8-10*

Net ATP produced/glucose

6-8*

(Mitochondria) PDH & TCA Cycle 1. Pyruvate dehydrogenase

-2

2 NADH

Oxidative phosphorylation

6

2. Isocitrate dehydrogenase

2 NADH

Oxidative phosphorylation

6

3. a-Ketoglutarate dehydrogenase

2 NADH

Oxidative phosphorylation

6

4. Succinate thiokinase

2 GTP

Substrate-level phosphorylation

2

5. Succinate dehydrogenase

2 FADH2

Oxidative phosphorylation

4

6. Malate dehydrogenase

2 NADH

Oxidative phosphorylation

6

Net ATP produced/glucose

30

Total ATP per glucose (aerobic oxidation) = 36-38* Total ATP per glucose (anaerobic oxidation) = 2 (Reactions 2 and 3 of glycolysis) *Transfer of electrons in cystolic NADH via malate shuttle produces mitochondrial NADH and 3 ATP, whereas transfer via a-glycerol-P shuttle produces FADH2 and 2 ATP.

GLUCONEOGENESIS Gluconeogenesis is the pathway for de novo synthesis of glucose from C 3 and C 4 precursors. This pathway is distinct from glycogenolysis, which gives rise to preformed glucose. Gluconeogenesis occurs mainly in the liver and kidney, with a small amount occurring in the epithelium of the small intestine. The pathway requires both mitochondrial and cytosolic enzymes. A. Function. The role of gluconeogenesis in homeostasis is to maintain proper blood glucose levels and to provide glucose for the body. The brain, central nervous system, and red blood cells are dependent on glucose for all or most of their energy. When fasting persists for more than 12 to 24 hours, liver glycogen stores are exhausted and gluconeogenesis provides glucose for these tissues. The primary precursors for de novo glucose synthesis are lactate (from KAPLAlf I meillea

49

Biochemistry

red blood cells and anaerobic muscle), glycerol (from triacylglycerol degradation in adipocytes), and amino acids (from protein degradation in skeletal muscle).

Clinical Correlate

B. Pathway and key enzymes. Gluconeogenesis uses the seven enzymes in the glycolytic pathway that catalyze reversible reactions. Additionally, there are four enzymes unique to gluconeogenesis that are required to bypass the three irreversible reactions in glycolysis (Figure 1-4-12).

A deficiency in any of the four key enzymes used in gluconeogenesis will result in hypoglycemia.

GLUCONEOGENESIS

GLYCOLYSIS

E

GK

Pyruvate Kinase

PFK-1

f \G6P~F6P f \F-1,6-P2~)

DHAP

Glu

"---/

G6Pase

G3P~ 3PG~

"---/

2PG~

Pyruvate

PEP

/

\

FBPase-1

Pyruvate Carboxylase

PEPCK

\

I

Oxaloacetate Figure 1-4-12. Gluconeogenesis and glycolysis.

Each of the four enzymes unique to gluconeogenesis also catalyzes an irreversible reaction, but in the opposite direction from the corresponding enzyme in glycolysis. To bypass the pyruvate kinase reaction, two enzymes are required: pyruvate carboxylase and phosphoenolpyruvate carboxykinase (PEPCK). 1. Pyruvate carboxylase, an enzyme located in the mitochondria, catalyzes the carboxylation

of pyruvate to oxaloacetate, the first step in gluconeogenesis (Figure I-4-13). Acetyl-CoA is an allosteric activator that must be present for the enzyme to function. Biotin serves as a carrier of the "activated carboxyl" group derived from bicarbonate. ATP hydrolysis to ADP and Pi provides the energy needed to generate the "activated carboxyl" group.

COOH

I + C=O I CH 3

Pyruvate

Pyruvate Carboxylase

COOH

I

C=O

I

CH 2

I

COOH Oxaloacetate

Figure 1-4-13. The pyruvate carboxylase reaction.

2. Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the second step in gluconeogenesis (Figure 1-4-14). PEPCK is a cytosolic enzyme that phosphorylates and decarboxylates oxaloacetate to form phosphoenolpyruvate. The phosphate group is derived from GTP, which is energetically equivalent to ATP. Thus, to bypass the irreversible pyruvate

50

meClical

Carbohydrates

kinase reaction, two enzymes are required and the equivalent of 2 mols of ATP are consumed in converting pyruvate to phosphoenolpyruvate.

COOH

I

C=O

I

CH2

I

Phosphoenolpyruvate Carboxykinase

f"\

GTP

COOH

•

I C-O-® + CO 2

II

CH 2

GOP

COOH Oxaloacetate

Phosphoenolpyruvate Figure 1-4-14. The PEPCK reaction.

3. Fructose-I,6-bisphosphatase (FBPase-l) catalyzes the hydrolysis offructose-l,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. This reaction bypasses the irreversible step in glycolysis catalyzed by PFK-1.

FBPase-l Fructose-l,6-bisphosphate + H 2 0

------~ ..~

fructose-6-phosphate + Pi

FBPase-l is a cytosolic enzyme that is activated by ATP and citrate and inhibited by AMP and fructose-2,6-bisphosphate. These same compounds alter the activity of the opposing enzyme, PFK-l, but in the opposite direction (see Table 1-4-2). Thus, glycolysis is inhibited when gluconeogenesis is occurring, and vice versa. 4. Glucose-6-phosphatase (G6Pase) catalyzes the last step in gluconeogenesis by removing the phosphate from glucose-6-phosphate and releasing free glucose. G6Pase Glucose-6-phosphate + H 2 0

------~ ..~

glucose + Pi

Clinical Correlate A deficiency in glucose-6phosphatase results in von Gierke disease, which is characterized by low serum glucose and high serum lactate. The elevated lactate results from both a compensatory increase in liver glycolysis and a decreased ability to use lactate as a substrate for gluconeogenesis.

This reaction bypasses the irreversible hexokinase step in glycolysis. G6Pase is associated with the endoplasmic reticulum and is found only in liver, kidney, and intestinal epithelium. The absence of G6Pase in skeletal muscle accounts for the fact that muscle glycogen cannot serve as a source of blood glucose. C. Overall reaction of gluconeogenesis. The conversion of pyruvate to glucose via gluconeogenesis is shown below. For every mol of glucose synthesized, six ATP equivalents are required. Four ATP equivalents are required to convert pyruvate to phosphoenolpyruvate, and two are required to reverse the 3-phosphoglycerate kinase step in glycolysis. The NADH is used to reverse the step catalyzed by glyceraldehyde-3-phosphate dehydrogenase.

2 pyruvate + 4 ATP + 2 GTP + 2 NADH

--7

glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD+

The ATP required for gluconeogenesis is derived from the oxidation of fatty acids. It should be noted that the reaction may begin with lactate rather than pyruvate because the lactate dehydrogenase reaction is reversible. KAPLA~.

meulCa

I

51

Biochemistry

I Blood I ~

Glucose

ILiver I

Glucose-6-phosphate

t t ..

. .. ...

Pyruvate

I Muscle I

Glycogen

Glycogen

Lactate

Lactate

. .. ollIE

, Glucose-6-phosphate

tt ... Pyruvate

~

I Blood I Lactate

""'-

Figure 1-4-15. The Cori cycle.

D. Precursors for gluconeogenesis. Any compound that can be converted to an intermediate in glycolysis or the citric acid cycle can serve as a precursor for gluconeogenesis. 1. Lactate. The function of the Cori cycle is to conserve glucose by recycling lactate

formed from anaerobic glycolysis in red blood cells and skeletal muscle. Lactate is released from these tissues into the blood and is taken up by the liver. It is then reconverted to glucose via gluconeogenesis to provide more glucose for energy. The recycling process is illustrated in Figure 1-4-15. 2. Alanine. Skeletal muscle releases large amounts of alanine formed by the transamination of pyruvate. The alanine is taken up by the liver and is reconverted to pyruvate, which is used for glucose synthesis. This recycling is known as the alanine cycle. The alanine cycle provides a means of conserving carbohydrate. Note that neither the alanine cycle nor the Cori cycle result in the net synthesis of glucose. 3. Glucogenic amino acids. All of the common amino acids except lysine and leucine are glucogenic, i.e., they can be degraded to intermediates in either glycolysis or the TCA cycle and thus can serve as precursors for glucose synthesis. The nitrogen arising from these amino acids is disposed of as urea. Lysine and leucine are strictly ketogenic and cannot be converted to glucose. 4. Glycerol. The hydrolysis of triacylglycerols in adipose tissue produces glycerol that is

In a Nutshell

released into the blood. The liver takes up glycerol and converts it to glycerol-3 phosphate, which can be oxidized to dihydroxyacetone phosphate, a gluconeogenic intermediate.

Enzyme

Activators Inhibitors

Pyruvate carboxylase

Acetyl-CoA

Phosphoeno~

ADP ADP

pyruvate carboxykinase Fructose-l,6ATP bisphosphatase Citrate Glucagon Epinephrine

AMP Fructose-2,6bisphosphate

Glucose-6phosphatase

Glucose

52

meCtical

5. Odd-numbered fatty acids. The oxidation of odd-numbered fatty acids produces one molecule of propionyl-CoA from the ffi-end. Propionyl-CoA can be converted to succinylCoA, an intermediate in the TCA cycle, and then to glucose. In contrast, degradation of even-numbered fatty acids produces only acetyl-CoA, which is not a precursor of glucose. 6. Fructose. Fructose (from dietary sucrose) can be phosphorylated to fructose-I-phosphate and cleaved to glyceraldehyde and dihydroxyacetone phosphate (DHAP), a glucogenic intermediate. Glyceraldehyde can be converted to glucogenic intermediates either by reduction to glycerol or by phosphorylation to glyceraldehyde-3-phosphate. 7. Galactose. Galactose can be phosphorylated by the liver to galactose-I-phosphate and then converted to glucose-6-phosphate by a series of reactions discussed below.

Carbohydrates

E. Regulation of gluconeogenesis. Gluconeogenesis is tightly regulated in order to maintain blood glucose at a normal level. Regulation is accomplished by three methods: substrate availability, enzymatic control, and hormonal control. l. Substrate availability. Under conditions of fasting, the primary substrates for gluconeo-

genesis are amino acids derived from protein degradation. The oxidation of fatty acids provides the energy required. All of these processes are coordinated by hormonal control. 2. Enzymatic control. The enzymes that are regulated are unique to gluconeogenesis. The opposing enzymes of glycolysis and gluconeogenesis at the irreversible steps in these pathways are regulated reciprocially so that when one is active the other is inactive. This prevents futile cycling of substrate by ensuring that glycolysis and gluconeogenesis are not occurring at the same time. a. Pyruvate carboxylase is activated by acetyl-CoA. The opposing enzyme in glycolysis, pyruvate kinase, is inhibited by acetyl-CoA. b. Fructose-l,6-bisphosphatase is activated by ATP and citrate and is inhibited by AMP and fructose-2,6-bisphosphate. The opposing enzyme in glycolysis, PFK-l, is inhi-bited by ATP and citrate and is activated by AMP and fructose-2,6-bisphosphate. c. Glucose-6-phosphatase and the opposing enzyme glucokinase are both regulated by

substrate availability. Both enzymes have high Km values and are not saturated under the conditions in the cell. Therefore, as glucose-6-phosphate and glucose concentrations increase, the activities of glucose-6-phosphatase and glucokinase increase, respectively. 3. Hormonal control. The major hormones involved in regulating blood glucose levels are glucagon and insulin, which have opposing effects. Glucagon promotes glucose synthesis and release into the blood, and insulin promotes glucose uptake and storage. Both of these effects are mediated by intracellular concentrations of cAMP. Cellular cAMP levels increase in response to glucagon and decrease in response to insulin. cAMP activates protein kinase-A. The active form of the protein kinase phosphorylates a subset of enzymes and alters their activities. Under conditions of low blood glucose, the secretion of glucagon initiates several responses that lead to restoring blood glucose levels. Responses to elevated glucagon include: a. Inhibition of pyruvate kinase. Phosphorylation of this enzyme leads to its inactivation, thereby decreasing the amount of glucose consumption by glycolysis. b. Decreased concentration of fructose-2,6-bisphosphate. The degradation of fructose2,6-bisphosphate is stimulated by glucagon. Therefore, its inhibitory effect on FBPase1 and its stimulatory effect on PFK-l are both relieved, resulting in increased gluconeogenesis and decreased glycolysis. c. Increased synthesis of key enzymes. The synthesis of PEPCK, FBPase-l, and glucose-

6-phosphatase are increased by glucagon. This is a slower response, taking hours rather that minutes or seconds to occur. Synthesis of these enzymes is also induced by glucocorticoids and catecholamines. d. Increased protein degradation. During gluconeogenesis, proteolysis in skeletal muscle is increased, thus providing an increased supply of amino acids for glucose synthesis. e. Increased lipolysis. The release of fatty acids from adipose triglyceride is stimulated by any hormone that increases intracellular cAMP. Fatty acids are taken up by the liver and oxidized to supply the energy required for gluconeogenesis.

In a Nutshell Increased glucagon ---+ increased cAMP ---+ increased protein kinase-A activity ---+ phosphorylation ---+ decreased pyruvate kinase activity ---+ increased gluconeogenesis

In a Nutshell Increased insulin ---+ decreased cAMP ---+ increased protein phosphatase activity ---+ dephosphorylation ---+ increased F-2, 6-P2 ---+ increased glycolysis

Clinical Correlate Ethanol-induced hypoglycemia is due to the oxidation of ethanol by the liver, leading to the accumulation of excessive NADH and a deficit of NAD+. This prevents the oxidation of lactate to pyruvate, a-glycerol-P to dihydroxyacetone phosphate, and malate to oxaloacetate. Gluconeogenesis therefore fails due to inadequate prescursors, resulting in hypoglycemia.

KAPLAlf _ I meillea

53

Biochemistry

GLYCOGENESIS AND GLYCOGENOLYSIS Glycogen is a highly branched polymer containing glucose molecules linked by a-1,4 glycosidic bonds, with a-1,6 glycosidic bonds between the two glucose molecules at the branch points. Branching increases the solubility of the molecule and facilitates breakdown of glycogen. At the core of the glycogen particle is the protein glycogenin, which serves as the initial primer in the synthesis of glycogen. Glycogenin is not only the primer for the first glucose molecule; it is also the catalyst for the synthesis of the first eight glucose residues of the glycogen molecule. The synthesis and degradation of glycogen occur mainly in the liver and skeletal muscle. The liver may store up to 10% of its wet weight as glycogen. All of the synthesis and degradation occurs at the ends of the branch points. A. Functions. Glycogen is the storage form for glucose in animal tissues. The function of glycogen in skeletal muscle differs from that in the liver.

Mnemonic The well-"red" person is slow, but complete: red muscle fibers are slow, but completely oxidize fuel (TCA cycle).

1. Skeletal muscle degrades glycogen and rapidly metabolizes glucose via glycolysis. This process generates ATP required for muscle contraction. Thus, the function of glycogen in muscle is to provide energy for contraction. In white ("fast") muscle fibers, glucose is released from glycogen and metabolized by glycolysis, with lactate being the major end product. In red ("slow") muscle fibers, pyruvate is completely oxidized by the TeA cycle and oxidative phosphorylation. 2. Liver uses glycogen mainly to regulate blood glucose levels. In response to hypoglycemia, liver glycogen is degraded, and glucose is released into the blood. However, liver glycogen stores become depleted after approximately 12 hours of fasting. In response to hyperglycemia, glucose is removed from the blood and stored as liver glycogen. The pathways of glycogenesis and glycogenolysis are shown in Figure 1-4-16.

y

UDP

Glycogen

UDP-glu

PFl

Glu-1-P

Gll.p

()

Glucose (blood) Figure 1-4-16. Overview of glycogen metabolism.

B. Key enzymes in glycogenolysis. Glycogen degradation occurs at the ends of the branches, where glucose-1-P units are sequentially released by glycogen phosphorylase, the most

54

KAPLAlfdme leaI

Carbohydrates

important enzyme in glycogenolysis. Total degradation requires an additional "debranching" enzyme system. 1. Glycogen phosphorylase cleaves a-I,4 glycosidic bonds by the addition of inorganic

phosphate, a process known as "phosphorolysis': This reaction is the rate-limiting step in glycogenolysis and is regulated both allosterically and hormonally. The glycogen phosphorylase reaction is shown below, where n is the number of glucose units in glycogen. glycogen phosphorylase (Glucose)n

------------.~

P'~

(Glucose)n_l + Glucose-I-P

1

Glycogen phosphorylase will remove glucose units until it gets to within 4-5 glucose units of a branch point, when it no longer binds efficiently to the partially degraded glycogen. 2. Debranching enzymes. The complete degradation of glycogen requires two additional enzymes that make up the "debranching system" (Figure 1-4-17). a[1--t4] chain

Ja·1,6-glucosidase +

o Figure 1-4-17. The glycogen debranching enzymes.

a. a-[1,4] --7 a-[1,4] glucan transferase removes three or four glucose units from a branch point and transfers them to the end of another chain. In this reaction, one a-I,4 bond is being cleaved, and another is being formed. The elongated chain now becomes a substrate for glycogen phosphorylase. b. a-I,6 glucosidase removes the single glucose unit remaining at the branch point and releases it as free glucose.

In a Nutshell

C. Key enzymes in glycogen synthesis. As shown in Figure 1-4-16, the synthesis of glycogen

begins with glucose phosphorylation to glucose-6-phosphate. This reaction is catalyzed by glucokinase in the liver and by hexokinase in other tissues. Glucose-6-phosphate is rapidly and reversibly converted to glucose-I-phosphate by phosphoglucomutase. Before glucose can be added to the glycogen polymer, it must be "activated" or energized. This is achieved by the formation ofUDP-glucose, which serves as the donor of glucose to a glycogen primer. Most monosaccharides are activated by reacting with UTP to form a UDP-sugar. 1. Formation of UDP-glucose. The phosphate group of glucose-I-phosphate reacts with

UTP to form UDP-glucose and pyrophosphate (PPi). Thus, one of the phosphate groups in UDP-glucose is derived from glucose-I-phosphate and the other from UTP, as shown

Enzyme

Activator Inhibitor

Muscle AMP ATP glycogen Ca2+ Glucose-Gphosphorylase Epinephrine phosphate Insulin Glucagon Glucose Liver glycogen Epinephrine Insulin phosphorylase Glycogen synthase

Glucose Insulin

Glucagon Epinephrine

KAPLA~. I meulca

55

Biochemistry

Clinical Correlate Glycogenesis is coupled to the influx of K+ in the cells. Insulin and glucose are therefore given to treat hyperkalemia (high serum K+), inducing glycogenesis and causing an influx of K+ into the cells.

below. The reaction is catalyzed by UDP-glucose pyrophosphorylase. The equilibrium of this reaction is "pulled" toward UDP-glucose formation by the hydrolysis of pyrophosphate to inorganic phosphate. UDP-glucose pyrophosphorylase Glu-1-P + Uridine-P-P-P

..

UDP-Glu + PP j

The linkage between glucose and UDP is a high-energy bond, making it a suitable donor in many biosynthetic reactions. 2. Elongation of glycogen chains. To be active, glycogen synthase requires an existing glycogen chain serving as a primer. If the chain has been totally degraded, then glycogenin, the protein found at the core of the glycogen particle, serves as a primer for glycogen synthesis and accepts the initial glucose residue. Glycogen synthase catalyzes the transfer of glucose from UDP-glucose to the end of a chain. The linkage created is an o,-1,4-glycosidic bond. This is the rate-limiting step in glycogen synthesis and is therefore the site of regulation. glycogen synthase .. (Glucose)n+l + UDP (Glucose)n + UDP-glucose

3. Branching enzyme. After approximately 10 glucose units have been added, a branch point is created by 0,-[1,4] 0,-[1,6] glucan transferase. Forming a branch point involves breaking an 0,-1,4 linkage and creating an 0,-1,6 linkage. The two shorter chains that are produced can now be elongated by glycogen synthase. There are always at least four glucose molecules between branch points. D. Coordinate regulation of glycogenesis and glycogenolysis. Glycogen phosphorylase and glycogen synthase are regulated in a reciprocal manner, such that when one of these enzymes is active, the other is inactive. The primary mode of regulation for both enzymes is hormonal and is mediated by phosphorylation/dephosphorylation. In addition, both glycogen synthase and phosphorylase activities can be affected allosterically.

56

meClical

------~~~-

-~~~

Epinephrine

Glucagon (liver cell)

ADENYLATE CYCLASE

..

"

0

& liver cells)

- . ---= ,R ADENYLATE '

ATP

GLYCOGEN PHOSPHORYLASE KINASE

cAMP-DEPENDENT PROTEIN KINASE

I

CYCI,ASE active

cAMP-

Phosphodiesterase active

Glucose

0(

0(

Glucose i-Phosphate

t

GLYCOGEN PHOSPHORYLASE

a active

Glycogen

5Ie CD; A,"'l

gO

~

.....

*Reactions catalyzed by PHOSPHOPROTEIN PHOSPHATASE Figure 1-4-18. Cascade for activating glycogenolysis.

*

..go ~

{

i

Biochemistry

1. Glycogen phosphorylase. A comprehensive diagram describing the regulation of this

In a Nutshell Hormone (glucagon and epinephrine)

~

G-protein

~

ieAMP

~

Activates PKA

I

Phosphorylates glycogen synthase

~

\

Phosp horylates phosphorylase kinase

~

Inactive Phosphorylates (no glycogenesis) glycogen phosphorylase

~

Active (iglycogenolysis)

enzyme is shown in Figure 1-4-18. Phosphorylation of a specific serine side chain activates the enzyme, resulting in glycogen degradation. The activation of phosphorylase is initiated by the binding of glucagon to liver cell receptors or by the binding of epinephrine to muscle receptors. Both of these hormones increase the intracellular synthesis of cAMP, resulting in the activation of a cAMP-dependent protein kinase (protein kinase A). A single molecule of hormone can generate many molecules of cAMP; each molecule of cAMPdependent protein kinase can phosphorylate (and activate) many molecules of phosphorylase kinase; each molecule of phosphorylase kinase can phosphorylate (and activate) many molecules of glycogen phosphorylase; and each molecule of glycogen phosphorylase can release many molecules of glucose-I-phosphate from glycogen. This "cascade" system amplifies the response initiated by the binding of a few hormone molecules by generating millions of molecules of glucose-I-phosphate. It is noteworthy that phosphorylase kinase can also be activated directly (without phosphorylation) by binding calcium. This is particularly important in skeletal muscle, where the coupling of excitation with contraction is mediated by calcium. Thus, the same regulatory molecule that activates contraction also activates glycogenolysis, which provides the energy for contraction. The active (phospho) form of glycogen phosphorylase can be allosterically inhibited by glucose and ATP, whereas the inactive (dephospho) form can be activated allosterically by AMP. Thus, although covalent modification of these enzymes is the primary mode of regulation, the activity of the phospho- and dephospho- forms can be fine-tuned by the accumulation of intracellular metabolites that act as allosteric effectors. 2. Glycogen synthase. The same signals that activate glycogen phosphorylase inactivate glycogen synthase. Glucagon and epinephrine (via cAMP-dependent protein kinase) stimulate the phosphorylation and inactivation of glycogen synthase. The coordinate regulation of these enzymes prevents futile cycling of substrate that would exist if both enzymes were active at the same time. The inactive (phosphorylated) form of glycogen synthase in muscle can be allosterically activated by glucose-6-phosphate. Many texts refer to glycogen synthase-1 and glycogen synthase-D. The D form is the inactive (phospho) enzyme, and the 1 form is the active (dephospho) form. E. Glycogen storage diseases. A number of enzyme deficiencies have been identified that result in the accumulation of an abnormal type or quantity of glycogen in tissues. Table 1-4-4 lists the major types of glycogen storage disease, the deficient enzyme, and the cardinal clinical feature of each type.

58

me&ical

Carbohydrates

Table 1-4-4. Glycogen storage diseases. Type

Deficient Enzyme

Cardinal Clinical Feature

von Gierke Pompe

Glucose-6-phosphatase a-I, 4-glucosidase

III

Cori

Glycogen debranching

IV

Andersen (amylopectinosis)

Branching enzyme

Severe hypoglycemia Cardiomegaly, muscle weakness, death by 2 years Mild hypoglycemia, liver enlargement Infantile hypotonia, cirrhosis, death by 2 years

McArdle

Muscle glycogen phosphorylase

Hers disease

Hepatic glycogen phosphorylase Muscle phosphofructokinase Hepatic phosphorylase kinase

I II

V VI

VII VIII

Muscle cramps and weakness on exercise Hypoglycemia, cirrhosis Muscle cramps No neuromuscular symptoms, hypoglycemia

It is clear from Table I -4-4 that the clinical consequences of an enzyme deficiency depend on

which enzyme is missing. For example, in type I deficiency, the absence of glucose-6phosphatase, impairs the ability of the liver to release free glucose by glycogenolysis, resulting in severe hypoglycemia. Other consequences include increased uric acid levels due to increased pentose phosphates, increased purine synthesis, and increased purine degradation; acidosis due to the accumulation of lactate; elevated serum lipids due to fat degradation in adipose; and growth retardation due to accelerated protein degradation. In type III deficiency, the absence of debranching enzyme only partially impairs glycogenolysis, resulting in mild hypoglycemia. Glycogen structure is normal in all of the types except III and Iv.

HEXOSE MONOPHOSPHATE SHUNT This pathway, also known as the pentose phosphate pathway, provides an alternative route for the oxidation of glucose. The products of the pathway are CO 2 , pentose phosphates, and NADPH. The shunt branches off of glycolysis at glucose-6-phosphate and re-enters at fructose6-phosphate (Figure 1-4-19). The hexose monophosphate (HMP) shunt is present in the cytosol of all cells. Unlike glycolysis, this pathway neither consumes nor produces ATP. A. Functions. This pathway supplies the cell with NADPH and pentoses. 1. NADPH. Almost all of the NADPH required in reductive biosynthetic processes, such as

the synthesis of cholesterol, fatty acids, and steroid hormones, comes from the hexose monophosphate shunt. Tissues that synthesize large amounts of these compounds have high levels of the NADPH-producing enzymes in the pathway. Red blood cells require large amounts of NADPH to maintain the reduced form of glutathione. Reduced glutathione helps prevent hemolysis by neutralizing the effects of strong oxidizing agents such as superoxide and hydrogen peroxide. Neutrophils, macrophages, monocytes, and other phagocytosing cells require large quantities of NADPH to generate superoxide. These cells use superoxide as a part of the "cidal" process for killing bacteria they engulf. NADPH oxidase reduces molecular oxygen to superoxide in these cells. A deficiency in either NADPH or NADPH oxidase can result in chronic infection.

Clinical Correlate Chronic granulomatous disease is caused by a defect in the NADPH oxidase complex, resulting in neutrophils that do not produce superoxide. Affected individuals therefore present with an inability to kill invading microbes that are engulfed.

meClical

59

Biochemistry

2. Pentoses. The synthesis of nucleotides and some coenzymes (NAD+, NADP+, FAD, CoA) requires ribose-5-phosphate, which is supplied by the HMP shunt. B. Pathway and key enzymes. As shown in Figure 1-4-19, the HMP shunt branches off glycolysis at glucose-6-phosphate. The two pathways equilibrate by having fructose-6-phosphate as a common intermediate. After the early steps in the pathway, which lead to NADPH and ribose-5-phosphate synthesis, a large number of intermediates (shown in brackets) allow excess pentoses to be reconverted to fructose-6-phosphate. The numbers at each reaction refer to the enzyme used. The reactions in the HMP shunt can be divided in two phasesthe oxidative and non oxidative phases. 1. The oxidative phase produces 2 mols of NADPH per glucose oxidized. This phase consists of three reactions starting with glucose-6-phosphate and resulting in ribulose-5phosphate. All of these reactions are essentially irreversible. In the first reaction, the carbon-1 of glucose-6-phosphate is oxidized to a cyclic acid (lactone), with the simultaneous production of NADPH (Figure 1-4-20). In the next reaction, the lactone of 6-phosphogluconate is hydrolyzed to the straight chain form by lactonase. In the last step of the oxidative phase, 6-phosphogluconate is oxidized and decarboxylated, with the formation ofNADPH, CO 2 , and ribulose-5-phosphate. Glucose

~

Glucose-6-P

I

Fructose-6-P

NADPH

NADPH

H2 0

~•

/ . 6-phosphoglucono-o-lactone

6-P-gluconate

2

CO 2

~

/

Ribulose-5-P

3 4

.....If-----~.~

Glyceraldehyde-3-P Erythrose-4-P Xylulose-5-P Fructose-6-P Sedoheptulose-7-P

5,6,7

.....E-----------___ • Ribose-5-P

1

1

Pyruvate

Nucleotide Synthesis

Figure 1-4-19. The hexose monophosphate shunt. (1. Glucose-6-phosphate dehydrogenase; 2. lactonase; 3. 6-phosphogluconate dehydrogenase; 4. pentose-P-isomerase; 5. pentose-P-epimerase; 6. transketolase; 7. transaldolase)

COOH

CH2-00~

~ OH

HO

OH

I

CH2 0@

NADPH

~

~ OH

0

0

H-C-OH

NADPH

I

~

HO-C-H

I

H-C-OH

HO

I

OH

H-C-OH

I

CH2 0@ Glucose-6-@

6-Phosphoglucono-1)-lactone

6-P-Gluconate

Ribulose-5-P

Figure 1-4-20. The oxidative phase of the hexose mono phosphate shunt.

60

meClical

Carbohydrates

2. The nonoxidative phase. All of these reactions are reversible. Ribulose-5-phosphate is isomerized to ribose-5-phosphate, the pentose required for nucleotide synthesis. The remaining reactions involve the transfer of Cz- and Crunits from one sugar to another. Intermediates having 3, 4, 5, 6, and 7 carbons are involved. The key enzymes in the transfer reactions are transketolase and transaldolase. Transketolase transfers C2 -units, using thiamine pyrophosphate as a Cz-carrier between donor and acceptor. Transal-dolase transfers C3 units. The major function of the non oxidative phase is to provide a pathway for recycling excess pentoses. This is particularly important in tissues that require larger amounts of NADPH than pentoses. The recycling is achieved by the formation of fructose-6-phosphate, a common intermediate in the HMP shunt and glycolysis. Fructose-6-phosphate can be isomerized back to glucose-6-phosphate and reused. For every 6 mols of glucose-6-phosphate used in the HMP shunt, 6 mols of CO 2 are produced and 5 mols of fructose-6-phosphate can be returned to glycolysis. C. Regulation of the HMP shunt. The rate-limiting step in the pathway is the initial reaction cat-

alyzed by glucose-6-phosphate dehydrogenase. The amount of this enzyme present in liver and adipose increases when the diet contains large amounts of carbohydrate, a condition that leads to fatty acid synthesis and an increased requirement for NADPH. Glucose-6-phosphate dehydrogenase is allosterically activated by NADP+ and inhibited by NADPH and palmitoyl-CoA.

FRUCTOSE METABOLISM Fructose is second to glucose as a dietary source of carbohydrates. It is mostly derived from the hydrolysis of sucrose in the brush border of the small intestine. The primary site of fructose metabolism is the liver. As the portal blood enters the liver, fructose is very efficiently removed and metabolized by the liver. The liver has three enzymes (fructokinase, aldolase B, and glyceraldehyde kinase) that convert fructose into triose phosphates, intermediates in glycolysis. Because these reactions bypass the rate-limiting step in glycolysis, fructose is metabolized to pyruvate and acetyl-CoA much more rapidly than is glucose. The enzymes of glycolysis, gluconeogenesis, and glycogenesis also allow dietary fructose to be converted to blood glucose or glycogen. The very rapid phosphorylation of fructose by fructokinase may result in a transient decrease in intracellular phosphate concentrations and a diminished ability to synthesize ATP. A. Pathway and key enzymes of fructose metabolism. The metabolism of fructose starts with the conversion to fructose-I-phosphate by fructokinase (Figure 1-4-21). Fructose-I-phosphate is split into two C3 fragments by aldolase B, producing dihydroxyacetone phosphate and glyceraldehyde. Glyceraldehyde can be phosphorylated to glyceraldehyde-3-phosphate by triose kinase. Alternatively, glyceraldehyde can be reduced to glycerol and used for gluconeogenesis or for triacylglycerol synthesis. Both dihydroxyacetone phosphate and glyceraldehyde-3phosphate are intermediates in glycolysis. They can be further degraded by glycolysis, or they be condensed to fructose-I,6-bisphosphate and used for gluconeogenesis or glycogenesis.

Note Transketolase is used to diagnose thiamine deficiency by measuring erythrocyte transketolase activity upon addition of thiamine pyrophosphate.

Clinical Correlate Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive disorder causing t NADPH production -7 a hemolytic anemia. Microscopic examination of erythrocytes reveals the presence of "Heinz bodies," small, dark inclusion bodies of precipitated hemoglobin. Oxidizing agents such as sulfonamides, aspirin, and antimalarial and antituberculous drugs t the amount of superoxide and hydrogen peroxide in the blood. The lack of NADPH -7 a lack of reduced glutathione, which is needed to neutralize the toxic effects of these substances. These agents can therefore aggravate the hemolytic anemia.

In a Nutshell Hexose Monophosphate Shunt • Produces ribose-5phosphate for nucleotide synthesis • Reactions occur in cytoplasm • Produces NADPH for use in anabolic processes (e.g., fatty acid biosynthesis) • Does not produce or consume ATP

meClical

61

Biochemistry

Dihydroxyacetone-P Fructokinase Fructose

)I

Aldolase-8

Fructose-1-P

-----<

ATP

I\

Glyceraldehyde

NADH

~

Glycolysis

ATP Triose kinase

Clinical Correlate Hereditary fructose intolerance (HFI) results from a deficiency of aldolase B, the enzyme that cleaves fructoseI-phosphate to glyceraldehyde and dihydroxyacetone phosphate. This results in an accumulation of fructose-Iphosphate in the liver, which inhibits glycogen phosphorylase and aldolase. Glycogenolysis and gluconeogenesis are thereby both impaired, resulting in severe hypoglycemia and vomiting. Symptoms are reversed after removing fructose and sucrose from the diet.

Clinical Correlate Galactosemia is an autosomal recessive disease resulting from a deficiency in either galactokinase or galactose-Iphosphate uridyl transferase. In both deficiencies, galactose accumulates and is converted to galactitol in the nervous system (retardation) and lens of the eye (cataracts). In the uridyl transferase deficiency, galactose-I-phosphate accumulates in the liver, resulting in hepato-megaly. Treatment requires excluding galactose (and lactose) from the diet.

62

meclical

Glycerol Glyceraldehyde-3-P

Figure 1-4-21. Fructose metabolism by the liver.

B. Abnormalities in frnctose metabolism arise from deficiencies in fructokinase and aldolase B, enzymes unique to fructose catabolism. A deficiency in fructokinase results in essential fructosuria, a benign condition in which fructose appears in the blood and urine and is excreted intact. A deficiency in aldolase B results in hereditary fructose intolerance.

GALACTOSE METABOLISM The primary source of galactose is milk, which contains high amounts of lactose. The concentration of lactose is higher in human milk than in cow's milk, although the caloric intake is about the same. Lactose is hydrolyzed in the small intestine to glucose and galactose. The metabolism of galactose occurs almost entirely in the liver. Three enzymes (galactokinase, galI-P:glu-I-P uridyl transferase, and UDP-galactose-4-epimerase) are required to assimilate galactose into the central pathways of carbohydrate metabolism. A. Pathway and key enzymes of galactose metabolism. Galactose metabolism starts by phos-

phorylation to galactose-I-phosphate by galactokinase. All dietary galactose is phosphorylated in the liver (Figure 1-4-22). Galactose-I-phosphate is then exchanged for the glucose-Iphosphate moiety of UDP-glucose. This reaction is catalyzed by "uridyl transferase" and results in glucose-I-phosphate and UDP-galactose. Finally, UDP-galactose is recycled to UDP-glucose by UDP-galactose-4-epimerase. The overall effect of these three reactions is to convert dietary galactose into glucose-I-P. Through pathways already considered in this chapter, glucose-I-phosphate can be incorporated into glycogen or converted to glucose-6-phosphate. The glucose-6-phosphate can be hydrolyzed by glucose-6-phosphatase to release glucose into the blood, or it can be used by glycolysis to produce pyruvate.

Galactokinase Galactose

( ATP

'lp

,.

Uridyl. transferase Galactose-1-P

7 '\ ,.

UDP-Glu

Glucose-1-P

UDP-Gal

V

4-epimerase

Figure 1-4-22. Metabolism of galactose by liver.

Lipids

Lipids are biologically important compounds that are structurally and functionally diverse. They can be classified into six major groups: fatty acids, triacylglycerols, ketone bodies, cholesterol, phospholipids, and sphingolipids. Each class of lipids plays a specific role in either energy production, transport, and storage, or as a structural component of cell membranes. Numerous diseases, including obesity, diabetes, hyperiipoproteinemias, and sphingolipidoses, are characterized by abnormalities in lipid transport and/or metabolism. Other minor classes of lipids, including bile acids, fat-soluble vitamins, and eicosanoids play important roles in normal body function as well as in certain disease states. This chapter describes the major classes of lipids, their cellular functions, and the metabolic pathways that connect them.

CLASSIFICATION OF LIPIDS The major classes of lipids and their functions are listed in Table 1-5-1. A brief description of the major structural features of each class follows the table. Table 1-5-1. Major classes of lipids and their metabolic functions. Lipid

Function

Fatty acids

Metabolic fuel; building block for triacylglycerol, phospholipids, and sphingolipids

Triacylglycerols

Storage depot and major transport form for fatty acids

Ketone bodies

Soluble metabolic fuel for skeletal muscle, cardiac muscle, kidney, and brain

Cholesterol

Structural component of plasma membrane; precursor of bile acids, vitamin D, and steroid hormones

Phospholipids

Major building block of membranes; storage site for polyunsaturated fatty acids; signal transduction pathways

Sphingolipids

Structural component of membranes; surface antigens

A. Fatty acids are composed of a long hydrocarbon chain with a carboxyl group at one end. They are amphipathic molecules, containing both polar and nonpolar ends. Fatty acids can be described using two different numbering systems. The C-numbering system starts at the carboxyl end, and the co-numbering system starts at the methyl end. The a-carbon is adjacent to the carboxyl group. Fatty acids are frequently identified as either "saturated" or "unsaturated" depending upon the absence or presence of double bonds, respectively.

meClical

63

Biochemistry

1. Saturated fatty acids contain no double bonds. The most common saturated fatty acids

are palmitic and stearic acid. Their formulae and those of other fatty acids found in human tissues are listed Table 1-5-2. Table 1-5-2. Length and structure of common saturated fatty acids.

Fatty acid

Length

Structure

Acetic

C2

CH3-COOH

Propionic

C3

CH3-CH-COOH

Butyric

C4

CH3-(CH2)2-COOH

Lauric

C l2

CH3-(CH 2)1O-COOH

Myristic

C l4

CH3-(CH2)12-COOH

Palmitic

C I6

CH3-(CHJI4-COOH

Stearic

CH3-(CH2)16-COOH

Arachidic

CIS C20

Lignoceric

C24

CH3-(CH2)22-COOH

CH3-(CH 2 )IS-COOH

2. Unsaturated fatty acids contain one or more double bonds in the cis-configuration. a. Monounsaturated fatty acids contain one double bond. The most common monounsaturated fatty acid is the I8-carbon oleic acid, which has one double bond between carbons 9 and 10: CH 3-(CH 2h-CH=CH-(CH 2h-COOH.

Clinical Correlate A deficiency of essential fatty acids (linoleic and linolenic acids) is seen in patients on long-term intravenous hyperalimentation with no intravenous fat supplementation.

b. Polyunsaturated fatty acids contain two or more double bonds. (1) Linoleic acid is an I8-carbon (0-6 fatty acid with two double bonds. It is an essential fatty acid because it must be obtained from dietary sources.

(2) Linolenic acid is an I8-carbon (0-3 essential fatty acid with three double bonds. CH3-CH2-CH=CH-CHz-CH=CH-CHz-CH=CH-( CH2)7-COOH

(3) Arachidonic add is a 20-carbon (0-6 fatty acid with four double bonds. It can be synthesized by humans from linoleic acid via elongation and desaturation reactions. If linoleic acid is deficient in the diet, arachidonic acid becomes an essential fatty acid. B. Triacylglycerols (TAG) contain a glycerol backbone with three fatty acids linked as esters

(Figure 1-5-1). This is the most common storage form for fatty acids.

64

meClical

Lipids

o II CH 2-O-C-R

~

I

R'-C-O-CH

~

I

CH2-0-C-R" Glycerol

Fatty Acids

Figure 1-5-1. Triacylglycerol.

Removal of one fatty acid generates a diacylglycerol, which acts as a second messenger in the phosphatidylinositol signal transduction pathway. Removal of fatty acids from both ends of the triacylglycerol molecule results in 2-monoglyceride, the end product of TAG digestion in the small intestine. C. Ketone bodies are C4 acids that have either a keto or a hydroxyl group attached to the p-

carbon atom. The two major ketone bodies are acetoacetic acid and p-hydroxybutyric acid (Figures 1-5-2 and 1-5-3).

o II

0

II

CH 3-C-CH 2-C- OH Figure 1-5-2. Acetoacetic acid.

H

0

I II CH -C-CH -C- OH 3 I 2 OH

Clinical Correlate Arachidonic acid is the major precursor for prostaglandins, thromboxanes, and leukotrienes, which act as local hormones and play critical regulatory roles in human physiology. The biologic action of these compounds is different for each organ system. Platelet aggregation is controlled by the antagonistic effects of prostaglandins and thromboxanes; leukotrienes are important mediators of inflammation and allergic responses. A deficiency in arachidonic acid and an overproduction of eicosanoids from arachidonic acid both result in a diverse group of symptoms.

Clinical Correlate Ketone bodies are synthesized from fatty acids and amino acids in the liver during prolonged starvation and diabetic ketoacidosis. They are excreted in the urine, but p-hydroxybutyric acid will not show up on a urine ketone test. Patients with excess ketones present with a fruity breath odor.

Figure 1-5-3. I3-Hydroxybutyric acid.

D. Cholesterol and sterol derivatives contain a common steroid nucleus-a fused fourmember ring system that contains 19 carbon atoms (Figure 1-5-4). Cholesterol, a 27-carbon compound (Figure 1-5-5), is the precursor for vitamin D (C 27 ), bile acids (C24 ), adrenocortical hormones (C21 ), progesterone (C21 ), androgens (C I9 ), and estrogens (CIS).

meClical

65

Biochemistry

18

16 2 3

4

6

Figure 1-5-4. Steroid nucleus.

Clinical Correlate Respiratory distress syndrome (ROS) occurs in preterm newboms due to the inability of the premature lung to synthesize dipalmitoylphosphatidylcholine, the active component of pulmonary surfactant. This lipid, which is normally synthesized shortly prior to parturition, forms a film that lines the alveoli and reduces the surface tension of the aqueous layer, thereby preventing the alveoli from collapsing during exhalation. Symptoms at birth include hypoxia, acidosis, and pulmonary edema. The maturity of the fetal lung can be assessed prenatally by measuring the ratio of dipalmitoylphosphatidylcholine/ sphingomyelin in amniotic fluid. Glucocorticoids (dexamethasone) can be used to induce production of dipalmitoylphosphatidylcholine and develop the lungs more quickly.

66

KAPLA~.

meulCa

OH Figure 1-5-5. Cholesterol.

E. Phospholipids are amphipathic molecules consisting of two alcohols linked by a phosphodiester bridge. Diacylglycerol, the alcohol common to all of the phospholipids, is the nonpolar structural component of membranes. The second alcohol forms the polar head of phospholipids. Each class of phospholipids is distinguished by the polar head. Choline, ethanolamine, and serine are amino alcohols, whereas inositol is a polyol. The four most important glycerol-based phospholipids can all be represented by the structure shown in Figure 1-5-6, where the nature of X is unique for each phospholipid. The names of these phospholipids, the nature of X, and its structure are given in Table 1-5-3.

o o II

II

CH -O-C-R

I

2

1

R -C-O-C-H 2

I

0

II

CH2 - 0- P- 0---0 Polar Head I

I

I 0-

I

I

Phosphodiester Bridge Figure 1-5-6. Glycerol-based phospholipid.

I -------------------

Lipids

Table 1-5-3. The unique polar heads of selected glycerol-based phospholipids. Phospholipid

x

Structure

Phosphatidylethanolamine (cephalin)

Ethanolamine

HO-CHz-CHz-NH 3

Phosphatidylcholine (lecithin)

Choline

HO-CHz-CHz-N(CH 3h

Phosphatidylserine

Serine

HO-CHz-CH-NH 3 tOOH

HoH OH

Phosphatidylinositol

Inositol

OH

HH OH

In membranes, the polar head group faces the aqueous environment, and the fatty acid groups of diacylglycerol constitute the lipid bilayer. F. Sphingolipids and glycolipids contain ceramide as a common structural component. Ceramide is composed of sphingosine, a long-chain amino alcohol with a saturated fatty acid linked to the amino group (Figure 1-5-7). The classes of sphingolipids and glycolipids can be differentiated on the basis of the X-group that is esterified to the terminal hydroxyl group of ceramide (Table 1-5-4).

Sphingosine OH

I

CH3 -(0-' 1'2 ) 12 -CH-CH-CH-CH-CH -01--1)(\ I 2 \C)

NH

[c=o I

Fatty Acid

~

Figure 1-5-7. Sphingolipids and glycolipids.

Table 1-5-4. The unique X-groups of selected spbingolipids and glycolipids. Sphingolipid or Glycolipid

X

Location

Sphingomyelin

Phosphocholine

Myelin sheath

Cerebrosides

Single hexose

Brain, peripheral nerve

Sulfatide

Galactose-SO 4

Nerve tissue

Globoside

Oligosaccharide

RBC membrane

Ganglioside

Oligosaccharide with

Ganglion cells

N -acetylneuraminic acid

IlAPLA~. I meulCa

67

Biochemistry

LIPOPROTEINS The metabolism of lipids frequently requires that a particular lipid be transported in the blood between different organs. Free fatty acids are transported by serum albumin, whereas the neutral lipids (triacylglycerol and cholesterol esters) are transported by lipoproteins.

Bridge to Pharmacology Fatty acids

t Bind strongly to serum albumin

A. Structure and composition. Ail lipoproteins consist of a hydrophilic shell and a hydrophobic core. The hydrophilic shell contains proteins, phospholipids, and unesterified cholesterolamphipathic molecules that interact favorably with both the aqueous environment and the inner core. The hydrophobic core contains the neutral lipids, triacylglycerols, and cholesterol esters, which are highly insoluble in water. A model for the structure of lipoproteins is shown in Figure 1-5-8.

Displace drugs that bind to albumin

phospholipid apoprotein cholesterol

Increase bioavailability of drug

Figure 1-5-8. Lipoprotein structure.

B.

Classes. The four major classes of lipoproteins in human serum can be separated by electrophoresis on the basis of their size and charge or by centrifugation on the basis of their density. A typical electrophoretogram is shown in Figure 1-5-9, and a typical centrifugal separation is shown in Figure 1-5-10.

HDL Density in g/mL Chylomicrons <1.006

c:

o

e

VLDL

~

'00 c:

C>

'E

VLDL

LDL

origin

Chylomicrons

"0

g/mL

LDL

1.006-1.063

HDL

1.063-1.210

e Figure 1-5-9. Lipoprotein electrophoresis.

68

meClical

0.95-1.006

Q)

Figure 1-5-10. Lipoprotein centrifugation.

Lipids

1. Chylomicrons are the least dense of the lipoproteins and do not migrate in an electric

field. They are formed in intestinal mucosa and transport dietary triacylglycerol (TAG) and cholesterol ester (CE). Chylomicrons are synthesized in the smooth ER of intestinal epithelial cells and secreted into the lymphatics. The TAGs in chylomicrons are hydrolyzed by lipoprotein lipase, an enzyme attached to the luminal surface of the vasculature of cardiac muscle, skeletal muscle, and adipose tissue. Chylomicrons contain several apoproteins, including apo B 48 , apo E, and apo C-II. Apo B48 is unique to chylomicrons; apo C-II activates lipoprotein lipase, resulting in fatty acid release to heart, skeletal muscle, and mammary glands. The presence of apo E facilitates the clearance of chylomicron remnants by the liver. Apo B48 is present in the nascent chylomicron; apo C-II and apo E are aquired from HDL following secretion. 2. Very low-density lipoproteins (VLDLs) are synthesized in the liver and transport TAG and CE. VLDLs are composed mainly of TAG, yet they are more enriched in CE than are chylomicrons. VLDLs contain apo B lOO , apo E, and apo C-IL Like chylomicrons, VLDLs are metabolized by lipoprotein lipase to produce remnants (intermediate-density lipoproteins [IDLs 1). These remnants can be further metabolized to particles of still lower density, or they can be internalized by the liver. 3. Low-density lipoproteins (LDLs) are generated from VLDLs and IDLs by the action of lipoprotein lipase, thus increasing the relative proportion of cholesterol esters in the neutral core. The major function of LDL is to transport cholesterol to extrahepatic tissues, where it is taken up by receptor-mediated endocytosis. The LDL particle retains onlyapo B lOO , and the uptake of LDL by cells is initiated by the interaction of apo BlOO with LDL receptors on the plasma membranes. 4. High-density lipoproteins (HDLs) are synthesized by the liver and are approximately 50% protein. When the particle is secreted by the liver, the core region is relatively empty. HDLs perform two major functions:

Clinical Correlate A deficiency in lipoprotein lipase will result in hyperchylomicronemia, or type I hyperiipoproteinemia.

Clinical Correlate Familial hypercholesterolemia (FH), or type II hyperiipoproteinemia, arises as a result of defective LDL receptors and is characterized by elevated LDL cholesterol. A positive correlation exists between elevated plasma concentration of LDL and coronary atherosclerosis.

a. Circulating reservoir for apoproteins. Members of the apo C and apo E families can be transferred back and forth between other lipoproteins. Newly synthesized chylomicrons and VLDL particles obtain some of their apoproteins from the HDL reservoir following secretion. b. Reverse cholesterol transport. HDLs are important in moving cholesterol from extrahepatic tissues to the liver. Elevated plasma levels ofHDL are associated with decreased incidence of coronary atherosclerosis. Cholesterol is taken up from the surface of cells by HDL, esterified to cholesterol esters, and ultimately returned to the liver either by uptake ofHDL particles by the liver or by the transfer of cholesterol esters to VLDL and chylomicron remnants followed by remnant uptake. Two proteins play important roles in reverse cholesterol transport: (1) Lecithin cholesterol acyl transferase (LCAT) is a plasma enzyme that esterifies HDL cholesterol. The fatty acid used for esterification comes from lecithin (phosphatidykholine). LCAT is activated by apo A-I, which is associated with HDL.

(2) Cholesterol ester transfer protein (apo D) is associated with HDL and facilitates the transfer of cholesterol esters to VLDL and chylomicron remnants in exchange for triacylglycerol.

In a Nutshell Apoprotein Function apo A

apo

B48

apo

B100

Reverse cholesterol transport Chylomicron clearance from serum LDL clearance from serum

apo C-II

Activation of lipoprotein lipase

apo D

Reverse cholesterol transport

KAPLA~.

meulCa

I

69

Biochemistry

C. Abnormalities of lipoprotein metabolism may result in either elevated or decreased plasma

concentrations of certain lipoproteins and associated lipids. 1. Hyperlipoproteinemias. Disorders of lipoprotein excess may be primary (hereditary) or secondary to a systemic illness such as diabetes, hypothyroidism, or extremely high saturated fat intake. Table 1-5-5 illustrates the different types of hyperlipoproteinemias, their mode of inheritance, the type of lipid and lipoproteins that accumulate, and the associated relative risk of atherosclerosis.

Table 1-5-5. Types of hyperlip oproteinemias. Type

Mode of Inheritance

1 IIa lIb

Autosomal recessive Autosomal dominant

III

?? Heterogeneous Heterogeneous

IV V

Increased Lipid and Lipoprotein Concentration

Atherosclerosis Relative Risk

TAG, chylomicrons, VLDL Cholesterol, LDL TAG, VLDL, LDL Cholesterol, TAG, VLDL TAG,VLDL TAG, VLDL, chylomicrons

+/+++ +++ ++ +/+/-

2. Hypolipoproteinemias. There are two major disorders originating from a deficit of lipoprotein. a. Abetalipoproteinemia is an autosomal recessive condition characterized by the absence of betalipop rote ins (apo B48 and apo BIQo). Chylomicrons and VLDLs are not formed, and lipid accumulates in intestinal cells and liver. Clinically, this results in ataxia, mental retardation, retinitis pigmentosa, and accumulation of acanthocytes in the blood. b. Familial alpha-lipoprotein deficiency (Tangier disease) is characterized by the absence of apo A. Serum levels of cholesterol and HDL are low, whereas triacylglyerol associated with chylomicion and VLDLs are elevated. Neurologic deterioration and hepatomegaly and large orange tonsils are the clinical sequelae.

FATTY ACID METABOLISM There are six major pathways of fatty acid and lipid metabolism. Fatty acids are synthesized from excess carbohydrate in liver and adipose tissue. They are stored as triacylglycerols in adipose tissue when nutrition is sufficient, and are mobilized from triacylglycerol and released into the blood during fasting or exercise. Most of the free fatty acids that are released from adipose tissue are carried by serum albumin to skeletal muscle, cardiac muscle, and liver, where they undergo P-oxidization to provide energy to the tissues. Cholesterol and ketones are synthesized from acetyl-CoA. Ketones are utilized as a metabolic fuel, and fatty acids are used to synthesize specialized products, such as phospholipids, sphingolipids, glycolipids, and prostaglandins. An overview of these pathways is outlined in Figure 1-5-11.

70

KAPLANct· •

me lea

I

--

----

------

---

Lipids

Carbohydrates -

Pyruvate

___~.I AcetYI_COA~ICitrateJ .#

l

/' /' Acetoacetyl-CoA

///////////

/' Oxidation for fuel __

L

()v!>I",

Oxaloacetate

\

TCAcyde

~-Hydroxy-~-methylglutaryl

(HMG)-CoA

-I

1 Lipolysis 1 ~ Triacylglycerols

===: I

Fatty acids 1

r.P~h:-o-s-p-:h""'o-:li:-p-:-id-:-S"'+ proteins_

Lipoproteins

ISphingolipids I IGlycosphingolipids I IProstaglandins I Figure 1-5-11. Interrelationship of pathways of fatty acid metabolism.

The pathways in Figure 1-5-11 that predominate at any time are determined largely by the insulin/glucagon ratio. An increase in the insulin/glucagon ratio (due to insulin secretion when blood glucose is high) favors lipid synthesis and storage, whereas a decrease in the insulin/glucagon ratio (due to glucagon secretion when blood glucose is low) favors fatty acid mobilization and l3-oxidation to generate energy. Many of these effects are mediated by the antagonistic effects of glucagon and insulin on the intracellular concentration of cAMP (glucagon increases cAMP and insulin decreases cAMP). Under conditions where excessive fatty acid oxidation is occurring and acetyl-CoA accumulates, ketone synthesis occurs as a means of converting acetyl-CoA into a soluble fuel that can be readily transported in the plasma. The key steps of these pathways, as well as the mechanism by which the pathways are regulated and coordinated, will be discussed separately. A. Fatty acid synthesis. In humans, fatty acids are synthesized from acetyl-CoA, bicarbonate, and NADPH. The reactions that occur are essentially a reversal of l3-oxidation, with two distinctions: the two processes use different enzymes and occur in different cellular compartments. Fatty acid synthesis occurs in the cytosol, whereas l3-oxidation occurs in mitochondria. Humans can synthesize all of the required fatty acids except linoleic acid and linolenic acid, which are therefore essential dietary requirements. Before fatty acid synthesis can begin, acetyl-CoA must be translocated from the mitochondria, where it is produced, to the cytosol, where it is utilized. Because the inner mitochondrial membrane is impermeable to acetyl-CoA, the citrate shuttle is used to carry acetyl-CoA from the mitochondrial matrix to the cytosol. The key steps of the citrate shuttle are shown in Figure 1-5-12.

KAPLAlf I med lea

71

---

Biochemistry

Inner mitochondrial membrane Mitochondrial matrix

pyruvate -

Cytosol

acetyl-co/citrate --t--+-..... citrate oxaloacetate \

\ )

~~

1~

oxaloretate ace.TI-coA -

fatty acids

malate t-NADPH

pyruvate,...-----t--+-- pyruvate

Figure 1-5-12. The citrate shuttle.

Citrate is formed in the mitochondria from oxaloacetate and acetyl-CoA and then transported into the cytosol. Once in the cytosol, citrate is cleaved by citrate lyase to form acetyl-CoA and oxaloacetate, which is converted through two sequential reactions to pyruvate. Pyruvate is returned to the mitochondria, where it is carboxylated to oxaloacetate to complete the shuttle. The citrate shuttle also produces NADPH, another factor required for fatty acid synthesis. About half of the NADPH for fatty acid synthesis is supplied by the pentose phosphate pathway. 1. Key enzymes in fatty acid synthesis

a. Acetyl-CoA carboxylase catalyzes the first step in fatty acid synthesis. The enzyme requires the presence of both biotin and bicarbonate for converting acetyl-CoA to malonyl-CoA (Figure 1-5-13). Malonyl-CoA is a three-carbon compound that serves as the donor of two-carbon units in the elongation of the fatty acid chain.

o

Acetyl-CoA 0 _ Carboxylase II II CH3 C- 5-CoA + HC03 + ATP • HOOC -CH2 - C -5 -CoA + ADP + Pi (Biotin) Acetyl-CoA

Malonyl-CoA

Figure 1-5-13. The conversion of acetyl-CoA to malonyl-CoA.

b. Fatty acid synthase (FAS) is a multienzyme complex that catalyzes all of the remaining reactions. It lengthens the fatty acyl-CoA being synthesized by the sequential addition of two-carbon units to the carboxyl end. After each addition, it reduces the product to a fully saturated acyl group. All of the reactions occur while the intermediates are covalently bound as thioesters to either acyl-carrier protein (ACP) or to the condensing enzyme (synthase). Each cycle adds a two-carbon unit and uses two molecules of NADPH to reduce the ketoacyl intermediate to a fully saturated fatty acid. Each cycle consists of the four sequential reactions shown in Figure 1-5-14.

72

iii8Cuca1 - - - _.._ - _ . _ - - - - - _. ._ - - - - - - _

..

__

..

_--

._--

Lipids

a

a

II

II

CH 3 -C -S -Synthase + HaaC -CH2 -C -S - ACP (1 )

Acetyl-S-Synthase

Malonyl-S-ACP

Acetoacetyl-S-ACP

t

r--NADPH

(2)

o II

NADPH

)

CH 3 -CH2 -CH2 -C -S - ACP ....."',...---""''-(4)

Butyryl-S-ACP

H

a

I

II

(3)

CH 3 -C =~ -C -S - ACP '"

co::

aH

a

I

II

CH 3 -CH-CH 2-C -S-ACP

H Crotonyl-S-ACP

[3-Hydroxybutyryl-S-ACP

Figure 1-5-14. The enzymatic actions of FAS.

At the end of the first cycle, butyryl-CoA is transferred onto the synthase so that the acyl carrier protein can be recharged with another molecule of malonyl-CoA and a new cycle can begin. Each cycle consists of the following four reactions: (1) Condensation of malonyl-CoA with fatty acyl-CoA to produce a ~-ketoacyl­ CoA intermediate.

(2) Reduction of ~-ketoacyl-CoA to reaction.

~-hydroxyacyl-CoA

in an NADPH-dependent

(3) Dehydration of ~-hydroxyacyl-CoA to produce enoyl-CoA with a double bond. (4) Reduction of the double bond by NADPH to produce a fatty acyl-CoA that has been lengthened by two carbons. This cyclic pattern is repeated until palmitoylCoA, the product of the FAS complex, is formed. 2. Elongation and desaturation reactions convert palmitate into a variety of fatty acids that are required for use by the normal cell. These reactions, which occur primarily in the endoplasmic reticulum, rely upon the use of malonyl-CoA for elongation. Elongation can also occur in the mitochondria, where acetyl-CoA, instead of malonyl-CoA, is used. Desaturation occurs in the endoplasmic reticulum and requires a multienzyme complex that contains cyt b s and cyt b s reductase. 3. Regulation of fatty acid synthesis depends on the availability of substrates and the activity of acetyl-CoA carboxylase, the enzyme that catalyzes the first step in the initial reaction converting acetyl-CoA to malonyl-CoA. This reaction is the rate-limiting step in fatty acid synthesis. Acetyl-CoA carboxylase is activated by citrate, insulin, and a high-carbohydrate, low-fat diet; it is inhibited by fatty acids, glucagon, and a high-fat diet. Acetyl-CoA carboxylase is inactivated by cAMP-dependent phosphorylation and activated by insulindependent dephosphorylation. The synthesis of both acetyl-CoA carboxylase and fatty acid synthase complex is induced by insulin. B. Fatty acid oxidation. Most fatty acid oxidation occurs in the mitochondria, although peroxisomes are important in the oxidation of very long-chain fatty acids and branched-chain fatty acids. A separate set of enzymes oxidizes fatty acids in peroxisomes.

In a Nutshell Fatty Acid Synthesis • Occurs in cytosol • Citrate shuttle (acetyl-CoA transport to cytoplasm, NADPH production) • Acetyl-CoA carboxylase: - first step/rate-limiting step - produces malonyl-CoA (C2 donor) - uses 1 ATP - activated by citrate, insulin - inhibited by palmitoylCoA, glucagon • Fatty acid synthase: - lengthens fatty acid chain by two carbons from carboxy terminus - reduces ketone to full saturation - uses 2 NADPH • Synthesis stops at C16 palmitoyl-CoA; needs microsomes from endoplasmic reticulum to elongate past C16 • Synthesis of palmitoyl-CoA requires 7 ATP and 14 NADPH KAPLA~.

meulCa

I

73

Biochemistry

1. Uptake and oxidation of fatty acids. The uptake and oxidation of fatty acids by cells can

be divided into three major stages: activation, the transfer of fatty acids into the mitochondria, and ~-oxidation of fatty acids. a. Activation. Fatty acids are converted to fatty acyl-CoA immediately upon entry into the cell. This reaction traps the fatty acid in the cell and generates a high-energy thioester bond with CoA-SH. The reaction is catalyzed by fatty acyl-CoA synthetase (thiolase) and is driven by the hydrolysis of pyrophosphate.

o

0 II

II

CH3-(CH2)n-C-OH + COA-SH + ATP

.. CH3-(CH2)n-C-S-CoA+AMP + P-P Fatty acyl-CoA Synthetase ,

I

2Pi

b. Transfer of fatty acids into mitochondria. Fatty acids having a chain length of C l2 or greater enter the mitochondria via the carnitine shuttle, which consists of two enzymes (i.e., carnitine-acyl transferase-land carnitine-acyl transferase-II) and one transporter (i.e., carnitine translocase).

Clinical Correlate

(1) Carnitine-acyl transferase-I (CAT-I) is associated with the outer surface of the inner mitochondrial membrane. It transfers the fatty acid from fatty acyl-CoA to

A deficiency in medium chain acyl dehydrogenase (MCAD) is the most common inborn error of fatty acid metabolism. Onset usually occurs in infancy. Symptoms include vomiting, lethargy, and hypo kinetic hypoglycemia. A deficiency in carnitine or CAT-l results in weakness, hypotonia, and hypo ketotic hypoglycemia

carnitine to form fatty acyl carnitine. (2) Carnitine translocase transports fatty acyl carnitine into the mitochondria and

transports free carnitine back out of the mitochondria. (3) Carnitine-acyl transferase-II (CAT-II) is associated with the inner surface of the inner mitochondrial membrane. It catalyzes the reformation of fatty acyl-CoA in the mitochondrial matrix. c. ~-oxidation of fatty acids involves the sequential removal of two-carbon fragments

from the carboxyl end. Each cycle of oxidation involves four reactions and generates one molecule each of acetyl-CoA, NADH, and FADH 2. The pathway is called ~­ oxidation because the two oxidation steps that generate FADH2 and NADH both involve the ~-carbon atom. The key reactions are shown in Figure 1-5-15. (1) Dehydrogenation of the fatty acid forms a double bond between the a and ~ car-

bons in a reaction that generates FADH 2. This reaction is catalyzed by a family of fatty acyl-CoA dehydrogenases that differ in specificity for fatty acid chain length: long-chain acyl dehydrogenase, medium-chain acyl dehydrogenase, and shortchain acyl dehydrogenase. The complete oxidation of long-chain fatty acids requires all three dehydrogenases. (2) Hydration of the double bond produces ~-hydroxy-acyl-CoA.

74

KAPLAlfilme leaI

-------

----

----

Lipids

o II

FAD"

R -CH2 -CH2 -C -S -CoA

7:

2

H

0

Hp

I

II

l

R -C =C -C -S -CoA

I

(1 )

0

--.:::-.----+-. (2)

H Fatty acyl-CoA

II

R -CH -CH -C -S -CoA

I

2

OH

Enoyl-CoA

I3-Hydroxyacyl-CoA

(3)

o

0

II

II

R-C-S-CoA

+

CH3-C-S-CoA

CoA-SH

..J "-"'---'""'--

o II

~

NAD+ NADH

0 II

R -C -CH2 -C -S -eoA

(4)

Shorter Acyl-CoA

Acetyl-CoA

[3-Ketoacyl-CoA

Figure 1-5-15.13-0xidation of fatty acids.

(3) Dehydrogenation of f3-hydroxyacyl-CoA in an NAD+-dependent reaction generates 13-ketoacyl-CoA. (4) Thiolytic cleavage by the addition of CoA-SH to the f3-carbon releases acetylCoA and completes the cycle. The resultant acyl-CoA can start at step (1) and proceed through the cycle until it is completely degraded to acetyl-CoA units. d. Oxidation of fatty acids with an odd number of carbon atoms results in the production of one molecule of propionyl-CoA from the co-end of the fatty acid. This molecule is released during the final thiolytic cleavage cycle. Propionyl-CoA can be carboxylated to methylmalonyl-CoA and then converted to succinyl-CoA for entry into the TCA cycle for further metabolism. The oxidation of unsaturated fatty acids is also important because nearly 50% of human fatty acids are unsaturated. Unsaturated fatty acids in natural fat contain cis double bonds only, but the enzyme enoyl-CoA hydratase, which catalyzes the second reaction in the f3-oxidation cycle, acts only on trans double bonds. An auxiliary enzyme, cisll.3 -transll.2 enoyl-CoA isomerase, converts natural cis bonds to the trans configuration, allowing continued oxidation of the unsaturated fatty acid. 2. Energetics of f3-oxidation. Oxidation of palmitic acid (C 16 ) produces eight molecules of acetyl-CoA, seven molecules of NADH, and seven molecules of FADH 2. Further oxidation of acetyl-CoA by the TCA cycle and the electron transport chain results in the following stoichiometry for the oxidation of palmitic acid:

The net yield of usable energy resulting from the complete oxidation of one molecule of palmitic acid is 129 molecules of ATP. Because the respiratory quotient (RQ) is defined as moles of C02 produced divided by moles of O 2 consumed, the RQ for fatty acid oxidation is approximately 0.7. In contrast, the RQ for the complete oxidation of glucose is approximately 1.0.

In a Nutshell f3-0xidation of Fatty Acids • Occurs in mitochondria • Oxidizes f3-carbon atom • Activation uses 2 ATP per molecule • Carnitine transports fatty acid from the cytosol to the mitochondria - inhibited by malonyl CoA and insulin • Oxidation removes 2 carbons per cycle - 1 acetyl-CoA ~ TeA cycle - 1 FADH2 and 1 NADH ~5ATP

• Odd-carbon fatty acids - propionate (0) is carboxylated to methylmalonyl-CoA (C4) - methymalonyl-CoA + vitamin B12 ~ succinylCoA ~ TeA cycle • Unsaturated fatty acids - cis bonds ~ trans bonds, then oxidation continues

meClical

75

Biochemistry

Note Oxidation of fatty acids produces more energy than carbohydrate oxidation, with 9 kcal/g of fat versus 4 kcal/g of glucose.

3. Regulation of fatty acid oxidation is achieved by controlling the rate at which fatty acids enter the mitochondria. CAT-I is inhibited by malonyl-CoA, the first compound committed to fatty acid synthesis. This ensures that a futile cycle is not generated by simultaneous synthesis and oxidation of fatty acids. The rate of oxidation is also controlled by the rate at which fatty acids are released from triacylglycerol stores in adipose tissue. 4. Comparison of fatty acid synthesis and oxidation. The key differences between synthesis and oxidation offatty acids are summarized in Table 1-5-6.

Table 1-5-6. Comparison between synthesis and oxidation of fatty acids. Synthesis

~-Oxidation

Final product

Palmitate

Acetyl-CoA

Tissues involved

Adipose, liver

Muscle, liver

Subcellular localization

Cytoplasmic

Mitochondrial

Membrane transport

Citrate shuttle carries acetyl groups from mito matrix into cytosol

Carnitine shuttle carries fatty acids from cytosol into mito matrix

Cofactors

NADPH

NAD+,FAD

Effect of high insulin/ glucagon ratio

Pathway preferred

Pathway suppressed

Other regulators

Activated by citrate

Inhibited by malonyl-CoA

Rate-limiting enzyme

Acetyl-CoA carboxylase

CAT-I

Clinical Correlate Ketosis, the overproduction of ketones, occurs as a result of a high glucagon/insulin ratio during carbohydrate deficiency states, such as starvation, severe diabetes, and alcoholism. The acidic properties of the ketones lower the pH of the plasma, causing metabolic acidosis and decreased plasma bicarbonate levels.

C. Ketone body metabolism. The major ketone bodies are acetoacetatic and f3-hydroxybutyric acids, weak acids that dissociate to acetoacetate and f3-hydroxybutyrate at physiologic pH. Spontaneous decarboxylation of acetoacetate gives rise to acetone. These compounds are synthesized from acetyl-CoA by liver mitochondria when excessive amounts of fatty acids are being oxidized and glucose availability is limited. Ketone synthesis is inhibited when adequate carbohydrate is available. Ketones are used as fuel by extrahepatic tissues. 1. Ketone synthesis occurs in liver mitochondria via the reactions shown in Figure 1-5-16.

GoASH 2 Acetyl-GoA

Acetyl-GoA

-~~"---.. a

Acetoacetyl GoA

\...

a

• HMG-GoA

NADH

~-HYdroXYbutyrate ..(-J-,,---c Figure 1-5-16. Ketone synthesiS.

76

meCtical ---~----~.---~.------

b

~Acetyl-COA

Acetoacetate

Lipids

a. Condensation of three molecules of acetyl-CoA in two sequential reactions generates ~-hydroxy-~-methylglutaryl-CoA (HMG-CoA). Acetoacetyl-CoA is formed in the first reaction and acts as the substrate for the second. b. Cleavage of HMG-CoA produces acetoacetate and acetyl-CoA.

c. Reduction of acetoacetate to

~-hydroxybutyrate

consumes NADH.

2. Ketone use by extrahepatic tissues occurs in the mitochondria. Following the uptake of ketones, ~-hydroxybutyrate is oxidized back to acetoacetate. The fate of acetoacetate involves two sequential reactions as shown in Figure 1-5-17.

a

p-Hydroxybutyrate ( ' . . ) Acetoacetate --('-,;:::_.--) Acetoacetyl-GoA NAD+

Succinyl GoA

b

I/'

CoA-SH

r

2 Acetyl-GoA Figure 1-5-17. Ketone use.

a. Conversion of acetoacetate to acetoacetyl-CoA is catalyzed by an enzyme that transfers CoA-SH from succinyl-CoA to acetoacetate. The absence of this transferase in liver accounts for the inability of liver to use ketones as fuel. b. Thiolytic cleavage of acetoacetyl-CoA releases two molecules of acetyl-CoA. This reaction is catalyzed by thiolase, an enzyme of the ~-oxidation pathway. D. Triacylglycerol metabolism. Triacylglycerols are the most common storage and transport form for fatty acids derived from either dietary sources or de novo synthesis. The synthesis and degradation of triacylglycerols involves formation and hydrolysis of the ester bonds between fatty acids and the glycerol backbone. The conditions that promote lipogenesis are a highcarbohydrate diet and elevated insulin/glucagon ratio. Fasting, exercise, and a decreased insulin/glucagon ratio promote lipolysis.

Note

The fruity breath odor commonly seen in patients with ketosis is caused by the ventilation of acetone (produced by the decarboxylation of acetoacetate).

1. Triacylglycerol synthesis occurs primarily in liver and adipose tissue. Synthesis requires glyc-

erol phosphate, which is formed by the reduction of dihydroxyacetone phosphate (DHAP), an intermediate in glycolysis. Fatty acids are transferred from fatty acyl-CoA to carbons 1 and 2 of the glycerol backbone by fatty acyl transferase. Hydrolysis of the phosphate bond at carbon3 generates diacylglycerol, which is then esterified with a third fatty acid, producing triacylglycerol. A summary of triacylglycerol synthesis is shown in Figure 1-5-18.

meClical

77

Biochemistry

E

o

o

GHpH

I

GH2 0H

I

NADH \.

G=O

I

-",",,--~.

HG-OH

I

GH 20-®

II

--C -R FA-GoA \. --...-.....---.. H OH ------.. FA-GoA \.

GH2 -O-®

O-®

Glycerol Phosphate

Dihydroxyacetone-®

t

II 0 -C -R'

O-®Phosphatidic

Lysophosphatidic acid

I

o-ColI -R

t

o

glycolysis

o ~ -R

FA-GoA

o ~ -R'

~O(-_/~­

t

II

o-c -R

®

o

II

glucose

acids

u

Triacylglycerol

t

II

O~_R II O-C -R' OH

Diacylglycerol

Figure 1-5-18. Triacylglycerol synthesis.

2. Triacylglycerol hydrolysis involves a family of lipases that differ in their localization and specificity. A summary of triacylglycerol hydrolysis is shown in Figure 1-5-19.

intestinal lumen (dietary) TAG intestinal lumen

jpa",..",

intestinal mucosal cell

llpas'

fatty acids + 2-monoglyceride

diffusion

lymphatics I

1 Iblood streaml endothelial lipoprotein lipase

I

+~------------~+~------~--~+ glycerol fatty acid chylomicron remnants

::c

~

t

glucose synthesis

I'We'1 bile Jnthesis

TAG synthesis sensitive lipase

Figure 1-5-19. Summary of triacylglycerol hydrolysis.

78

me leaI KAPLAlfd-

Lipids

a. Pancreatic lipase hydrolyzes dietary triacylglycerol in the small intestine. Hydrolysis occurs at positions 1 and 3, resulting in the release of two molecules of fatty acid and 2-monoglyceride. These compounds diffuse into the intestinal mucosal cells, where they are re-esterified to triacylglycerol and incorporated into chylomicrons. The chylomicrons are released into the lymphatics and enter the bloodstream. b. Lipoprotein lipase (LpL) is produced by the endothelial cells of the vasculature in adipose and muscle tissue. It hydrolyzes triacylglycerols in chylomicrons and VLDLs into free fatty acids and glycerol. The fatty acids are taken up by adipose and muscle tissue. In adipose tissue they are re-esterified into triacylglycerols for storage. In muscle, the free fatty acids are oxidized for energy. The glycerol produced by the action of lipoprotein lipase is taken up by liver. The chylomicron remnants, emiched in cholesterol, are taken up by the liver where the cholesterol can be converted to bile acids or repackaged into VLDLs. c. Hormone-sensitive lipase is found primarily in adipose tissue where it hydrolyzes the first fatty acid from triacylglycerol. This is the rate-limiting step in mobilization of fatty acids from adipose stores. The enzyme is activated by cAMP-dependent phosphorylation. A number of hormones, including epinephrine and norepinephrine, stimulate cAMP production in adipose tissue and initiate lipolysis. The fatty acids released from adipose are carried in the plasma bound to albumin. Glycerol is released and extracted by the liver, where it can be used for gluconeogenesis or resynthesis of triacylglycerol. The adipocyte cannot recycle glycerol due to the absence of glycerol kinase.

Brid~eto

Pharmacology

Gemfibrozil and clofibrate are cholesterol-lowering drugs that increase the activity of lipoprotein lipase. They are discussed in the Cardiovascular Pharmacology chapter in Organ Systems Book 1 (Volume III).

CHOLESTEROL METABOLISM There are two routes by which cholesterol is introduced into the body: It can originate from the diet, or it can be synthesized de novo by any nucleated cell. Cholesterol is an integral structural component of plasma membranes and serves as a precursor of vitamin D, bile acids, and steroid hormones. A. Cholesterol synthesis. The major site of cholesterol synthesis is the liver. The adrenal cortex, the ovaries, and the testes also produce significant amounts of cholesterol and use it for steroid hormone synthesis. The enzymes required to synthesize cholesterol are extramitochondrial and are localized in the cytosol and the endoplasmic reticulum. The substrates required for cholesterol synthesis are acetyl-CoA, NADPH, ATP, and Oz. The overall equation for cholesterol synthesis is:

18 acetyl-CoA + 18 NADPH + 18 ATP + 4 Oz

~

cholesterol (CZ7) + 18 NADP+ + 18 ADP + 18 Pi + 9 COz

1. Stages of cholesterol synthesis. The synthetic pathway occurs in four stages. a. Mevalonic acid synthesis. The first stage of cholesterol synthesis is the sequential condensation of three molecules of acetyl-CoA to produce HMG-CoA, which is then reduced to mevalonic acid. 2NADPH 3 acetyl-CoA ----,l.--~.~HMG-CoA ------..".".-=--l.~mevalonic acid HMG-CoA reductase The key enzyme in the overall pathway is HMG-CoA reductase. This enzyme reduces HMG-CoA to mevalonic acid in a reaction that requires two molecules of NADPH. This is the rate-limiting step in cholesterol synthesis and is therefore the site at which KAPLAlf I medlea

79

Biochemistry

Bridge to Pharmacology Lovastatin and pravastatin are cholesterol-lowering drugs that block the action of HMG-CoA reductase, the ratelimiting step in cholesterol synthesis. These drugs are discussed in the Cardiovascular Pharmacology chapter in Organ Systems Book 1 (Volume III).

synthesis is controlled. The synthesis of HMG-CoA involves the same steps as were described for ketone synthesis with two exceptions: synthesis of HMG-CoA in the cytosol and mitochondria require different sets of enzymes, and the cytosolic HMGCoA pool used for cholesterol synthesis does not mix with the mitochondrial pool used for ketone synthesis. b. Synthesis of activated isoprene units. The second stage of cholesterol synthesis is the conversion of mevalonic acid (C 6 ) into activated isoprene units (Cs ) that are designed to condense with one another (Figure 1-5-20). Two types of activated isoprene units are synthesized: isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DPP). Synthesis of an activated isoprene unit requires three molecules of ATP. These compounds have many synthetic uses; they are substrates for dolichol, ubiquinone, and fat-soluble vitamin synthesis.

Isopentenyl Pyrophosphate

3,3 Dimethylallyl Pyrophosphate

Figure 1-5-20. Activated isoprene units.

c. Formation of squalene. In the third stage, squalene, a C30 linear compound, is formed by condensation reactions that use six activated isoprene units. Squalene is generated via three condensation reactions (Figure 1-5-21): (1) Condensation of two Cs units

IPP + DPP -7 geranyl pyrophosphate + PPi (2) Condensation of Cs and C lO units IPP + geranyl-PP -7 farnesyl pyrophosphate + PPi (3) Condensation of two CIS units farnesyl-PP + PP-farnesyl-7 squalene + PPi

Figure 1-5-21. Squalene.

d. Conversion of squalene to cholesterol. The fourth and final stage of cholesterol synthesis involves the cyclization of squalene to form lanosterol, which is converted via a number of reactions to cholesterol (Figure 1-5-22).

80

meClical

Lipids

Lanosterol

Cholesterol

Figure 1-5-22. Conversion of squalene to cholesterol.

2. Cholesterol ester hydrolysis and formation. Approximately 70% of the cholesterol found in humans exists as cholesterol ester. The most common fatty acid found esterified to the 3-hydroxyl group of the A ring of cholesterol is oleic acid. a. Hydrolysis of cholesterol esters (1) Pancreatic cholesterol esterase hydrolyzes dietary cholesterol esters in the intestinal lumen. Re-esterification occurs after diffusion into the intestinal mucosal cell but prior to incorporation into chylomicrons. (2) Intracellular cholesterol esterases are found in all tissues but are in highest concentrations in liver and steroid hormone-producing glands. b. Synthesis of cholesterol esters. Two enzymes contribute to the synthesis of cholesterol esters. (1) Lecithin cholesterol acyl transferase (LCAT), found in the plasma, participates in reverse cholesterol transport. After HDL acquires cholesterol from extrahepatic tissues, it is esterified by LCAI. The fatty acid used for esterification comes from the 2-position of phosphatidylcholine. (2) Acyl cholesterol acyl transferase (ACAT), found inside the cell, is involved in cholesterol storage. Esterification involves the transfer of a fatty acid from fatty acyl-CoA to cholesterol. 3. Regulation of cholesterol synthesis. Cholesterol synthesis is highly regulated. Diets high in fat and carbohydrate stimulate HMG-CoA reductase, the rate-limiting enzyme in the pathway. The activity is suppressed by high dietary cholesterol and by fasting, which limits the available acetyl-CoA and NADPH required for synthesis. HMG-CoA is also under hormonal control: it is stimulated by insulin and thyroxine and inhibited by glucagon. Finally, cholesterol is constantly recycled between hepatic and extrahepatic tissues, either via the enterohepatic bile circulation or from cholesterol-rich chylomicron remnants, IDLs, and LDLs. These lipoproteins bind to specific receptors on cell membranes and are internalized and hydrolyzed, yielding free cholesterol. This increase in free cholesterol in the cell has two major regulatory effects: it suppresses the synthesis of LDL receptors ("down regulation"), thereby decreasing additional uptake of LDL-cholesterol from plasma, and it suppresses the synthesis of HMG-CoA reductase, thus decreasing the de novo synthesis of cholesterol by the cells.

Mnemonic lCAT is involved in the leaving of cholesterol via HDL; ACAT is involved in the accumulation of cholesterol in cells.

KAPLAN· . I meiIlea

81

Biochemistry

Bridge to Pharmacology Cholestyramine and colestipol are cholesterol-lowering drugs that bind bile acids in the intestine and promote their excretion. This increases the production of bile acids from cholesterol and ultimately lowers LDL cholesterol. These drugs are discussed in the Cardiovascular Pharmacology chapter in Organ Systems Book 1 (Volume III).

B. Cholesterol derivatives. Cholesterol is the precursor for bile acid and steroid hormone synthesis. 1. Bile acids are 24-carbon compounds derived from cholesterol. Synthesis occurs in the

liver and involves four reactions, each of which makes bile acids more amphipathic and more suitable for their roles in lipid emulsification. Bile acids are the major excreted form of cholesterol. a. Side chain cleavage of cholesterol releases a three-carbon fragment and results in a C24 compound in which the terminal carbon of the side chain has been oxidized to an acid. b. Conjugation of the side chain carboxyl group with either glycine or taurine adds a more acidic group and ensures complete ionization at physiologic pH. c. Reduction of the double bond in the ring system uses NADH as a source of reducing power. d. Hydroxylation at carbons 7 and 12 is catalyzed by microsomal mixed-function hydroxylases. These reactions require both O 2 and NADPH. An example of a bile acid (cholic acid) and its conjugated derivative (taurocholic acid) are shown in Figure I-5-23. The term "bile salt" indicates that at physiologic pH, the side chain group is negatively charged and is associated with a counterion. 2. Steroid hormones are derived from cholesterol in the adrenal cortex, the ovaries, and the testes. The structures of the major steroid hormones are shown in Figure I-5-24. Steroid hormone synthesis involves two reactions that are common to all pathways and numerous additional reactions that have tissue and cell specificity.

H

I

C-N-CH CH

II

o

HO H

H Cholic acid

Taurocholic acid

Figure 1-5-23. Cholic acid and taurocholic acid.

82

meClical

2

2

-so 3 H

Lipids

Adrenal Cortical Steroids

Cortisol

Aldosterone

Gonadal Steroids

OH

o Progesterone

OH

HO

Testosterone

17~- Estradiol

Figure 1-5-24. Major steroid hormones.

a. Common synthetic reactions. The initial reaction in the synthesis of all steroid hormones is cleavage of the side chain to generate pregnenolone, a C21 intermediate. Pregnenolone is converted to progesterone by the action of 3-~-hydroxysteroid dehydrogenase (3-~-HSD). Both pregnenolone and progesterone are C21 intermediates in the synthesis of all steroid hormones. The rate-limiting step in steroid hormone synthesis is the initial cleavage of the side chain. The enzyme 20-22 desmolase is located in the mitochondria, and its activity is increased in response to hormone binding to membrane receptors. In cells that synthesize glucocorticoids, ACTH stimulates desmolase activity; in mineralocorticoid synthesis, the activity is stimulated by angiotensin II; in androgen and estrogen synthesis, the activity is stimulated by LH. b. Tissue-specific synthetic reactions include the following four types of reactions. (1) Hydroxylation reactions can occur at various positions on the steroid nucleus. Cortisol synthesis by the adrenal cortex requires hydroxylation at carbons 11, 17, and 21, whereas aldosterone synthesis requires hydroxylation at carbons 11, 18, and 21. (2) Side chain cleavage by 17-20 desmolase removes the remainder of the side chain and converts the steroid to a C 19 androgen. (3) 5-a-reductase reduces the double bond in testosterone to form dihydrotestosterone (DHT). This enzyme is present in tissues that use DHT as the major androgen. (4) Aromatase removes the methyl group that extends up between the A and Brings of the steroid nucleus and makes the A ring aromatic. This enzyme is present in tissues that convert androgens to estrogens.

Clinical Correlate The most common cause of congenital adrenal hyperplasia is an inherited deficiency in 21-hydroxylase, leading to decreased cortisol and aldosterone and increased adrenal androgens. ACTH levels are elevated, and patients present with salt wasting and virilization.

KAPLA~. I meulCa

83

Biochemistry

PHOSPHOLIPIDS Phospholipids are the major building blocks of membranes. They also participate in signal transduction pathways and serve as reservoirs for polyunsaturated fatty acids needed for eicosanoid synthesis. A. Synthesis. The synthesis of phosphatidylethanolamine (PE) will be used to illustrate the synthetic strategy used to form these compounds. The two major substrates required for PE synthesis are activated ethanolamine and diacylglycerol. Activation of ethanolamine occurs in a two-step reaction. Free ethanolamine is first phosphorylated and then condensed with cytidine triphosphate to generate CDP-ethanolamine (Figure 1-5-25). The formation of phosphatidylethanolamine involves the transfer of P-ethanolamine from CDP-ethanolamine to the free hydroxyl group of diacylglycerol (Figure 1-5-26).

ATP Ethanolamine

ADP

_\......::::.._...L~__•

Cytidine-P-P-P Ethanolamine-P

P-P

_\.....:::..._L...::....._. CDP-ethanolamine

Figure 1-5-25. Activation of ethanolamine.

o

II O-C-O-R

o II

cytidine-P-P-ethanolamine + DAG ~R'-C-O CMP

0 {

o -PII I o

0 -ethanolamine

Figure 1-5-26. Formation of phosphatidylethanolamine.

In a Nutshell Phospholipid Synthesis • ATP phosphorylates ethanolamine • CTP activates phosphoethanolamine • CDP-ethanolamine combines with diacylglycerol to form phosphatidyl-ethanolamine

84

iiieilical

B. Interconversion of phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidylcholine (PC). Three types of reactions allow interconversion between these three classes of phospholipids. 1. Polar head group exchange. The ethanolamine moiety of phosphatidylethanolamine can undergo an exchange with free serine, resulting in phosphatidylserine. 2. Decarboxylation of phosphatidylserine results in phosphatidylethanolamine. 3. Methylation of phosphatidylethanolamine in three consecutive reactions results in phosphatidylcholine. S-adenosylmethionine (SAM) is used as a methylating agent. These interconversions are illustrated in Figure 1-5-27.

Upids

Phophatidylethanolamine

Phosphatidylserine

Phosphatidylcholine

Figure 1-5-27. Interconversion of PE, PS, and PC.

C. Phosphatidylinositol synthesis uses a variation of the above pathway in which the diacyl-

glycerol moiety is activated by the formation of CDP-diacylglycerol. DAG-phosphate is subsequently transferred from CDP-DAG to one of the hydroxyl groups of inositol.

SPHINGOLIPIDS AND GLYCOLIPIDS This group of lipids is classified into five major categories: sphingomyelin, cerebrosides, sulfatides, globosides, and gangliosides. All five classes are categorized as sphingolipids, containing ceramide (N-acyl-sphingosine). Additionally, all of the classes except sphingomyelin are glycolipids due to the presence of carbohydrate. A. Synthesis of sphingolipids and glycolipids occurs in two major steps. The first step is the synthesis of the ceramide core. The common ceramide core is synthesized from three precursors: palmitoyl-CoA, serine, and a long-chain fatty acyl-CoA. All classes of sphingolipids and glycolipids are formed by the transfer of groups from their activated carriers to a hydroxyl group on the terminal carbon of ceramide (see Figure 1-5-7). Activated carriers used in sphingolipid and glycolipid synthesis include UDP-sugars, CDP-choline, phosphoadenosine phosphosulfate (PAPS), and CMP-N-acetylneuraminic acid (CMP-NANA). B. Classes of sphingolipids and glycolipids (see Table 1-5-4) 1. Sphingomyelin is a major component of membranes of the central nervous system. It is

the only sphingolipid that contains phosphate. The transfer of phosphocholine from UDP-choline to ceramide forms sphingomyelin. 2. Cerebrosides and sulfatides. Cerebrosides contain either glucose or galactose. The addition of sulfate to galactocerebroside generates a sulfatide. Synthesis requires UDP-glu, UDP-gal, and PAPS as "active sulfate:' 3. Globosides. The addition of two or more sugars to ceramide results in globosides. The sugars may be glucose, galactose, or N-acetylgalactosamine. These compounds are important constituents of red blood cell membranes. 4. Gangliosides. Glycolipids containing neuraminic acid, the most common form of sialic acid, are classified as gangliosides. These lipids are found in very high concentration in ganglion cells of the central nervous system. C. Sphingolipidoses (Table 1-5-7)

~1caI

85

Biochemistry

Table 1-5-7. Common sphingolipid disorders. Disease

Mode of Inheritance

Mechanism of Disease

Characteristics

Gaucher

Autosomal recessive

Deficiency of

Hepatosplenomegaly Neurologic deficits Mental retardation

~-glucocerebrosidase

t Glucocerebrosides in brain, liver, spleen, bone marrow Fabry

X-linked recessive

Deficiency of a-galactosidase t Ceramide trihexoside

Renal failure Telangiectasias Skin rash Pain in lower extremities

Tay-Sachs

Autosomal recessive

Deficiency of hexosaminidase A t GMz gangliosides

Mental retardation, blindness, muscular weakness Cherry red spot on macula Death by age 3 Common in Ashkenazic Jews

Deficiency of sphingomyelinase

Mental retardation Hepatosplenomegaly Neurologic deficits Foam cells in bone marrow Cherry red spot in 40% of cases Death by age 3

Niemann-Pick Autosomal recessive

t Sphingomyelin and cholesterol in reticuloendothelial tissues

Farber

Autosomal recessive

Deficiency of ceramidase t Ceramide

Hoarseness, dermatitis Skeletal deformations Mental retardation Hepatomegaly

PROSTAGLANDINS, THROMBOXANES, AND LEUKOTRIENES These compounds, collectively known as the eicosanoids, are derived from arachidonic acid and play crucial regulatory roles in human physiology. They bind to cell surface receptors and act as autocrine and paracrine hormones. They exert their effects at very low concentrations, have extremely short half-lives, and have many diverse functions. A. Prostaglandins are 20-carbon hydroxy fatty acids containing a five-membered ring. Prostacyclin, PGlz, is the major prostaglandin and is synthesized by endothelial cells. It promotes vasodilatation of the coronary arteries and antagonizes platelet aggregation. B. Thromboxanes are 20-carbon hydroxy fatty acids with a six-membered ring containing an

oxygen atom. Thromboxane A2 is the major thromboxane. It antagonizes prostacyclin, leading to platelet aggregation and vasoconstriction. It is synthesized primarily by platelets. C. Leukotrienes contain no ring. They play a major role in inflammatory responses and

chemotaxis. The slow-reacting substance of anaphylaxis (SRS-A) is a combination of leukotrienes that acts as a potent smooth muscle constrictor.

86

meClical

Lipids

D. Synthesis of prostaglandins, thromboxanes, and leukotrienes. Arachidonic acid (AA) is the major precursor of these compounds. AA is stored in membrane phospholipids, where it is esterified to carbon-2 of the glycerol backbone. The release of AA from the membrane is catalyzed by the action of phospholipase Az. The major enzymes involved in eicosanoid synthesis and their functions are summarized in Table 1-5-8. Linoleic acid, an omega-6 fatty acid, is the dietary precursor of arachidonic acid, which is used to synthesize all of the series-2 eicosanoids (PGI 2, T~, etc.). Linolenic acid, an omega-3 fatty acid, is the dietary precursor of eicosapentaenoic acid, which is used to synthesize the series-3 eicosanoids. The most important eicosanoid in this series is TXA3, which is synthesized by platelets and antagonizes the effects of T~.

Table 1-5-8. Key enzymes in eicosanoid metabolism. Enzyme

Action

Phospholipase A2

Releases arachidonic acid from membrane stores; inhibited by glucocorticoids (e.g., cortisone)

Cyclooxygenase

Catalyzes initial step in the pathway that converts arachidonic acid to prostaglandins and thromboxanes; inhibited by nonsteroidal antiinflammatory drugs

5-Lipoxygenase

Catalyzes the initial step in the pathway that converts arachidonic acid to leukotrienes

I KAPLAlf medlea

87

Amino Acid Metabolism One of the major functions of the amino acid pool is to provide a source of building blocks for protein synthesis. Humans can synthesize only 10 of the 20 amino acids found in proteins. The remainder are termed the "essential" amino acids and must be supplied by dietary protein. Both the quantity and the composition of the amino acid pool in cells remain remarkably constant under a wide variety of conditions. Processes that contribute to the pool of amino acids include degradation of body protein, digestion and absorption of dietary protein, and the de novo synthesis of nonessential amino acids. Processes that remove amino acids from the pool include the synthesis of body protein, the oxidation of excess amino acids to CO2 and H20, and the synthesis of a large number of specialized products, including heme, neurotransmitters, purines, pyrimidines, and catecholamines. This chapter reviews protein digestion and absorption, pathways for synthesis of nonessential amino acids, mechanisms for disposing of a-amino groups and the "remnant" carbon skeletons, and the use of amino acids as precursors for a large number of specialized products.

ESSENTIAL AMINO ACIDS An essential amino acid is one that cannot be synthesized in adequate amounts to meet the needs of the cell and therefore must be supplied by dietary protein. There are 10 essential amino acids (Table 1-6-1). Two of these (arginine and histidine) are essential only in infants and children. Humans are unable to synthesize the aromatic ring system in amino acids, the branched chain amino acids, or the basic amino acids. Table 1-6-1. Essential amino acids. Arginine* Histidine* Isoleucine Leucine Lysine

Methionine Phenylalanine Threonine Tryptophan Valine

*Essential only in early childhood

Mnemonic In sentence form: Any help in leaming these little molecules proves truly valuable (arg, his, ile, leu, thr, Iys, met, phe,

A. The amino acid pool. There are approximately 100 grams of free amino acids in the body, of which nearly 50% is glutamine and alanine. Approximately 10% of the total pool consists of essential amino acids. The major drain on the amino acid pool is protein synthesis, with 200-300 grams of protein synthesized per day. In anabolic states, when insulin is acting on liver and muscle, this rate is increased. Additionally, specific amino acids are removed from the pool to support the synthesis of specialized compounds, including heme,

trp, val)

In name form: Pvt. Tim Hall, U.s. Army (phe, val, thr, trp, ile, met, his, arg, leu, Iys) KAPLA~. I meulca

89

Biochemistry

neurotransmitters, creatine, polyamines, carnitine, and nucleotides. Therefore, an exogenous protein source is required to replenish and maintain the amino acid pool. The recommended protein requirement for a healthy adult is 0.8 g/kg body weight/day. B. Digestion of dietary protein. Protein digestion starts in the stomach, where the acidic gastric juice denatures the protein and activates pepsin. Proteins are hydrolyzed by pepsin to a mixture of large peptides. Further degradation occurs in the small intestine by the pancreatic proteases. These enzymes (trypsin, chymotrypsin, elastase, carboxypeptidases) are secreted as inactive precursors and are activated in the small intestine. Activation is initiated by enteropeptidase, an intestinal enzyme that converts inactive trypsinogen to trypsin. Trypsin, in turn, activates the other pancreatic proteases. These enzymes continue the hydrolysis of dietary protein, producing a mixture of free amino acids, dipeptides, and tripeptides. C. Absorption of amino acids. Specific transport systems absorb amino acids and small peptides into the epithelial cells where the dipeptides and tripeptides are hydrolyzed to free amino acids before they leave the cell. The transport systems for amino acids in the intestinal cells are similar to those for glucose, where transport across the luminal membrane is Na+ -dependent and transport across the contraluminal membrane is Na+ independent. Several different carriers have been identified on the basis of their specificity. Genetic deficiencies in each of these have been reported. Hartnup disease and cystinuria are clinical problems arising from faulty transport mechanisms. 1. Hartnup disease is a rare autosomal recessive disorder characterized by the excretion of large quantities of neutral amino acids in the urine. The clinical symptoms of this disease can be attributed to the loss of tryptophan, a precursor of nicotinamide, in the urine. Similar symptoms are seen in pellagra, a condition caused by niacin or nicotinamide deficiency. Up to 50% of nicotinamide is normally supplied by the metabolism of tryptophan.

Mnemonic Cystinuria results from an inability to reabsorb COAL in the renal tubules: cystine, omithine, arginine, and lysine.

Note Pyridoxal phosphate is a derivative of pyridoxine (vitamin 86 ) and acts as a coenzyme for all transaminases.

90

meCtical

2. Cystinuria is one of the most common amino acidurias. It is an autosomal recessive disease caused by a defect in the transport protein for lysine, arginine, cystine, and ornithine, resulting in their excretion in the urine. The low solubility of cystine leads to the precipitation and formation of kidney stones.

AMINO ACID DEGRADATION The catabolism of amino acids will be reviewed in two parts: the disposal of the a-amino group and the disposal of the carbon skeletons. Many of these reactions require the actions of pyridoxal phosphate. A. Disposal of a-amino groups. The degradation of amino acids usually begins with removal of the a-amino group. Approximately 85% of the amino nitrogen ultimately gets excreted as urea. The two most important reactions in the flow of nitrogen from amino acids to urea are transaminations and the oxidative deamination of glutamate. All of the amino acids except lysine and threonine have specific transaminases that are involved in the disposal of the a-amino group. 1. Transaminases are a family of enzymes that transfer the a-amino group from an amino acid to an acceptor, an a-keto acid. Pyridoxal phosphate (PLP) acts as a transient carrier in the reaction (Figure 1-6-1). Transaminases are specific for the amino acid but are nonspecific for the acceptor. Most transaminases use a-ketoglutarate as an acceptor, providing a mechanism for funneling the amino groups from many amino acids into a common pool of glutamate.

Amino Acid Metabolism

2. Fate of glutamate. Two routes for disposal of the a-amino group of glutamate exist in the liver. It may be released as ammonia or transferred to oxaloacetate.

Glutamate

Amino acid N~

N~

I R -CH -COOH)( E",-PLP

R -C -COOH II

o

E",-PLP I NH2

I HOOC -CH, -CH, -CH -COOH

)C

HOOC -CH -CH -C -COOH 2 2 II 0

a-Ketoacid

a-Ketoglutarate

Figure 1-6-1. Transfer of a-amino groups from amino acids to glutamate.

a. Oxidative deamination of glutamate. This reaction is catalyzed by glutamate dehydrogenase, a mitochondrial enzyme that releases an ammonium ion from glutamate, making it available for urea synthesis.

Glutamate Dehydrogenase Glutamate + NAD+ ...

• a-Ketoglutarate + NADH + NH/

b. Transamination to oxaloacetate. Some of the a-amino groups that have been collected in glutamate are transferred to oxaloacetate to form aspartate. Aspartate, in turn, acts as an amino group donor in the synthesis of urea. This reaction occurs mainly in the cytosol of liver, and is catalyzed by aspartate transaminase (AST), also known as glutamate:oxaloacetate transaminase.

Aspartate Transaminase • Glutamate + Oxaloacetate ...

a-Ketoglutarate + Aspartate

3. Urea synthesis. Urea synthesis occurs only in the liver. Urea contains two amino groups that are linked by a carbonyl group. One amino group comes from ammonia and the other is donated by aspartate. The carbonyl group comes from bicarbonate. The synthesis of one mol of urea requires five enzymes and 3 mols of ATP.

NH/ + HC0 3- + Aspartate + 3 ATP ...... Urea + Fumarate + 2 ADP + AMP + 2 Pi + PP i a. Reactions and enzymes of the urea cycle. The first two steps in the urea cycle occur in the mitochondria, and the remaining steps occur in the cytosol. An overview of the urea cycle is shown in Figure 1-6-2, with a brief description of the enzymes and the reactions. KAPLAlf _ I meillea

91

Biochemistry

--------~----------------------------------------------------------------------------------

Mitochondrial Matrix

NH: + HCO"3 + 2 ATP

I

Ca"'amoyl phosphale syolhelase I

r

Carbamoyl phosphate

Omllhloe I,"osca"'amylase

Om"hloe + -

-

-

-

-

I

Citrulline

I

Cytosol

I

I

:

:

Citrulline Arginosuccinate synthetase

~spartate ATP MP+ PPj

Arginosuccinate

~osuccloale lyase Fumarate

!

TCAcycle

Arginine

A Urea

Arginase

Ornithine -

-

-

-

Figure 1-6-2. The urea cycle.

In a Nutshell

(1) Carbamoyl phosphate synthetase I (CPS-I) catalyzes the condensation of

ammonia and bicarbonate to form carbamoyl phosphate. The energy for creating the high-energy bond in carbamoyl phosphate is supplied by 2 mols of ATP. This is the rate-limiting step in urea synthesis. The activity of CPS- I is allosterically activated by N-acetylglutamate (NAG), a compound formed when glutamate accumulates in the mitochondria. This occurs when the diet is high in protein. The synthesis of NAG is activated by arginine. Carbamoyl phosphate synthetase II is a cytosolic enzyme that participates in pyrimidine synthesis. It uses glutamine as a donor of amino groups.

High protein diet

J, Glutamate accumulation

J, Increase in NAG

J, CPS-I activated

(2) Ornithine transcarbamoylase (OTCase) transfers the carbamoyl group from carbamoyl phosphate to ornithine, resulting in citrulline. Citrulline is then transported across the inner mitochondrial membrane into the cytosol, where the remaining reactions in the urea cycle occur. (3) Argininosuccinate synthetase condenses citrulline with aspartate to form argininosuccinate. The a-amino group of aspartate is linked directly to citrulline in this reaction. The energy for synthesizing arginosuccinate is provided by ATP hydrolysis to AMP and PPi.

92

KAPLAlfdme leaI

-

-----------

-------

Amino Acid Metabolism

(4) Argininosuccinate lyase cleaves argmmosuccinate to form argmme and

fumarate. All of the aspartate molecule except the a-amino group is released as fumarate; the a-amino group becomes a part of the arginine side chain. (5) Arginase releases urea from the side chain of arginine. The other product is ornithine, which is transported back into the mitochondria for participation in another cycle of urea synthesis. The urea is excreted in the urine. Note that urea contains two amino groups, one from aspartate and the other from ammonia. b. Abnormalities in the urea cycle. Inherited deficiencies have been reported for each of the enzymes of the urea cycle. These can be distinguished from one another on the basis of the products excreted (Table 1-6-2). Ammonia accumulates in all of the deficiencies but is most pronounced in deficiencies of CPS-I and OTCase and at least pronounced in an arginase deficiency.

Note Arginase is found only in the brain, liver, and kidney.

Table 1-6-2. Defects in urea cycle enzymes. Defective enzyme

Condition

Metabolites Accumulated

Carbamoyl-P synthetase Ornithine transcarbamoylase Argininosuccinate synthetase Argininosuccinate lyase Arginase

Type I hyperammonemia Type II hyperammonemia Citrullinemia Argininosuccinic aciduria lIyperargininemia

Ammonia, glutamine, alanine Ammonia,glutamine,orotate Citrulline Argininosuccinate Arginine

4. Alanine. Skeletal muscle uses alanine as a carrier for transporting a-amino groups to the

liver, where urea synthesis occurs. As shown in Figure 1-6-3, the amino groups that have been collected in glutamate are subsequently transferred to pyruvate with the formation of alanine. This reaction is catalyzed by alanine aminotransferase (also known as glutamate:pyruvate transaminase). Skeletal muscle releases large amounts of alanine into the circulation. Alanine is taken up by the liver, where the reverse reaction occurs. This cycle allows amino groups from skeletal muscle to be transferred to the hepatic pool of glutamate. The alanine aminotransferases of both skeletal muscle and liver are cytosolic enzymes. Liver recycles pyruvate by using it for gluconeogenesis.

Amino acids a-Ketoacids

X

Muscle a-Ketoglutarate

Glutamate

X

In a Nutshell • Amino groups in muscle are transferred to pyruvate to form alanine. • Alanine is picked up by liver, where it is converted back to pyruvate. • Liver uses pyruvate for gluconeogenesis and amino groups for urea synthesis.

Liver

Alanine -+---11-~ Alanine

PY"Nate

Pyruvate

t

~

a-Ketoglutarate

X

Glutamate _

Urea

Glucose..,I-+-- Glucose

Figure 1-6-3. Alanine as a carrier of a-amino groups from muscle to liver.

5. Glutamine. All tissues produce some free ammonia that can be "detoxified" by the formation of glutamine. This reaction is important in extrahepatic tissues, where urea synthesis does not occur, and it is of particular importance in the brain, where the toxic effects of ammonia are severe.

Glutamate + NlI4 + + ATP

Glutamine synthetase ..

Glutamine + ADP + Pi KAPLAN"dme leaI

93

Biochemistry

Skeletal muscle and brain release large amounts of glutamine into the circulation, with most of the glutamine being taken up by the kidney and the liver. The nitrogen is released from glutamine as ammonium ions in two sequential reactions catalyzed by glutaminase and glutamate dehydrogenase (Figure 1-6-4). Both of these reactions occur in mitochondria. In liver, the ammonia is incorporated into urea; in kidney, it is excreted as ammonium ion. Ammoniagenesis in kidney is an important mechanism for titrating protons (60% of H + is excreted through NH4+). The activity of glutaminase is markedly increased in states of prolonged acidosis. In prolonged fasting and starvation, the a-ketoglutarate in this reaction is an important substrate for renal gluconeogenesis.

Glutamine

Glutamate dehydrogenase

Glutaminase

-_'),.----'~

NHt

Glutamate

----=--::::------o:o--~,

NAO~

::O(P)H

a-Ketoglutarate

JHt

Figure 1-6-4. Glutamine as a carrier of amino groups.

In a Nutshell • Strictly ketogenic: leu, Iys • Both ketogenic and glucogenic: ile, phe, tyr, trp • Strictly glucogenic: everything else

94

mectical

B. Disposal of carbon skeletons. Carbon skeletons are the "remnants" of amino acids after the amino groups have been removed. The carbon skeletons can be oxidized to C02 and H20, with the generation of energy, or they can be used to synthesize either glucose or ketones. As shown in Figure 1-6- 5, all of the amino acids can be degraded to seven common metabolites: acetyl-CoA, acetoacetyl-CoA, pyruvate, oxaloacetate, fumarate, succinyl-CoA, a-ketoglutarate, and propionyl-CoA (which is converted to succinyl-CoA). The ketogenic amino acids (leucine and lysine) are degraded to acetyl-CoA or acetoacetyl-CoA, both of which can be converted to ketone bodies. Four amino acids are both ketogenic and glucogenic (isoleucine, phenylalanine, tyrosine, and tryptophan). The remaining amino acids are strictly glucogenic.

Amino Acid Metabolism

~

Glycine, Alanine, Cysteine, Serine, Threonine, Tryptophan

Leucine, 'so'.u';".

Asp"ag;". Aspartate

I___~~

-----.;.~ pyruvate

1 _ _ _ _ _... ~

Acetyl-CoA .....e----- Acetoacetyl-CoA .....i------- Leucine Lysine Phenylalanine Tryptophan Tyrosine

Oxaloacetate

Citrate

Tyrosine~

Proline a-Ketoglutarate __ Glutamate - - Arginine Histidine Glutamine

Phenylal:::J .... Fumarate

Succinyl-CoA

t'--______

Propionyl-CoA .....i------- Isoleucine Methionine Threonine Valine

Figure 1-6-5. Metabolic intermediates from amino acids.

C. Degradation of branched-chain amino acids. Liver has little, if any, transaminase activity for valine, isoleucine, and leucine. Therefore, in contrast to the other amino acids, the degradation of the branched-chain amino acids starts in skeletal muscle, where transaminase activity is high. The first two reactions in the degradation of these three amino acids are catalyzed by common enzymes (Figure 1-6-6). Branched-chain amino acid transaminase (BCAA transaminase) transfers the a-amino group to a-ketoglutarate to form glutamate and a-keto acids corresponding to the carbon skeletons valine, isoleucine, and leucine. This enzyme is present in high concentrations in skeletal muscle and very low concentrations in the liver. In the next step, the a-keto acids are decarboxylated by a single branched-chain ketoacid dehydrogenase (BCKA-DH), resulting in branched-chain acylCoA analogs. The products of the BCKA-DH reaction proceed along different pathways. The end products for the degradation of each amino acid are shown in Figure 1-6-6. The end products of branched-chain amino acid degradation are used by nerve tissue for the synthesis of lipid.

Clinical Correlate Maple syrup urine disease results from an autosomal recessive deficiency of branchedchain ketoacid dehydrogenase. Affected children have elevated plasma and urine levels of the branched-chain amino acids leucine, valine, and isoleucine, as well as elevated levels of their corresponding a-keto acids and a-hydroxyacids. Clinically, the urine has an odor of maple syrup or burnt sugar; if untreated, brain darnage and death occur by the end of the first year.

meclical

95

Biochemistry

Valine, Isoleucine, Leucine

1

BCM "",samlnase

Branched-chain ketoacid analogs

CO'1

BCKA-dehydwgenase

Branched-chain acyl-CoA analogs

/

I

Val

lie

~

Leu

~

/

Succinyl-CoA

~

Propionyl-CoA Acetyl-CoA

Acetoacetyl-CoA

Figure 1-6-6. Degradation of branched-chain amino acids.

The BCKA-DH is a multienzyme complex structurally homologous to pyruvate dehydrogenase and a-ketoglutarate dehydrogenase. All three catalyze the oxidative decarboxylation of a-ketoacids, and they are composed of three distinct enzymes that require the actions of five coenzymes (NAD+, FAD, CoA, thiamine-PP, and lipoic acid). They are all located in the mitochondria of most cells. D. Assimilation of propionyl-CoA into the TeA cycle. Propionyl-CoA results from the degradation of isoleucine and methionine and from the ill-end of odd-chain fatty acids. The pathway for converting propionyl-CoA into succinyl-CoA is shown in Figure 1-6-7. A deficiency in either propionyl-CoA carboxylase or methylmalonyl-CoA mutase results in organic aciduria. This condition is usually seen in children who present with signs of acidosis. Chromatographic analysis of blood and urine are an important part of the diagnosis. Treatment protocols include measures to counteract the acidosis, followed by restriction of dietary protein. Individuals excreting excessive amounts of organic acids are at risk for developing a secondary carnitine deficiency, leading to impaired fatty acid oxidation. These secondary effects can be prevented by supplementation with L-carnitine. Odd-chain fatty acids

ISOI~

7n,neb'Otin

Propionyl-CoA + HC0 3-

I

Threonine

vitamin B"

~Methylmalonyl-CoA

(

~

) Succinyl-CoA

Methylmalonyl-CoA mutase

ATP ADP + Pi Propionyl-CoA carboxylase

Figure 1-6-7. Conversion of propionyl-CoA to succinyl-CoA.

96

metlical

Amino Acid Metabolism

1. Propionic aciduria results from a deficiency of biotin, propionyl-CoA carboxylase, holo-

carboxylase synthase, or the enzyme that covalently attaches biotin to all carboxylases. In the latter case, additional organic acids accumulate. 2. Methylmalonic aciduria results from a deficiency in vitamin B12 or a defect in methylmalonyl-CoA mutase. Some patients respond favorably to megadoses of vitamin B12 .

SYNTHESIS OF NONESSENTIAL AMINO ACIDS The pathways for amino acid synthesis are complex. All of the nonessential amino acids are synthesized from intermediates in glycolysis or the TCA cycle, from essential amino acids, or from interorgan pathways. Only the key concepts of these pathways will be reviewed. A. Synthesis from intermediates in glycolysis or the TCA cycle. Eight of the nonessential amino acids can be synthesized from intermediates in glycolysis or from the TCA cycle intermediates. This is illustrated in Figure 1-6-8. The transamination of pyruvate, oxaloacetate, and a-ketoglutarate give rise to alanine, aspartate, and glutamate, respectively. The addition of an amide group to the side chains of aspartate and glutamate results in asparagine and glutamine. Phosphoglycerate is a precursor of serine and glycine. Glycolysis

TCA cycle

Glucose ----. Phosphoglycerate ---+- Pyruvate --+ a-Ketoglutarate - - Oxaloacetate

~

~

Serine

~

Alanine

Aspartate

/\..

{-

/\.. GlYCine Cysteine

~

Glutamate

Proline Glutamine Asparagine

Figure 1-6-8. Precursors for the nonessential amino acids.

B. Synthesis from essential amino acids. Phenylalanine is the precursor for tyrosine, and methionine provides the sulfur for cysteine synthesis. Therefore, if either phenylalanine or methionine is deficient in the diet, tyrosine and cysteine also become essential amino acids. This situation may occur transiently in premature babies. The enzyme systems for synthesizing tyrosine and cysteine are developed late in gestation. 1. Tyrosine. The conversion of phenylalanine to tyrosine is catalyzed by phenylalanine

hydroxylase (Figure 1-6-9). This enzyme requires 02 and tetrahydrobiopterin (THB). During the hydroxylation reaction, THB is oxidized to dihydrobiopterin (DHB). For the synthesis of tyrosine to continue, DHB must be reduced back to THE. The regeneration of THB requires NADPH and dihydrobiopterin reductase. Phenylalanine hydroxylase

Phenylalanine

--7-:::;:;O-7-::::-..

""'\:::-----~)

02

K

THB

NADP+

Tyrosine

DHB

D;hydrob;opterio reductase

NADPH

Clinical Correlate Phenylketonuria (PKU) results from either a deficiency of phenylalanine hydroxylase or dihydrobiopterin reductase. There is therefore a buildup of phenylalanine, phenyl pyruvate, phenylacetate, and phenyllactate in both plasma and urine, and tyrosine becomes an essential amino acid. Clinically, there is a musty body odor and mental retardation. Treatment is to remove phenylalanine from the diet (including products containing NutraSweet®, a dipeptide containing phenylalanine and aspartic acid). If the deficient enzyme in PKU is dihydrobiopterin reductase, the ability to synthesize catecholamines and serotonin is also impaired.

Figure 1-6-9. Hydroxylation of phenylalanine to tyrosine. KAPLA~.

meulCa

I

97

Biochemistry

2. Cysteine. Serine and methionine are required for the synthesis of cysteine. Key steps in the pathway are shown in Figure 1-6-10. The sulfur atom in cysteine originates in dietary methionine, and the remainder of the cysteine molecule is derived from serine. Methionine is first converted to homocysteine through several steps. Cystathionine synthase, an enzyme requiring pyridoxal phosphate, condenses homocysteine with serine to form an adduct of the two amino acids. Cystathionase then cleaves the adduct so that the sulfur atom is attached to serine, generating cysteine. Excess homocysteine can be converted back to methionine by the addition of a methyl group from tetrahydrofolate. Methionine synthase is the only enzyme in human biochemistry known to use THF as a methylating agent. This is also one of the two reactions in humans that requires vitamin Bl2.

/~

! Methionine

Serine

\

___\..:..0.-_ _ _• Cystathionine

Homocysteine

Cystathionine synthase Vitamin B6

H20 \.

• Cysteine + a-Ketobutyrate + NH/

Cystathionase Vitamin B6

Vitamin B12 CH 3 -THF THF Methionine synthase Figure 1-6-10. Synthesis of cysteine from methionine and serine.

Clinical Correlate Epidemologic studies suggest that elevated plasma homocysteine is a risk for coronary heart disease that is independent of the risk associated with elevated plasma cholesterol.

a. Homocystinuria is characterized by excretion of large amounts of homocystine in the urine. It is most frequently seen in children who present with failure to thrive and visual problems due to displacement of the lens. Homocystinuria may be either acquired or inherited. A deficiency in pyridoxine, folate, or vitamin Bl2, or an inherited defect in either cystathionine synthetase or methionine synthase all result in the accumulation of homocysteine, which is readily oxidized to its disulfide form, homocystine. Cystathionine synthase requires pyridoxal phosphate (the activated form of vitamin B6) as a cofactor, and methionine synthase requires coenzymes derived from both folate and Bl2. b. Cystathioninuria results from a deficiency in pyridoxine or from a genetic defect in cystathionase. Large amounts of cystathionine are found in the urine and blood. C. Interorgan synthesis of arginine. The synthesis of arginine in humans is dependent on

cooperation between the intestinal mucosa and the kidney. The first two enzymes of the urea cycle are found in intestinal cells, and the third and fourth enzymes of the cycle are found in the kidney. Only liver has all of the enzymes. Intestinal cells synthesize citrulline and release it into the blood. Citrulline is extracted by the kidney and converted to arginine. The kidney has low levels of arginase activity, thus arginine accumulates; however, it does not accumulate in liver due to the high level of arginase activity in the urea cycle.

SPECIALIZED PRODUCTS DERIVED FROM AMINO ACIDS The synthesis of many of the specialized products, especially the biogenic amines, uses three types of reactions: decarboxylation, hydroxylation, and methylation. Decarboxylation reactions require pyridoxal phosphate; most of the hydroxylations require tetrahydrobiopterin (THB) as a reductant; the methylation reactions require S-adenosylmethionine as a methylating agent. Other specialized products derived from amino acids are melanin, creatine, heme, and nitric oxide.

98

KAPLAlfdme leaI

Amino Acid Metabolism

A. S-adenosylmethionine (SAM). Almost all methylation reactions require SAM as a methylating agent. The methyl transfer potential of SAM is much higher than that of methyl-THE SAM is synthesized by the condensation of methionine and ATP (Figure 1-6-11). A high-energy bond attaches the sulfur atom to the methyl group.

eaOH I

H2N -CH

I

CH 2

Mg

+

2+

ATP

I

CH2

I

CH 3 -S

High-Energy Bond

:Y

Methionine

o SAM

OH methyl transferase

r

OH

:-CH,

S-adenosyl homocystein e

Figure 1-6-11. Synthesis and use of S-adenosylmethionine (SAM).

B. Histamine. Decarboxylation of histidine results in histamine, an important mediator of allergic and inflammatory responses. Histamine is synthesized in mast cells of connective tissue near blood vessel (Figure 1-6-12). Histidine Decarboxylase Histidine - - - - - - -.....~ Histamine + CO 2 Pyridoxal-P

Figure 1-6-12. Synthesis of histamine. C. y-Aminobutyric acid (GABA). Decarboxylation of glutamate results in GABA, an important

inhibitory neurotransmitter (Figure 1-6-13). Glutamate Decarboxylase Glutamate _ _ _ _ _ _ _ _....... y-Aminobutyric acid (GABA) + CO2 Pyridoxal-P

Figure 1-6-13. Synthesis of "t"aminobutyric acid.

D. Serotonin and melatonin. Hydroxylation of the aromatic ring of tryptophan followed by its decarboxylation results in serotonin, a potent vasoconstrictor and stimulator of smooth muscle contraction. As shown in Figure 1-6-14, tryptophan hydroxylase requires 02 and tetrahydrobiopterin (THB). Dihydrobiopterin reductase and NADPH are required to regenerate THB. The end product of serotonin degradation is 5-hydroxyindoleacetate (5-HI). In the pineal gland, serotonin can be converted to melatonin by sequential acetylation and methylation reactions.

Clinical Correlate Parkinson disease results from a decrease of dopamine in the substantia nigra. It is found in approximately 1% of the U.s. population above the age of 55. Symptoms include tremors, postural instability, rigidity, and bradykinesia. The symptoms are treated with L-dopa and carbidopa; the latter selectively inhibits the aromatic acid decarboxylase outside of the central nervous system, but it cannot cross the blood-brain barrier and does not inhibit the conversion of L-dopa to dopamine.

KAPLAlf I me illea

99

Biochemistry

02 \. Tryptophan

r

THB

Tryptophan hydroxylase

"*"")'

DHB

Aromatic amino acid decarboxylase • 5-0H-Tryptophan -_~,......-----~. Serotonin

CO2

Figure 1-6-14. Synthesis of serotonin.

E. Catecholamines, melanin, and thyroxine are all derived from tyrosine.

1. Catecholamines act as neurotransmitters when synthesized by the brain and act as hormones when produced by the adrenal medulla. The pathway for synthesis is the same in both tissues (Figure 1-6-15). It includes hydroxylation, decarboxylation, and methylation reactions. Tyrosine hydroxylase and dopamine-~-hydroxylase have different cofactor requirements.

02 Tyrosine \ . hydroxylase Tyrosine

-7

tDHB

THB

Clinical Correlate Neoplastic transformation of enterochromaffin cells results in carcinoid tumors that secrete excessive serotonin, and high levels of s-hydroxyindole acetate are excreted in the urine.

Note There is only one difference in position between T3 and T4 :

T3: X = H T4 :

x= I

Bridge to Pharmacology The ability of nitroglycerin and other nitrates to relieve pain associated with angina is due, at least in part, to the spontaneous formation of NO from these compounds.

100

maC-ical

•

Aromatic acid 02 Dopamine-~Phenylethanolamine-Ndecarboxylase \.. hydroxylase methyl transferase (PNMT) ""'"'-_ _ _~. Norepinephrine • Epinephrine DOPA • Dopamine __ ?' "") Cu+ CO2 Ascorbate SAM S-Adenosylhomocysteine

"Y

Figure 1-6-15. Catecholamine synthesis.

Catecholamines are rapidly degraded by two enzymes that are widely distributed in many tissues: catechol-O-methyl transferase (COMT) and monoamine oxidase (MAO). 2. Melanins are the pigments of skin and hair that are formed in melanocytes. The initial reaction is catalyzed by tyrosine hydroxylase. The isozyme present in melanocytes, also known as tyrosinase, requires copper and ascorbate as cofactors. It hydroxylates tyrosine at two positions, creating a very reactive molecule that polymerizes and gives rise to a series of compounds collectively known as "melanins:' Some forms of albinism result from a deficiency in tyrosine hydroxylase. 3. Thyroxine synthesis occurs in the follicle cells of the thyroid. Synthesis requires the tyrosine-rich protein, thyroglobulin, and the uptake of iodide. The iodide must be oxidized to 1+ before it can be incorporated into tyrosine side chains. The introduction of one molecule of 1+ gives monoiodinated tyrosine (MIT), whereas introduction of two molecules ofI+ produces diiodinated tyrosine (DIT). Covalent cross-linking of two DIT molecules results in tetraiodothyronine (T4), whereas cross-linking of MIT and DIT produces tri-iodothyronine (T 3)' Stimulation by thyroid-stimulating hormone (TSH) results in excision and release of T 3 and T 4. F. Nitric oxide, also known as "endothelium-derived relaxing factor," is synthesized from arginine in endothelial cells of small blood vessels. Nitric oxide diffuses into the smooth muscle cells, where its direct effect is to stimulate cyclic GMP synthesis. cGMP activates a cascade of events resulting in relaxation of smooth muscle. Nitric oxide is synthesized from arginine by the enzyme nitric oxide synthase. This enzyme requires 02 and NADPH. The reaction catalyzed by NO synthase is shown in Figure 1-6-16. Its synthesis is stimulated by calcium ions. The regeneration of tetrahydrobiopterin requires NADPH.

Amino Acid Metabolism

EB Ca2 + Arginine + O2

NO synthase

;=-

NADPH

~.

Nitric oxide + ornithine

NADP+

Figure 1-6-16. Synthesis of nitric oxide.

Nitric oxide is also synthesized on nerve cells where it functions as an excitatory molecule and in macrophages where it is a potent toxin. Nitric oxide synthesis in endothelial and nerve cells is stimulated by calcium; synthesis in macrophages is stimulated by lipopolysaccharides and cytokines. G. Creatine synthesis requires arginine, glycine, and S-adenosylmethionine (Figure 1-6-17). Creatine phosphate is a storage form for high-energy phosphate in muscle. It can be used to rapidly regenerate ATP from ADP by the enzyme creatine phosphokinase (CPK).

Glycine amidinotransferase Arginine + Glycine \ Guanidinoacetic acid Ornithine

Methyl transferase

;=-

SAM

"=').

Creatine' .-----

CPK

)

S-Adenosylhomocysteine ATP

• Creatine phosphate ADP

Figure 1-6-17. Synthesis of creatine and creatine phosphate.

Both creatine and creatine phosphate spontaneously cyclize to form creatinine, which is excreted in urine. Under normal circumstances, the amount of creatinine excreted is constant and is directly proportional to muscle mass. Blood levels of creatinine are used clinically to assess kidney function or to estimate muscle mass. The amount of creatinine in a 24-hour urine specimen can also be used to validate the completeness of a 24-hour specimen. H. Heme synthesis requires the assembly of a porphyrin ring, which is derived entirely from glycine and succinyl-CoA (Figure 1-6-18). The initial step in the synthesis is catalyzed by L1-aminolevulinic acid synthase (L1-ALA synthase), an enzyme that requires pyridoxal phosphate. This is the rate-limiting step in heme synthesis and is inhibited by heme, the product of the pathway. Condensation of 2 mols of L1-ALA produces a porphobilinogen, the first intermediate in the pathway with a pyrrole ring. Condensation of four molecules of porphobilinogen gives a linear pyrrole, which can be cyclized to produce a porphyrinogen. Cyclization is catalyzed by uroporphyrinogen I synthase, but formation of the isomer that leads to heme requires another protein, uroporphyrinogen III cosynthase. The immediate precursor to heme is protoporphyrin IX. Insertion of iron by the enzyme heme synthase, also known as ferrochelatase, results in heme.

Clinical Correlate Porphyria refers to any abnormality in the pathway of heme synthesis. If the block is early in the pathway, the intermediates that build up are excreted in the urine; if the block is late in the pathway, they are excreted in the urine and feces and accumulate in the skin. Lead poisoning can be considered an acquired porphyria because it inhibits ALA dehydratase and heme synthase (ferrochelatase).

iiieCtical

101

Biochemistry

Glycine Succinyl-CoA

ALA synthase

ALA dehydratase 1 - - - - - - L'l-Aminolevulinate ;. Porphobilinogen

Uroporphyrinogen-I synthase ------;;.~

Uroporphyrinogen-III

Uro-III cosynthase

Protoporphyrin IX

Heme synthase (ferrochetolase)

Heme Figure 1-6-18. Heme synthesis.

Heme degradation begins in the reticuloendothelial celis, where heme oxygenase opens the ring system to give the linear pyrrole, biliverdin, and carbon monoxide (Figure 1-6-19). This is the only reaction in human biochemistry that generates carbon monoxide. Biliverdin is reduced to bilirubin, which is further metabolized in the liver. The addition of either one or two molecules of glucuronate to bilirubin greatly increases its solubility. The major forms of heme that are excreted are stercobilin (feces) and urobilin (urine). Free bilirubin is known as "unconjugated" or "indirect" bilirubin, whereas bilirubin glucuronide is known as "conjugated" or "direct" bilirubin.

Clinical Correlate Hyperbilirubinemia can result from massive hemolysis, a block in the catabolism of heme, bile obstruction, or liver damage. Whatever the cause, patients will present with jaundice.

102

meClical

Biliverdin reductase

Heme oxygenase Heme

, ,J

02

NADPH

Biliverdin

,

NADPH

J

UDP-glucuronyl transferase Bilirubin ........... ~ J Bilirubin diglucuronide UDPglucuronate

UDP

Figure 1-6-19. Degradation of heme.

Summary of Metabolism

Fuels burned by the body include sugars, fatty acids, ketones, and amino acids. Each fuel has a unique storage form, transport form, and intracellular metabolites. These three forms can be interconverted via separate synthetic and degradative pathways, thus allowing fuel to be stored or mobilized independently. The pathways for storage and mobilization are regulated in a reciprocal fashion, avoiding futile cycles created by simultaneous synthesis and degradation. This chapter reviews the major storage forms of energy, the types of fuels used by each organ, the hormones that regulate fuel metabolism, and the transitions that occur in going from a well fed state to an overnight fast and finally to a state of prolonged fasting.

PATHWAYS OF FUEL METABOLISM Each metabolic fuel has three primary forms: a storage form, a transport form, and unique intracellular intermediates. The relationships among these different forms of each fuel are shown in Figure 1-7-1.

I _ Triglycerides

,.-_--,

~. d~"----

~ooe~ Bodies

Glycogen

pyruvate ......f----------',

Fatty

_L_A_Ci_dS--,~

GilT I ._--.. ~

y Acet ; CoA

I

Amino . Acids

~

I~proteins

I

TCA Cycle ......f------------'

~

I

Electron Transport and ------.. Oxidative Phosphorylation

ATP

Figure 1-7-1. Summary of fuel metabolism.

A. Relationship between storage and transport forms. Fatty acids, glucose, and amino acids serve as the transport forms of triglycerides, glycogen, and proteins, respectively. The transport forms are found in highest concentration in the blood. They can either be released or taken up by cells, depending on the nutritional status of the organism. The formation of ketone bodies provides an overflow pathway for excess acetyl-CoA. When fatty acid oxidation is producing acetyl-CoA faster than it can be used, the excess acetyl-CoA is converted to KAPLAlf

-

med lea

I

103

Biochemistry

In a Nutshell Separate pathways for reversible processes prevent the futile cycling of opposing pathways.

ketones. The ketones can then enter the blood and be taken up by other tissues and used as fuel. The pathways by which storage forms of fuel are converted to transport forms and vice versa are unique and have been reviewed in detail in other chapters. For example, the incorporation of fatty acids into triglyceride and the release of fatty acids from triglyceride occur by different pathways rather than by the reversal of a common pathway. The same relationship exists for glycogen synthesis and degradation and for protein synthesis and degradation. These different and opposing pathways between storage and transport forms of fuel are usually regulated in a reciprocal manner, ensuring that when one pathway is in operation, the other is dormant. B. Relationship between transport forms and intracellular metabolites. Inside the cell, each of the fuels is converted to one or more characteristic intracellular metabolites. For example, amino acids are degraded to glycolytic or TCA intermediates, glucose is degraded to pyruvate, and fatty acids are oxidized to acetyl-CoA (Figure 1-7-2). Opposing but separate pathways are also present in cells, allowing these fuel-specific intermediates to be converted to their corresponding transport forms and released into the blood. A more detailed metabolic map of these interconversions is shown in Figure 1-7-3. Once again, the opposing pathways are tightly regulated so that intermediates are not being synthesized and degraded simultaneously. As shown in Figure 1-7-2, all of the metabolic fuels are eventually degraded to acetyl-CoA, which is oxidized to C02 and H20 by common pathways. The energy for synthesizing most of the cellular ATP comes from the TCA cycle, the electron-transport chain, and oxidative phosphorylation. C. Transitions between metabolic fuels. Certain transitions among the various fuels are permissible and others are nonpermissible. Both carbohydrates and proteins can be converted to fatty acids. Carbohydrate can be used for the synthesis of the nonessential amino acids and protein. However, acetyl-CoA cannot be converted to pyruvate and, therefore, cannot Glycogen (liver and muscle)

Ribose 5-Phosphate

~

Glucose

HEXOSE MONOPHOSPHATE SHUNT

~__In_te_rc_on_ve_rs_io_n_of_-, 3,4,5,6,& 7

Glucose - - - - . Glucose 6-P hosPhate -

1 I

Carbon Sugars

NADPH

GLYCOLYSIS

i/

Pyruvate

GIYOOgen/ Amino

~~'\

I

____ Vitamin 0

Acetyl CoA - - - - - - - - - - Cholesterol--Steroid Hormones ---Bile Acids

Fatty Acids Ketone Bodies

ATP

1

Ketogenic Amino Acids

Figure 1-7-2. Relationship of carbohydrate, fat, and amino acid metabolism.

104

me'lieal

Summary of Metabolism

act as a precursor for carbohydrate or protein synthesis. The reaction catalyzed by pyruvate dehydrogenase (pyruvate to acetyl-CoA) is an irreversible reaction, and there is no opposing enzyme in human tissues that will get around this irreversible step. Therefore, the conversion of fat to carbohydrate and protein is considered a nonpermissible transition. D. Regulation of fuel metabolism. The pathways that are operational in fuel metabolism depend on the nutritional status of the organism. Shifts between storage and mobilization of a particular fuel, as well as shifts between the type of fuel being used, are very pronounced in going from the well fed state to an overnight fast and finally to a prolonged state of starvation. The shifting metabolic patterns are regulated mainly by the insulin/glucagon ratio. Insulin is an anabolic hormone that promotes fuel storage_Its action is opposed by a number of hormones, including glucagon, epinephrine, cortisol, and growth hormone. The major function of glucagon is to respond rapidly to decreased blood glucose levels by promoting the synthesis and release of glucose into the circulation. 1. Well fed state. Immediately after a meal, the blood glucose level rises and stimulates the release of insulin. The three major target tissues for insulin are liver, muscle, and adipose tissue. Insulin promotes glycogen synthesis in liver and muscle. After the glycogen stores are filled, liver converts excess glucose to fatty acids and triglycerides (Figure 1-7-3). Insulin also promotes triglyceride synthesis in adipose tissue and protein synthesis in muscle. Following a meal, most of the energy needs of the liver are met by the oxidation of excess amino acids.

Ribulose 5-P04 Nucleotides+-Ribost 5-P04

Glucose 1-P04

NADPH~

!- -

==x.:::

L-UDP-GIUCOSe

6-P-Gluconolactone

-

Galactose 1-P04 UDP-Galactose

----------------j
JGl!L~ugC~0~S~EJI I rl I

GIUCOS16-Po4...

-Fructost6-P04----1 MANNOSE

! !

FRUCTOSE

Fructose 1-P04

Fructose 1,6-bis-P0 4'-...... ~

/

'\..

Glyceraldehyde

Glyceraldehyde 3-1' 0 4 - - Di~YdrO~ • I ace one 4 • PhosPhoenr pyruvate

Glycerol 3-P04 ~ GLYCEROL I ~ FATTY Fatty AcidCOA. ACID

Lactate +----+- Pyruvate

!

Acetyl-CoA

~

NH3yC02 ASPARTATE

oxaloacetate

Carbamoyl P04

I

V

I

--------Acetoacetate~ LEUCINE

Citra\te

Argininosuccinate )

ur~

i

CoA

a-Ketoglutarate

Arginine

Glutamate

Meth~malonyl-

Ornithine

\

ALANINE SERINE CYSTEINE GLYCINE

• M~~~YI-

/ Fumarae t

Citrulline

HMP shunt activity varies with the body's need for reducing power (NADPH). Activity increases during the well fed state because of the need for NADPH for fatty acid or cholesterol synthesis.

IGAlACTOS' I

IGLYCr'" I

NAD~-P-GIUConat'

Note

Succinyl-Co

~~~~~~________

°t

~

PropionylCoA r---.JC::::==i-_Acetyl CoA FATTY ACYL

od~~!ber

Figure 1-7-3. Inter-relationships of metabolism.

mectical

105

Biochemistry

Note HMP shunt activity decreases with an overn ight fast as gluconeogenesis and ketone body formation begin.

2. Overnight fast. Glucagon and epinephrine levels rise during an overnight fast. These hormones stimulate the release of glucose from the liver and fatty acids from adipose tissue. In liver, glycogen degradation and the release of glucose into blood are stimulated. Hepatic gluconeogenesis is also stimulated, but the response is slower than that of glycogenolysis (Figure 1-7-4). The release of fatty acids from adipose tissue is stimulated by both glucagon and epinephrine. The ATP required for gluconeogenesis is supplied by oxidation of fatty acids. The carbon skeletons for glucose synthesis are derived primarily from amino acids that are released from skeletal muscle.

""C

Q)

/

Exogenous glucose

Protein sparing. Brain adapts to ketone bodies using 2/3 ketone bodies and 1/3 glucose as its energy source.

(J)

:J Q) (J)

o (.)

:J

a

. Llve~ glycogen

/"_" 'i.,•...../----1'.------ ......•. / --- ............... . .. ' ......\ Gluconeogenesis 10 hours

20 hours

2 days

24 days

40 days

Time fasting Figure 1-7-4. Source of blood glucose during fasting.

Note HMP shunt activity is almost nonexistent during the prolonged fasting or starvation state.

3. Prolonged fast (starvation). Levels of glucagon and epinephrine are markedly elevated during starvation. Lipolysis is brisk, resulting in excess acetyl-CoA that is used for ketone synthesis. Levels of both lipids and ketones are therefore increased in the blood. Muscle uses fatty acids as the major fuel and the brain adapts to using ketones for some of its energy. After 1-2 weeks of fasting, the brain derives approximately two thirds of its energy from ketones and one third from glucose. The shift from glucose to ketones as the major fuel diminishes the amount of protein that must be degraded to support gluconeogenesis (Figure 1-7-4). There is no "energy-storage form" for protein because each protein has a specific function in the cell. Therefore, the shift from using glucose to ketones during starvation spares protein. Red blood cells and renal medullary cells that have few, if any, mitochondria continue to be dependent on glucose for their energy.

ENERGY STORAGE AND UTILIZATION Fats are much more energy rich than carbohydrates, proteins, or ketones. Complete combustion of fat results in 9 kcal/g compared with 4 kcal/g derived from carbohydrate, protein, and ketones. The storage capacity and pathways for use of fuels varies with different organs and with the nutritional status of the organism as a whole. A. Storage. Most carbohydrate storage occurs in skeletal muscle and liver. Muscle glycogen accounts for approximately two thirds of the carbohydrate storage. A small fraction of the total carbohydrate is found as glucose in the circulation. Fats are stored in adipose tissue, which comprises anywhere from one fourth to one third of the human body weight. Proteins are found in all body tissues where, on the average, they account for three fourths of the

106

meClical

Summary of Metabolism

tissue mass. Degradation of more than approximately one third of the protein in the body is incompatible with life. Therefore, protein sparing by shifting patterns of fuel use is essential to survival during prolonged fasting. B. Utilization. Patterns of fuel use depend both on the metabolic state (well fed versus fasting)

and on the tissue being studied. Regardless of which fuel is used, they are all degraded to acetyl-CoA, which is oxidized by common pathways to C02 and H20. The fact that all metabolic fuels give rise to a common intermediate (acetyl-CoA) provides a mechanism for establishing priorities in fuel use. 1. Priorities. The fasting state promotes fat use and inhibits carbohydrate and protein use.

In the fasting state, gluconeogenesis, proteolysis, and lipolysis are all activated. The amino acids are supplying carbon skeletons for glucose synthesis and the oxidation of fatty acids to acetyl-CoA provides the energy for driving glucose synthesis. The accumulation of acetyl-CoA inhibits pyruvate dehydrogenase, the enzyme that irreversibly converts pyruvate to acetyl-CoA. When pyruvate dehydrogenase is inhibited, the ability to use carbohydrate and protein as sources of fuel is decreased. Under these conditions, pyruvate (derived from either carbohydrate or amino acid precursors) can be diverted away from oxidation and toward gluconeogenesis. Thus, when fat is being used as a metabolic fuel, there is no need to oxidize carbohydrate or protein.

In a Nutshell The increase in acetyl-CoA leads to a decrease in the activity of pyruvate dehydrogenase, which in turn leads to a decrease in the use of carbohydrates and proteins.

2. Organ specificity. Different organs use different fuels. The organ-specific patterns of fuel

use in the well fed and fasting states are summarized in Table 1-7 -1. Table 1-7-1. Preferred fuels in the well fed and fasting states. Organ

Postfeeding

Fasting

Skeletal muscle Cardiac muscle Brain Kidney cortex Kidney medulla Liver Adipose tissue Red blood cells

Glucose Glucose and fatty acids Glucose Fatty acids Glucose Glucose, amino acids Glucose Glucose

Fatty acids, ketones Fatty acids, ketones Ketones, glucose Fatty acids Glucose Fatty acids Fatty acids Glucose

a. Skeletal muscle. The main fuels of skeletal muscle are glucose and free fatty acids. Due to the enormous bulk, skeletal muscle is the body's major consumer of fuel. After a meal, glucose is used to replenish the glycogen stores. In the post-prandial state, elevated levels of insulin promote the uptake of glucose and amino acids by muscle. Excess amino acids are oxidized for energy, and excess glucose is broken down through glycolysis. If the muscle is anaerobic due to intense contraction, the production of pyruvate by glycolysis may exceed the capacity of the citric acid cycle to carry out terminal oxidation. In this case, lactic acid accumulates. Pyruvate is converted to lactate so that the NAD+ needed by glyceraldehyde-3-phosphate can be regenerated. In the fasting state, muscle uses fatty acids and ketones as the primary fuel. Any available glucose is spared for use by brain and red blood cells. b. Cardiac muscle. After a meal, glucose and fatty acids are the major fuels consumed by heart muscle. After a brief period of fasting, fatty acids are used, and after a longer fast, ketones are used. Thus, cardiac muscle behaves much like skeletal muscle in terms of fuel use, except it has a much higher oxidative capacity and many more mitochondria.

In a Nutshell • Glucose and fatty acids are the main fuels in skeletal muscle. • Cardiac muscle uses glucose, fatty acids, and ketone bodies for fuel. • The brain uses glucose for fuel and will resort to ketone bodies during prolonged fasting only if it's desperate. KAPLA~. I meulca

107

Biochemistry

c. Brain. Glucose is the primary fuel for brain. Fatty acids cannot cross the blood-brain barrier and are therefore not used at all. Between meals, brain relies on blood glucose supplied by either hepatic glycogenolysis or gluconeogenesis. Only in prolonged fasts does the brain gain the capacity to use ketones for energy, and even then ketones supply only approximately two thirds of the fuel supply. The remaining one third of the fuel continues to be supplied by glucose.

In a Nutshell The kidney isn't picky: it'll use whatever is available for fuel.

d. Kidney. The kidney is unique in that it utilizes virtually all fuels: fatty acids, glucose, lactate, amino acids, citrate, glycerol, and ketones. The renal cortex relies primarily on fatty acids and to a lesser extent on ketones but will also use lactate, amino acids, and glycerol. The renal medulla preferentially uses glucose. During states of fasting, the rate of gluconeogenesis by the kidney is increased. e. Liver. The two major roles of liver in fuel metabolism are to maintain a constant level of blood glucose under a wide range of conditions and to synthesize ketones when excess fatty acids are being oxidized. After a meal, the glucose concentration in the portal blood is elevated. The liver extracts excess glucose and returns the concentration in the blood to normal. The extracted glucose is used by the liver to replenish its glycogen stores, and any glucose that remains is then converted to acetyl-CoA and used for fatty acid synthesis. The increase in insulin following a meal stimulates both glycogen synthesis and fatty acid synthesis in liver. The fatty acids are converted to triglycerides and are released into the blood as VLDLs. In the well fed state, the liver derives most of its energy from the oxidation of excess amino acids. Between meals and during prolonged fasts, the liver releases glucose into the blood. The increase in glucagon during fasting promotes both glycogen degradation and gluconeogenesis. Lactate, glycerol, and amino acids provide carbon skeletons for glucose synthesis. f. Adipose tissue. After a meal, the elevated insulin stimulates glucose uptake by adipose tissue. Insulin also stimulates fatty acid release from VLDL and chylomicron triglyceride. Lipoprotein lipase, an enzyme found in the capillary bed of adipose tissue, is activated by insulin. The fatty acids that are released from lipoproteins are taken up by adipose tissue and re-esterified to triglyceride for storage. Insulin is also very effective in suppressing the release of fatty acids from adipose tissue. During fasting, the increase in glucagon and epinephrine activates hormone-sensitive lipase in fat cells, allowing fatty acids to be released into the circulation.

HORMONAL REGULATION OF METABOLISM The hormones that regulate fuel metabolism can be divided into two major groups: anabolic hormones that promote storage and catabolic hormones that promote mobilization. A. Hormones promoting fuel storage. In humans, insulin is the only hormone that promotes fuel storage. Insulin is produced by the ~-cells in the islets of Langerhans in the pancreas. The release of insulin from the ~-cells is stimulated by an increase in the concentration of blood glucose. B. Hormones promoting fuel mobilization. The action of insulin is antagonized by a number

of hormones that are collectively referred to as "catabolic hormones:' These hormones and their sites of synthesis are listed in Table 1-7-2. They all increase the rate at which fatty acids, glucose, and amino acids are released from their storage forms.

108

meClical

----~.--~~

-------~~--

Summary of Metabolism

Table 1-7-2. Fuel-mobilizing hormones. Hormone

Site of Synthesis

Glucagon Epinephrine Norepinephrine Cortisol Growth hormone *

Alpha cells of pancreas Adrenal medulla Adrenal medulla Adrenal cortex Anterior lobe of pituitary

*Growth hormone increases gluconeogenesis in liver and increases liver glycogen.

C. Mechanisms of hormone action. Hormones mediate the transfer of information from one

group of cells to another. There are two major types of mechanisms by which informational transfer occurs, depending on where the hormone exerts its effect. 1. Hormones acting at the plasma membrane of cells. The binding of hormones by cell surface receptors initiates a chain of events that results in the phosphorylation of specific intracellular proteins. There are several intermediate steps that occur between hormone binding and protein phosphorylation. Most of these hormones generate "second messengers" (cAMP, cGMP, Ca2+, diacylglycerol) inside the cell that stimulate specific protein kinases. An example is shown in Figure 1-7-5. In this case, the hormone stimulates adenylate cyclase to synthesize cAMP. The synthesis of cAMP requires interaction between three types of proteins: hormone receptors, G-proteins that bind GTP or GDP, and adenylate cyclase. The Gprotein acts as a communication link between the receptor and adenylate cyclase. When the receptor binds hormone, this information is transmitted to adenylate cyclase via the G-protein, and cAMP synthesis begins. (The G-protein must bind GTP in order to communicate with adenylate cyclase. The hydrolysis of GTP to GDP will terminate the communication.) The increased cAMP activates a protein kinase that then phosphorylates a few specific enzymes in the cell. The activity of the phosphorylated enzyme may be either increased or decreased. The enzyme activity can be restored to its original level by removing the phosphate group, a reaction catalyzed by a family of protein phosphatases. Extracellular

Hormone

Intracellular

Reor,or G-Protein

1

Adenylate Cyclase

ATP (

i

Inactive Protein Kinase

cAMP

Enzyme-OH Active Protein Kinase (

y

p. I

Protein Phosphatase

Enzyme-O-P03 (Altered activity)

Plasma Membrane

Figure 1-7-5. Mechanism of peptide hormones that activate adenylate cyclase.

meCtical

109

Biochemistry

2. Hormones that act in the nucleus. The other major site where hormones exert their effects is in the nucleus of cells, where the hormone-receptor complex binds to DNA and alters the rate of gene expression. Steroid hormones, thyroid hormones, and 1,25-dihydroxycalciferol (activated vitamin D) all bind to specific receptors and alter the rate of mRNA synthesis. Binding of the hormone-receptor complex to "enhancer" sequences in DNA results in increased mRNA synthesis, whereas binding to "silencer" sequences results in decreased mRNA synthesis. The mechanism by which steroid hormones stimulate the transcription of a particular gene is illustrated in Figure 1-7-6. The enhancer region in the DNA alone has no stimulatory effect on transcription. However, when the hormone-receptor complex binds to the enhancer region, transcription of a specific coding sequence is stimulated.

I"

••

DNA

-----1 Enhancer

Promoter

H

Coding Sequence

1-1---

(!) Hormone-Receptor Complex

DN"----1

E5'

Pmmote'

\

ICod;ng Sequence I

'fVVVV\J\

' , , ____ ®____ / / /

mRNArnthes;s Protein synthesis

Figure 1-7-6. Hormonal stimulation of gene expression.

D. Mechanisms of specific hormones that regulate fuel metabolism. The hormones primarily responsible for fuel mobilization are glucagon, catecholamines (epinephrine and norepinephrine), and cortisol. The only hormone that promotes fuel storage is insulin. A brief description of the mechanism for each of these hormones and how they coordinate the various pathways of carbohydrate, fat, and protein metabolism is discussed below.

In a Nutshell

1. Hormones that activate adenylate cyclase. Three of the four hormones that mobilize fuel II!'

Glucagon, epinephrine:~ and norepinephrine all increase cAMP.

110

mellical

(glucagon, epinephrine, and norepinephrine) bind to cell surface receptors and activate adenylate cyclase, resulting in the synthesis of cAMP and the subsequent effects previously described in Figure 1-7-5. a. Glucagon release is stimulated by low blood glucose and by elevated levels of epinephrine and several amino acids, particularly alanine and arginine. The primary target for glucagon is the liver, where it stimulates glycogenolysis and gluconeogenesis and inhibits glycolysis. It also activates lipolysis and the release of fatty acids from adipose tissue. The binding of glucagon to receptors in these tissues results in the phosphorylation of a number of enzymes by cAMP-dependent protein kinase. The enzymes that are phosphorylated in response to glucagon are listed in Table 1-7-3, together with the pathway they participate in and the effect that phosphorylation has on the enzyme.

Summary of Metabolism

Table 1-7-3. Effect of phosphorylation on key enzymes in carbohydrates and lipid metabolism. Pathway

Effect of Phosphorylation

Glycogen phosphorylase

Glycogenolysis

Activation

Enzyme Carbohydrate metabolism Glycogen synthase

Glycogenesis

Inhibition

Pyruvate kinase

Glycolysis

Inhibition

Pyruvate dehydrogenase

Links glycolysis with TCA cycle

Inhibition

Phosphofructokinase-2

Synthesis of F-2,6-Pz*

Inhibition

Fructose-2,6-bisphosphatase Degradation of F-2,6-P z

Activation

Lipid metabolism Hormone-sensitive lipase

Lipolysis

Activation

Acetyl-CoA carboxylase

Fatty acid synthesis

Inhibition

HMG-CoA synthase

Cholesterol synthesis

Inhibition

•

*Fructose-Z,6-bisphosphate is both an allosteric activator of glycolysis and an inhibitor of gluconeogenesis.

b. Epinephrine and norepinephrine are released from the adrenal medulla in response to hypoglycemia. These hormones have two major types of receptors, ex and ~. When the ~ receptors are occupied, adenylate cyclase is activated and cAMP levels are increased. Glycogenolysis in cardiac and skeletal muscle and lipolysis in adipose tissue are all increased by cAMP-dependent mechanisms. Binding of epinephrine to ex receptors on liver cells results in other second messengers (diacylglycerol and calcium) and stimulation of other protein kinases, ultimately resulting in the activation of glycogenolysis and gluconeogenesis. Epinephrine also increases the heart rate and the force of heart contraction, stimulates contraction of bronchial smooth muscle, and increases blood pressure. 2. Insulin lowers cAMP levels. Insulin antagonizes the effects of glucagon by mechanisms that are not entirely understood. For example, glucagon promotes phosphorylation of the intracellular proteins described previously, and insulin promotes dephosphorylation of these same proteins. In liver, insulin activates cAMP phosphodiesterase, the enzyme that degrades cAMP. As cAMP levels in cells drop, protein phosphatases begin to shift the equilibrium from the phospho- to the dephospho- forms of these enzymes.

cAMP+HzO

cAMP phosphodiesterase

- - - - - - - - - - - - - - - - : I.. ~

5'AMP

(±linsulin

3. Hormones that modulate gene expression. Steroid and thyroid hormones exert their effects by altering the rate of mRNA synthesis for specific proteins or sets of proteins. Glucagon and insulin can also alter the rate of mRNA synthesis for key regulatory enzymes in glycolysis and gluconeogenesis (discussed below).

KAPLAlf I meillea

111

Biochemistry

a. Cortisol functions mainly to increase de novo synthesis of glucose. Cortisol is synthesized and secreted by the adrenal cortex. Its synthesis is stimulated by ACTH, a hormone released from the anterior pituitary in times of stress. Cortisol has three major target tissues: liver, adipose tissue, and skeletal muscle. The enzymes whose synthesis is induced by cortisol are summarized in Table 1-7-4. Cortisol results in increased lipolysis and proteolysis in adipose tissue and muscle, respectively. The synthesis of glucose from amino acid precursors is increased in liver, as are the glycogen stores. Glucose use is decreased in both muscle and adipose tissue. b. Thyroid hormones (T3 and T4) also exert their action in the nucleus by altering the rate of gene expression. Some of the metabolic effects of thyroid hormones are increased oxygen consumption, lowering of blood cholesterol, hyperglycemia, and thermogenesis. Receptors for the thyroid hormones are found in virtually all cells of the body, and the interaction of hormone with receptor stimulates numerous pathways of protein, lipid, and carbohydrate metabolism.

In a Nutshell

c. Glucagon and insulin can also alter the rate of gene expression. The mechanism by which glucagon alters gene expression involves cAMP-dependent phosphorylation of nuclear proteins that interact with the DNA. The mechanism for insulin is unclear, although its effects are clearly antagonistic to the effects of glucagon. Glucagon increases mRNA synthesis for enzymes that are specific for gluconeogenesis (PEPCK, FBPase-I, and glucose-6-phosphatase) and represses the synthesis for key glycolytic enzymes (glucokinase, PFK-l, and pyruvate kinase). Insulin stimulates the synthesis of mRNA for the key glycolytic enzymes and represses mRNA synthesis for gluconeogenic enzymes.

Glucagon

· t

PEPCK

·t ·t

FBPase-1 Glucose-6-phosphatase

J- Glucokinase • J- PFK-l • J- Pyruvate ki nase

112

Table 1-7-4. Induction of enzyme synthesis by cortisol. Tissue and enzyme

Pathway

Consequence

Liver FBPase-l PEPCK

Gluconeogenesis Gluconeogenesis

Increased glucose release Increased glycogen stored Increased glucose release

Adipose tissue Hormone-sensitive

Lipolysis

Increased release of fatty acids (fuel for gluconeogenesis)

Skeletal muscle Proteases

Protein degradation

Increased amino acid release (substrate for gluconeogenesis)

meclical ----

- - - - -----------------------------

---

SECTION II

Molecular Biology

---

~~~------~

..

---.~~~--~~~~~-

Nucleic Acids The nucleic acids DNA and RNA are the informational macromolecules of the cell. The building blocks for nucleic acids are nucleotides, which are arranged in a specific linear sequence and are linked together by phosphodiester bonds between the 3'-OH of one sugar and the 5' phosphate ester of the adjacent sugar. All of the information that specifies the properties and functions of a cell is programmed into the nucleotide sequence of DNA. DNA normally exists as a double helix. The helix is stabilized by noncovalent interactions between the purine and pyrimidine bases in opposite strands of the DNA duplex. This chapter reviews the chemical structure and the nomenclature of the DNA and RNA building blocks, the types of interactions that stabilize the secondary structure of nucleic acids, the conformations of DNA, and the metabolism of the purine and pyrimidine bases. Disease states associated with purine and pyrimidine metabolism are also reviewed.

In a Nutshell

NUCLEIC ACID STRUCTURE Nucleic acids are polymers of nucleotides covalently linked to one another. The primary structure of nucleic acids refers to the sequence of the nucleotides in DNA or RNA. Higher orders of structure are stabilized by noncovalent interactions between purine and pyrimidine bases.

A. Chemical structure of nudeotides. The nucleotides are the building blocks of nucleic acids. As shown in Figure II-I-I, nucleotides consist of three components: a nitrogenous base, a pentose, and a phosphate group. The linkage of the nitrogenous base to the pentose generates a nucleoside. The addition of a phosphate group to the pentose generates a nucleotide. The genetic information is carried in the sequence of the nitrogenous bases, and the sugar and phosphate groups form the backbone of the polynucleotide chain.

Nucleotide • Nitrogenous base • Pentose • Phosphate(s) Nucleoside • Nitrogenous base • Pentose

1. Nitrogenous base. Nucleic acids contain two types of nitrogenous bases: purines and

pyrimidines (Figure II -1-1). a. Purines contain two rings, both with nitrogen atoms. There are two types of purines found in nucleic acids: adenine (A) and guanine (G). b. Pyrimidines contain a single nitrogen-containing ring. The three types of pyrimidines found in nucleic acids are cytosine (C), thymine (T), and uracil (U). Thymine is found only in DNA, and uracil is found only in RNA.

Note Important nucleotide bonds often refer to the pentose carbons: • 5'-(

phosphate

2. Pentose sugar. RNA contains ribose, and DNA contains deoxyribose. Deoxyribose differs from ribose in that there is a hydrogen atom instead of a hydroxyl group attached to the carbon-2. The carbon atoms of the pentoses in the nucleic acids are numbered with primes. For example, the pentose in DNA is 2'-deoxyribose.

• 3'-( hydroxyl

3. Phosphate groups form the linkages between nucleotides in nucleic acids. The phosphate group is esterified to the 5' carbon of the pentose. In nucleic acids, phosphodiester bonds

• 1'-( glycosidic bond to base

OH on RNA and 2'-( H on DNA

• 2'-(

I KAPLAlf medlea

115

Molecular Biology

are formed between adjacent nucleotides. There are three types of phosphodiester linkages found in nucleic acids. a. 3'-5' phospho diester linkage is the most common linkage and forms the backbone of both DNA and RNA molecules. b. 2'-5' phospho diester linkage is involved in RNA splicing.

Pyrimidines

Purines

0

o

HNC)rH HN~CH' N&H JZ ~1 :1 ~16 N N

o

H

I

H Adenine (6-aminopurine)

Guanine (2-amino-6-oxypurine)

Nucleoside

N

H

I

H Uracil (2,4-dioxypyrimidine)

Cytosine (2-oxy-4aminopyrimidine)

:x

Also,

61

o

H

I

H Thymine (5,6-methyluracil)

0

cytidine H and ...... N deoxycytidine I

I

O-?-.......N

Also, adenosine and deoxyadenosine

0

H

H

5'CH 2 0H

o 4,V"~1' H

H

3'

H

2'

H

bH 6H e.g., URI DINE

e.g., GUANOSINE

e.g., DEOXYTHYMIDINE

Nucleotide

H

II

e.g., guanylic acid (GMP)

HO-P-OH

I o

4'

(Also, adenylic acid AMP (5'-AMP) R Adenosine-5' -phosphate Adenosine monophosphate l' Adenosine ribonucleotide H

5'~IH2 0 4'

H

5'CH 2 0H

H

3'

OH

2'

H

OH

R

~

H

H

3'

o

H

2'

H

H

I

HO-P-OH

II

o

Figure 11-1-1. Nucleic acid structure.

116

meclical

l'

Depending on R, uridylic acid Thymidylic acid (TMP) Deoxycytidylic acid 3'-D CMP Deoxycytidine-3'-phosphate cytosine-3'deoxyribonucleotide may be formed

Nucleic Acids

c. 5'-5' phosphodiester linkage is found in the cap structure at the 5' end of eukaryotic

messenger RNA.

B. DNA and RNA are polymers of nucleotides. 1. Phospho diester linkage. Nucleotides are linked together by phosphodiester bonds formed between the 3' hydroxyl group on one sugar and the 5' phosphate group of the adjacent nucleotide (Figure II -1-2). The specificity of the nucleotide at a particular posi-

tion is determined by the complementary base in the template strand.

2. Polarity. Polynudeotides have polarity. At one end of the polymer, the ribose has a free 3' hydroxyl group, and on the other end the ribose has a 5' phosphate group, .. OH 3'. i.e., 5' p

5'pp 5'pp

G>

~

:r

(Q

3'

Sugar

rot 3

"0

3'OH

a CD

o I

0

II

II

II

0

0

0

I

0

!e-

+

iil :l

a.

I

0- P - 0 - P - 0 - P -05- - CH

3'

rot

PPI

3

"C

t•

~

r

!e-

iil

:l

a.

Phosphodiester bond

2

Sugar

3' OH

5'

5'

Figure 11-1-2. Phosphodiester linkage in DNA and RNA.

C. Major differences in DNA and RNA. DNA and RNA differ in both structure and function.

In a Nutshell

1. Pentose. DNA contains 2' deoxyribose, whereas RNA contains ribose.

2. Nitrogenous bases. DNA contains A, T, G, and C, whereas RNA contains A, U, G, and C. 3. Number of strands. DNA is double stranded, whereas RNA is single stranded. 4. Function. DNA is the repository of genetic information, whereas RNA represents "working copies" of the DNA that are used in the transfer of genetic information from DNA sequences to protein sequences.

DNA • H at pentose 2'-( ("deoxy") • Bases are A, T, G, and ( • Double stranded

RNA • OH at pentose 2'-( ("ribo") • Bases are A, U, G, and C • Single stranded

I KAPLAlr me d lea 117

Molecular Biology

Note

D. Nomenclature and conventions

Glycosidic bond connects pentose l'-Cto base; phosphodiester bond connects one pentose to another pentose.

1. Nomenclature of nitrogenous bases, nucleosides, and nucleotides: The terminology that

distinguishes a free nitrogenous base from a base attached to a pentose (nucleoside), and a base attached to a ribose-S'-phosphate (nucleotide) is summarized in Table II-I-I. 2. Linkage between nitrogenous base and sugar. In purine nucleosides and nucleotides, the nitrogen-9 of the ring system is linked to the l' -carbon of the sugar. In pyrimidine nucleosides and nucleotides, the nitrogen -1 of the ring is linked to the I' -carbon of the sugar (see Figure II-I-I). Table II-I-I. Nomenclature of bases, nucleosides, and nucleotides. Base

(Deoxy) Ribonucleoside*

(Deoxy) Ribonucleotide*

Adenine (A) Guanine (G) Cytosine (C) Thymine (T)t Uracil (U)t

(Deoxy) (Deoxy) (Deoxy) (Deoxy) Uridine

(Deoxy) adenylate (Deoxy) guanylate (Deoxy) cytidylate (Deoxy) thymidylate Uridylate

adenosine guanosine cytidine thymidine

*Nomenclature for deoxyribonucleosides and deoxyribonucleotides is indicated in parentheses. tThymine is found only in DNA, whereas uracil is found only in RNA.

3. Conventions for writing nucleic acid sequences. The nucleotide sequence is represented by the single letter codes corresponding to the bases (A, C, G, T, U). Unless otherwise specified, the orientation is written in the direction of synthesis (5' to 3'). ACCTG If polarity (directionality) is being emphasized, the numbers may be added to the beginning and the end of the letter sequence. 5' ACCTG 3' If the presence of the phosphate groups is being emphasized, the structure may be written as follows. In the first structure below, there is a free hydroxyl group on the 3' end. In the second structure, there is a phosphate at the 3' end.

Note 5' pApCpCpTpG-OH 3' Nuc1eases cut phosphodiester bonds. Exonuc1eases remove terminal nuc1eotides only, whereas endonuc1eases cut between internal nuc1eotides.

5' ApCpCpTpGp 3' The presence of a 5' triphosphate and a 3' hydroxyl group is indicated by either of the following structures. If a phosphate group is not shown at the 3' end, as in the first sequence, it is assumed that there is a free hydroxyl group. In the second structure, the hydroxyl group is drawn in. 5' pppApCpCpTpG 3' 5' pppApCpCpTpG-OH 3'

118

meClical

Nucleic Acids

E. Secondary structure of nucleic acids. The secondary structure of DNA refers to its doublestranded duplex structure. Although RNA is almost always a single-stranded molecule, it can bend back on itself to form regions of double-stranded secondary structure.

In a Nutshell 10 structure = nucleotide sequence

1. Base pairing. The double-stranded structure is stabilized by base pairing between purines

20 structure = double helix, which can form between any antiparaliel and complementary sequences:

and pyrimidines. Complementary bases that pair with one another are G and C (in both DNA and RNA), A and T (in DNA), and A and U (in RNA). 2. Antiparallel strands. As shown in Figure 11-1-3, the two strands of DNA are always oriented in an antiparallel fashion, where one strand runs in the 3' to 5' direction and the other runs in the 5' to 3' direction.

5'

• Double-stranded DNA • Inverted complementary sequences within an RNA

3'

• RNA and DNA

3'

5'

Figure 11-1-3. Double-stranded DNA.

3. Stabilizing forces. The two strands are held together by two types of forces: hydrogen bonding and base stacking. a. Hydrogen bonding between base pairs. A-T and A-U base pairs are stabilized by two hydrogen bonds, and the G-C pair is stabilized by three hydrogen bonds (Figure 11-1-4). Because of the extra hydrogen bond, G-C base pairs provide more stability to the DNA duplex structure than do A-Tor A-U pairs.

liN

b

Cytosine

H

/N

?

c~

HO--r=O

H

fN

0 -
~'i--r~ OH

H

-...::::::

O==j--OH 0

H

£ . N~N~HHOo~

Guanine

I

I

H

c(

I

•• H

I

H N/ "

... O

A N ....... H· N I H

I

HH ~ OH

(3 H-BONDS)

H

b

Thymine

CH,

.-t . . o=iT "I

I

?

H N H-<

C~O¥"N

I

.......N y N " Q C H ,

~N'•••• H

A

N.b (2

~'i--r~ OH

I

Adeninex: ........ N

HO-j=O

o

,.........H·

OH

H

•••• 0_.....

H 0

~-BONDS)

H

H OH

H H

H

Figure 11-1-4. Hydrogen bonding between base pairs.

b. Base stacking interactions. The ring structures of the purines and pyrimidines are stacked over one another in the interior of the double helical structure. Hydrophobic interactions between adjacent purine and pyrimidine rings add stability to the double helix. The base stacking interactions are interrupted by a number of intercalating KAPLA~.

meulca

I

119

Molecular Biology

agents, such as actinomycin D, which have been shown to "slide" in between neighboring base pairs in DNA. These agents prevent DNA from serving as an effective template, thus inhibiting replication (DNA -7 DNA) and transcription (DNA -7 mRNA).

Note Localized "melting" is needed to initiate DNA replication and RNA transcription. DNA sequences needed to initiate DNA replication or RNA transcription are usually A-T rich.

4. Destabilization of the double helix. Double-helical DNA can be denatured by conditions that disrupt hydrogen bonding and hydrophobic interactions, resulting in the "melting" of the double helix into two single strands that separate from one another. No covalent bonds are broken in this process. Some conditions that promote denaturation are: a. Changes in pH. Addition of sodium hydroxide leads to the ionization of protons associated with the nitrogen atoms in the purine and pyrimidine rings. Ionization disrupts the hydrogen bonds that hold complementary bases together. b. High temperatures. Temperatures above 80-90°C result in the "melting" of the double helix. c. Chemicals. Both formamide and urea interfere with hydrogen bonding between bases.

TOPOLOGY OF NUCLEIC ACIDS Double-stranded DNA may be either linear or circular. Most DNA forms a right-handed, antiparallel helix.

In a Nutshell • B DNA is the common, right-handed, antiparallel helical DNA with approximately 10.5 base pairs per helical turn. • Z DNA is a left-handed helical DNA that occurs occasionally within a B DNA chromosome.

A. Conformations of linear DNA. Linear DNA is the form of double-stranded DNA found in most eukaryotic chromosomes. Three conformations have been identified. 1. B DNA is the most common form of DNA. It is the classic Watson-Crick right-handed antiparallel helical structure of DNA in which the bases are stacked upon one another and are perpendicular to the axis of the helix. The helix contains two grooves-a major and a minor groove-that provide sites where DNA-protein interactions occur. 2. Z DNA is a rare form of DNA that is found in G-C rich sequences and in DNA sequences that have long tracts of alternating purines and pyrimidines. In contrast to B DNA, this form of DNA forms a left-handed antiparallel helix. The biologic function is unknown. 3. A DNA is a form of DNA in which the bases are not perfectly perpendicular to the helical axis but are tilted about 20 degrees away from the axis. This type of DNA is the anhydrous form of B DNA. Like B DNA, it is a right-handed antiparallel helix. This type of structure is found in RNA-DNA hybrids.

B. Circular DNA has no ends. This is an advantage because circular DNA is resistant to exonucleases. Mitochondrial DNA and the DNA of most prokaryotes and some viruses are closed circular structures. Circular DNA may exist in a relaxed form (no supercoils), or it may be converted to more-compact structures by supercoiling.

In a Nutshell Negative supercoils stabilize partially unwound DNA, promoting replication and transcription. Both circular prokaryotic chromosomes and linear eUkaryotic chromosomes contain negative supercoils.

1. Supercoiling. One of the important properties of closed circular DNA is that it can form supercoils, which are important for DNA packaging. The supercoils may be either positive or negative. a. Negative supercoiling twists the DNA in the opposite direction from the clockwise turns of the right-handed double helix. DNA is usually negatively supercoiled, the form required for most biologic reactions. b. Positive supercoiling adds torsional pressure and allows the DNA to be wound more tightly. In positive supercoils, the DNA is twisted in the same direction as the intrinsic winding of the double helix. 2. Topoisomerases are enzymes that relieve the tension caused by supercoils in replicating DNA. These enzymes make transient breaks in the DNA by cutting and resealing phosphodiester bonds in the DNA strands.

120

meClical

Nucleic Acids

PURINE AND PYRIMIDINE METABOLISM A. Synthesis of nudeotides. Nucleotides are synthesized by either salvage pathways or by the

pathway for de novo synthesis. In salvage pathways, the nucleosides and the nitrogenous bases that are released by the degradation of nucleic acids are salvaged and reutilized. In the pathway for de novo synthesis, the starting materials are more elemental precursors, such as amino acids, ammonia, bicarbonate, and ribose-S' -phosphate. Tetrahydrofolate is an important coenzyme in purine and pyrimidine synthesis, acting as a donor of Cl fragments in the de novo assembly of the nitrogenous ring systems. In the adult, the salvage pathway is the major route for synthesis, and the de novo pathway is used primarily by rapidly dividing cells. Deoxyribonucleotides are synthesized by the reduction of ribonucleoside diphosphates. This reaction is catalyzed by ribonucleotide reductase and requires thioredoxin and NADPH as sources of reducing power. 1. Synthesis of purines. As shown in Figure II-1-S, the purine ring system is synthesized

from amino acids, bicarbonate, and Cl fragments carried bytetrahydrofolate. The purine ring is attached to ribose throughout the assembly. A parent purine nucleotide, IMP, gives rise to both AMP and GMP. The synthesis of purines is subject to feedback inhibition by GMP and AMP.

CO 2- 6 ---------- .. - - Glycine ~C"i-.5 7 N ,,' Asparate - 1 N , ~ I : II Ca - - Folate Folate-2 C~ ~C~ / ~N"'i 4: N / 3 : __ 9~

C-":'

f

Note Deoxynucleotide synthesis inhibitors are used as antineoplastic agents. • 5-Fluorouracil inhibits thymidylate synthetase. • Hydroxyurea inhibits ribonucleotide reductase. These drugs are discussed in the Pharmacology section of General Principles Book 2 (Volume II).

1

Glutamine

Glutamine

Figure 11-1-5. Origin of atoms in the purine ring system.

2. Synthesis of pyrimidines. In contrast to purines, the pyrimidine ring is completely assembled before it is attached to ribose-S' phosphate. As shown in Figure II -1-6, the precursors for the pyrimidine ring are carbamoyl phosphate and aspartate. The parent nucleotide in the pyrimidine pathway is orotidylate (OMP). Both UTP and CTP are derived from OMP. Thymidine nucleotides (required only for DNA synthesis) are synthesized by methylation of dUMP in a reaction that uses Cl-tetrahydrofolate as a Cl donor. Pyrimidine biosynthesis is inhibited by CTP and UMP.

In a Nutshell Purines are assembled while attached to ribose-5' phosphate; the pyrimidine ring is completed before it is attached to ribose-5' phosphate.

:- -4" --- ---:

,yC,C5: I: II: -- Aspartate O=C' C, 3

From carbamoyl phosphate [

N,

I

2 ~N/6 :, , .JI ',_________ 1

Figure 11-1-6. Origin of atoms in the pyrimidine ring.

KAPLA~.

I

meulC8

121

Molecular Biology

B. Degradation of nudeotides. The degradation of nucleotides starts with the removal of the

phosphate group and the pentose, yielding free purine and pyrimidine bases. The amino groups attached to the ring system are removed as ammonia by the action of adenosine deaminase and guanine deaminase. 1. Degradation of purines. The purine ring system cannot be opened up and degraded.

Therefore, the strategy is to make the ring as soluble as possible so that it can be excreted. The end product of purine degradation is uric acid. 2. Degradation of pyrimidines. In contrast to purines, the pyrimidine ring can be opened and partially degraded. Thymidine is degraded to butyrate, whereas uracil and cytosine are degraded to ~-alanine. C. Disease states are associated with defects in purine and pyrimidine metabolism. There are a

number of clinical conditions that arise from abnormalities in the degradation of purine nucleotides. 1. Gout results from the excessive production of uric acid. Elevated levels of uric acid can

occur secondary to other diseases that are characterized by high turnover of nucleic acids (i.e., polycythemia and leukemia). In gout, the joints become inflamed because of the precipitation of uric acid crystals. Kidney disease is also seen because of accumulation of uric acid in the tubules. The disease affects mostly males and is frequently treated with allo-purinol, an inhibitor of xanthine oxidase. Xanthine oxidase catalyzes the sequential oxidation of hypoxanthine to xanthine to uric acid. 2. Lesch-Nyhan syndrome arises from a deficiency in the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT). This enzyme is a part of the salvage pathway for purines. A deficiency leads to the inability to recycle purines and an overproduction of uric acid. Individuals with Lesch-Nyhan syndrome are mentally retarded, have hyperuricemia, suffer from gout, and have compulsive self-destructive behaviors. The basis for the involvement of the central nervous system is unknown. 3. Severe combined immunodeficiency disease (SCID). A deficiency in adenosine deaminase, an enzyme in the salvage pathway for purines, results in defective development of T and B lymphocytes. Individuals with a deficiency in this enzyme must live in a sterile environment to survive. Large amounts of dATP accumulate in red blood cells. This compound allosterically inhibits ribonucleotide reductase, an enzyme essential for DNA synthesis.

122

meClical

The Nucleus, the Cell Cycle, and Meiosis Most of the cellular events originate in the nucleus. In eukaryotic cells, the nuclear compartment is defined by the nuclear envelope, containing pores that allow macromolecules to pass in and out of the nucleus. The reproduction of cells involves duplication of cellular contents followed by cell division, or mitosis. The process of cell division occupies a small part of the total cell cycle. Most of the cell cycle is spent in interphase, a period of growth and preparation for cell division. Meiosis is a special type of cell division that produces sperm and ova, each of which contains half of the original complement of chromosomes. Each of the gametes produced by meiosis is genetically distinct from one another and from the parent cell. This chapter reviews the structure and function of the cell nucleus, the events occurring in interphase and mitosis, and the reductive cell division that occurs during meiosis.

NUCLEUS The nucleus of the cell serves as a reservoir of genetic information. A. Function. Three cellular functions are carried out in the nucleus. 1. DNA synthesis and repair. The process of DNA replication is both rapid and accurate.

The nucleus contains all of the enzymes and proteins required for DNA synthesis. Additionally, there are several proofreading and repair mechanisms that correct mistakes that are made spontaneously or by environmental insult. 2. RNA synthesis. Transcription of DNA into RNA occurs in the nucleus of eukaryotic cells by three distinct RNA polymerases, each synthesizing a different class of RNA. 3. RNA processing. In eukaryotic cells, RNA is synthesized as a precursor that is converted to mature RNA in the nucleus by a number of steps collectively known as "processing". B. Structure. The nucleus is the most conspicuous organelle in eukaryotic cells. It contains

almost all of the cellular DNA, and microscopy reveals the following structural features. 1. Nuclear envelope. The nucleus is separated from the cytoplasm by a double membrane (inner and outer). The outer membrane is continuous with the rough endoplasmic reticulum and, therefore, is a part of the endomembranous system. The inner and outer membranes form two concentric rings and are separated by a perinuclear compartment. 2. Nuclear lamin. The inner surface of the nuclear envelope is lined by an electron-dense layer containing three major proteins. This network of proteins plays an important role in the structural organization of the nucleus by providing connections between the inner nuclear membrane and the chromatin.

In a Nutshell Nuclear lamin proteins are important in nuclear disassembly and reassembly during mitosis and meiosis.

KAPLAN'dme leaI

123

Molecular Biology

3. Nuclear pores. The inner and outer nuclear membranes are fused together at the pores, providing channels of communication between the nucleus and the cytosol. Water, ions, amino acids, and small proteins can freely pass through the pores. The pores are associated with proteins that are required for transport of large macromolecular complexes into and out of the nucleus. The number of nuclear pores is usually fIxed in a particular cell type but may vary at times in relation to the transcriptional activity in the cell. 4. Chromatin. The chromosomes and chromatin are composed of DNA that is associated with histones, a family of positively charged proteins. 5. Nucleolus. The nucleolus is a very dense region containing large amounts of RNA and functioning as the site of ribosome assembly. The nucleolus is associated with the nuclear organizer region (NOR) on the chromosome. The NOR of the DNA contains many copies of the genes for ribosomal RNA. Electron microscopy reveals three distinct zones in the nucleolus. The periphery of the nucleolus has a granular zone that contains ribosomal precursor particles in various stages of assembly. The central region has a fIbrillar zone containing ribonuclear protein fIbrils. The DNA in the NOR region of the chromosome is a pale-staining region of the nucleolus.

CELL CYCLE The cell cycle begins when a cell comes into existence and ends when the cell divides into two daughter cells. The cell cycle can be divided into two major components: interphase and cell division (Figure II -2-1).

Go

In a Nutshell • Cells perform their differentiated functions while in G1 (or Go). • Mitochondria and centrioles, which contain DNA, divide during S phase. • During G2, each chromosome consists of two sister chromatids, connected at the centromere.

124

meclical

Ctk = Cytokinesis M = Mitosis Interphase

Figure 11-2-1. The cell cycle.

A. Interphase is the period of the cell cycle that precedes mitosis. It can be subdivided into three parts (G b S, and G2) based on discrete events occurring during interphase. l. Gl phase (Gap phase) is a period of cellular growth that precedes DNA synthesis. In most

cells, Gl lasts approximately 12 hours. During this time, lipids, carbohydrates, and proteins are synthesized. In some cells, Gl may be very long. For example, in muscle and nerve cells, Gl is so long that it seems the cells may have completely stopped cycling. Such resting cells are said to be in a special Gl state called Go.

---------

The Nucleus, the Cell Cycle, and Meiosis

2. S phase (synthesis phase) usually lasts for 6-8 hours and includes the entire period of DNA synthesis and chromosomal replication. The rate of RNA synthesis increases, and the cell prepares for mitosis. 3. G2 phase lasts between 3 and 4 hours and resembles the Gl phase in terms of cellular activity, except that the cell is now tetraploid (diploid X 2), whereas in the Gl phase it was diploid. B. Mitosis is the nuclear division of the cell, resulting in the reduction of the chromosome

complement from the tetraploid to the diploid number found in each daughter nucleus. Cytokinesis is the division of the cytoplasm, resulting in the enclosure of each daughter nucleus within a separate cell. As shown in Figure 11-2-2, mitosis is divided into six phases.

NUcl~::::us

Mitotic Spindle

Preprophase

Prophase

Metaphase

Telophase (Cytokinesis)

Late Anaphase

Early Anaphase

Figure 11-2-2. Phases of mitosis.

1. Preprophase. The chromosomes condense into recognizable thread-like structures in the

nucleus. A pair of centrioles (barrel-like structures) are visible in the cytoplasm. 2. Prophase. During prophase, the two copies of each chromosome are separated lengthwise into two single chromosomes, called chromatids. The main structural component of the mitotic apparatus is the mitotic spindle, which begins to form near the end of prophase. When the centrioles begin to separate, bundles of microtubules assemble between them, creating a spindle. At the end of prophase, the nuclear envelope begins to rupture. 3. Metaphase. The nuclear envelope and the nucleolus disappear during metaphase. The spindle moves into the region formerly occupied by the nucleus. The chromosomes move toward the midpoint of the spindle, and each chromosome attaches to bundles of spindle microtubules. 4. Early anaphase. The chromatids begin to split longitudinally and start migrating toward the poles of the cell. Once the chromatids split, they are referred to as chromosomes. 5. Late anaphase. The chromosomes aggregate at the poles, and a cleavage furrow begins to form, marking the beginning of cytokinesis.

In a Nutshell Until anaphase, each chromosome contains two sister chromatids. After anaphase, each chromatid is a separate chromosome. KAPLAlf I medlea

125

Molecular Biology

Note • Cells entering meiosis I are called primary gametocytes (spermatocytes or oocytes); they have the same DNA content as a cell entering mitosis. • Cells entering meiosis II are called secondary gametocytes; they contain 23 chromosomes, each consisting of two sister chromatids.

6. Telophase. A nucleolus forms, and nuclear envelopes appear around each group of daughter chromosomes. The condensed chromatin expands again and begins to reappear. The cytoplasm divides by deepening the cleavage furrow until it forms two daughter cells, thus completing cytokinesis.

MEIOSIS Meiosis is the type of cell division that occurs in sperm and ova and reduces the chromosome number by half. It occurs before maturation of the gametes. Genetic recombination also occurs in meiosis and involves the exchange of segments of chromosomes, resulting in a mixing of allelic linkages into new combinations. This is one of the ways in which deleterious mutations are introduced into the chromosome. As shown in Table II-2-1, meiosis involves two complete cell divisions, each resembling the mitotic division previously described. There is no DNA replication during either of the two meiotic divisions. DNA replication occurs before the first meiotic division. Table 11-2-1. Summary of the stages in meiosis. Meiotic Division I

Meiotic Division II

Prophase I Metaphase I Anaphase I Telophase I

Prophase II Metaphase II Anaphase II Telophase II

A. First meiotic division. The six stages of the first meiotic division are illustrated in Figure II-2-3 (steps A-F). 1. Meiotic prophase occurs in three stages: A, B, and C. a. Stage A. The chromosomes condense into visible threads, and pairing of homologous chromosomes occurs. The pairing is precise, except for the X-Y chromosome combination in males. The centro meres of homologous chromosomes do not pair. b. Stage B. Homologous chromosome pairing is complete, and four chromatids appear in each pair; this is a tetrad.

Note Recombination occurs between the chromatids within a tetrad; it changes allelic linkages but not gene sequence.

c. Stage C. This is the recombination, or cross-over, stage where the interchange of chromatid segments between two paired homologous chromosomes can occur. The point of exchange has an X-like appearance and is called a chiasma. At the chiasma, the chromosome pairs are held together, and at this time large segments of genes are exchanged between homologous chromosomes. 2. Meiotic metaphase I (stage D). Paired chromosomes line up on the mitotic spindle. The two chromosomes of each homologous pair form connections with microtubules leading to opposite ends of the cell.

In a Nutshell During meiosis II, sister chromatids of the 23 chromosomes separate. The final cells resulting from meiosis II are spermatids and ova (plus polar body).

126

meClical

3. Meiotic anaphase I (stage E). Both chromatids migrate toward the same end of the cell. 4. Meiotic telophase I (stage F). Each daughter cell gets one member of each chromosome pair, for a total of 23 double-stranded chromosomes.

B. Second meiotic division. This is shown in Figure II-2-3, step G. The steps are similar to those in a mitotic division, except that no DNA synthesis occurs before this division. The 23 chromosomes divide at the centromere, and each of the newly formed daughter germ cells receives 23 chromatids. Therefore, each germ cell now has a haploid number of chromosomes and half the amount of DNA of a diploid somatic cell.

The Nucleus, the Cell Cycle, and Meiosis

A

B

c

o

First meiotic division

G

Second meiotic division

Figure 11-2-3. First and second meiotic divisions.

meClical

127

.--_._---

DNA Replication and Repair

Genetic information is transmitted from parent to progeny by replication of parental DNA. The process of replication requires a number of proteins. Some participate as enzymes in the polymerization of nucleotide; others are responsible for unwinding the double-stranded DNA helix, holding it apart at the point of replication and preventing it from tangling. Maintaining the stability of the genome depends on a number of repair systems that correct occasional mistakes made during replication or that occur after replication due to environmental insult. This chapter reviews the basic concepts underlying DNA replication, the proteins and enzymes involved in replication, and some of the mechanisms that have evolved for repairing DNA. Inhibitors of DNA replication used as chemotherapeutic agents are reviewed, and some clinical abnormalities associated with DNA repair are also discussed.

DNA REPLICATION Most of the detailed information about DNA replication has been obtained from in vitro studies with Escherichia coli DNA. Although DNA replication in eukaryotic systems is more complex, the same basic concepts and mechanisms are involved. A. General concepts. The purpose of DNA replication is to create two daughter DNA molecules that are identical to the parental DNA. This is achieved by semi-conservative replication (Figure II -3-1). 1. Semi-conservative replication uses each of the parental strands of DNA as a template for directing the synthesis of one new daughter DNA molecule. 2. Base-pairing is the process by which each nucleotide in the parental strand is recognized and the complementary base is incorporated in the newly synthesized strand. As shown in Figure II-3-1, adenine (A) always base pairs with thymine (T), and guanine (G) always base pairs with cytosine (C).

Daughter DNAs

5'-GTTACG-3' 5'-GTTACG-3' 3'-CAATGC-5' - - -... 3'-CAATGC-5

L

+

I strands~

5'-GTTACG-3' 3'-CAATGC-5'

NeW'y synthesized

Figure 11-3-1. Semiconservative replication.

meClical

129

Molecular Biology

B. Enzymes involved in DNA replication. The properties of the E. coli enzymes required for

DNA replication are described below. 1. DNA polymerase I (Kornberg enzyme). The polymerization of nucleotides is catalyzed by DNA polymerase I, which also has exonuclease activities that function in proofreading and repair mechanisms.

In a Nutshell All DNA polymerases (prokaryotic, eukaryotic, and viral): • Must synthesize 5' to 3' • Must use an antiparallel template • Require a nucleotide (or short nucleic acid sequence) with a free 3'_ OH as a primer • Are associated with proofreading enzymes

In a Nutshell In both prokaryotes and eukaryotes, each replication fork requires: • Primase • DNA polymerases • Helicase • SSBs

• Ligase

a. Requirements for polymerization. DNA polymerase I requires all four deoxyribonucleotides (dATP, dTTP, dCTP, and dGTP) and Mg2+ for activity. Additional requirements are a DNA template (either single- or double-stranded DNA) and a primer with a free 3'-OH group that can react with the first nucleotide. The primer is subsequently erased, and the gaps are filled in by the polymerase. Chain elongation proceeds in the direction of 5' to 3'. b. Proofreading function (3' to 5' exonuclease activity). Occasionally polymerases add a nucleotide to the 3'-OH end that cannot hydrogen bond to the corresponding base in the template strand. Correct hydrogen bonding is required for polymerization to continue. Thus, the polymerase stops and removes the unpaired base from the 3' end so that polymerization can begin again. c. Repair function (5' to 3' exonuclease activity). The main function of the 5' to 3' exonuclease activity is to erase the nucleotides in the primer. 2. DNA polymerase II and III. There are two additional DNA polymerases in E. coli. The properties of DNA polymerase III are similar to those of DNA polymerase I. DNA polymerase III is a part of a multiprotein complex and is the major replicating enzyme in E. coli. The function of DNA polymerase II is unknown. 3. DNA ligase. This enzyme catalyzes the formation of a phospho diester bond between the 3' -OH of one fragment of DNA and the 5' -monophosphate group of an adjacent DNA fragment. These fragments are synthesized as a discontinuous strand in the DNA duplex. The formation of the phospho diester bond is an endergonic reaction and requires the input of energy that is supplied by NAD+ in bacteria and by ATP in animal cells and viruses. 4. Primase. The primer molecule that is required by DNA polymerase is a short strand of RNA (4-10 bases) that is complementary to the template strands of DNA. The primer is synthesized by a specific RNA polymerase known as primase. The growing end of the RNA primer is a free 3' -OH group. The primase does not require a primer for initiation of polynucleotide synthesis. 5. Helicases. Unwinding of the two strands in the DNA helix must occur before DNA replication can begin. This process requires energy (ATP) and enzymes known as helicases that force the two parental strands of DNA to separate at the replication fork. 6. Single-strand DNA binding (SSB) proteins. The single strands of DNA resulting from the action of helicase are prevented from reannealing by binding to SSB proteins. These proteins also protect the single-stranded DNA from cleavage by nucleases. 7. DNA topoisomerases. These enzymes produce a number of "swivel" points in the DNA molecule that relieve the strain induced by the replication fork. These enzymes cut and reseal the DNA. There are two major classes of topoisomerases: class I and II. a. Class I topoisomerases relax superhelical DNA in front of the replication fork by creating a transient nick in one of the strands. Class I topoisomerases do not require ATP. b. Class II topoisomerases introduce negative supercoils in the DNA molecule. This process requires ATP and involves transient breaking and resealing of both strands of DNA.

130

iiieClical --------~.---------

DNA Replication and Repair

c. Gyrase is the class II topoisomerase used by E. coli to promote DNA helix unwinding during replication. C. Events occurring at the replication fork. As the two strands of parental DNA unwind, a

fork-type structure is formed that is referred to as the "replication fork" (Figure II-3-2). As DNA synthesis proceeds along the DNA duplex, the replication fork continues to move. The synthesis of one of the daughter strands of DNA occurs continuously (the leading strand) whereas the other strand (the lagging strand) is made in short fragments (Okazaki fragments) that are then joined together.

5'

3'

Helicase

In a Nutshell 5'

Figure 11-3-2. Events at the replication fork.

l. Unwinding of parental DNA. At the site of replication, the two parental strands do not

completely separate but are unwound in a localized region known as the replication fork. The replication fork can move in either direction from the origin of replication (ori). Unwinding of the helix at the replication fork is accomplished by helicase, and the separation of the single strands is maintained by the binding of several molecules of singlestrand binding proteins. 2. Synthesis of leading strand. At the replication fork, one strand is synthesized continuously in the 5' to 3' direction as the parental DNA duplex molecule unwinds. Thus, the leading strand is complementary to the parental strand that runs in the 3' to 5' direction. 3. Synthesis of the lagging strand. The lagging strand is also synthesized in the 5' to 3' direction, but the synthesis occurs discontinuously, producing small fragments (approximately 1,000-2,000 bases) known as Okazaki fragments. These fragments are later joined by DNA ligase to form a continuous lagging strand. 4. RNA primers. There is no known DNA polymerase that can initiate the polymerization of a deoxyribonucleotide chain. Thus, the synthesis of both the leading strand and each of the Okazaki fragments in the lagging strand starts with a short RNA primer (approximately 10 bases). The primer is complementary and antiparallel to the 5' to 3' parental strand. The RNA primers are synthesized by a protein complex containing both singlestrand binding proteins and primase. This complex binds near the replication fork and moves along the lagging strand as it is opened up by helicase. The 3' OR of the last

Both prokaryotic and eukaryotic DNA synthesis is bidirectional: • DNA replication begins at an "ori" sequence in the DNA. • Two replication forks form at each ori site and move in opposite directions.

• E coli DNA contains a single ori site, whereas eukaryotic chromosomes each contain many ori sites.

In a Nutshell In both prokaryotes and eukaryotes, DNA replication is semidiscontinuous: • The leading strand is made continuously. • The lagging strand is made in discontinuous Okazaki fragments and then joined.

meClical

131

Molecular Biology

ribonucleotide in the primer serves as the site at which the first deoxyribonucleotide is added by DNA polymerase. Elongation of the DNA fragment occurs until it approaches another RNA primer. 5. Conversion of Okazaki fragments to a continuous strand. The precursor fragments are eventually joined to form a continuous strand that contains no RNA. This is accomplished by two sequential reactions. When DNA polymerase III approaches another RNA primer, it dissociates and DNA polymerase I enters. DNA polymerase I removes (erases) the ribonucleotides one at a time from the 5' end (5' to 3' exonuclease activity) and adds complementary deoxyribonucleotides to fill in the gaps. Finally, DNA ligase forms a phosphodiester bond between two adjacent DNA fragments. D. Inhibitors of DNA replication. The synthesis of DNA is inhibited by a number of compounds, most of which are either analogs of purines, pyrimidines, or folic acid. These compounds are frequently used as chemotherapeutic agents because of their ability to decrease the rate of division of rapidly growing cancer cells. l. Purine analogs. The most commonly used purine analog inhibitors are 6-mercapto-

purine, 8-azaguanine, and 6-thioguanine.

Note A nucleotide analog lacking a 3'-( OH will terminate elongation when it is inserted; e.g., cytosine arabinoside or other "dideoxynucleotides."

2. Pyrimidine analogs. Analogs such as S-fluorodeoxyuridine and S-bromodeoxyuridine inhibit thymidylate synthase, an enzyme critical for DNA synthesis. Cytosine arabinoside is an analog of cytosine nucleosides in which the sugar moiety is altered. Insertion of this analog into a DNA chain stops elongation of the chain. 3. Folic acid analogs. The synthesis of both purines and pyrimidines requires folic acid as a carrier of Cl fragments. The transfer of a Cl fragment from tetrahydrofolic acid to an acceptor results in the oxidation of tetrahydrofolic acid to dihydrofolic acid. In order to recycle the coenzyme, the cell requires NADPH and dihdyrofolic acid reductase. There are a number of folic acid analogs, such as methotrexate and aminopterin, that specifically act as powerful competitive inhibitors of dihydrofolic acid reductase.

DNA REPAIR The structure of DNA can be altered in a number of ways. Incorrect bases may be inserted during replication, or changes can result from exposure to ultraviolet radiation or chemicals found in the environment. Several repair systems have evolved that correct many of these changes. A. General terminology and definitions l. Mutation is a heritable change in the nucleotide sequence of DNA.

2. Mutagen is a chemical that alters the structure of a base in DNA. 3. Point mutation is a change in a single nucleotide. There are two types of point mutations: transitions and transversions. a. Transition is the change of one purine for another purine or of one pyrimidine for another pyrimidine. Transitions occur either from mispairing of bases during DNA replication or from chemical insult. For example, nitrous oxide converts cytosine to uracil by the removal of the amino group on the cytosine ring. b. Transversion is the exchange of a purine for a pyrimidine or vice versa. B. Types of DNA repair. Many of the mistakes occurring during replication are corrected by

the proofreading (3' to 5' exonuclease) activity of DNA polymerases. Additional types of repair mechanisms exist, which correct alterations that occur because of chemical or environmental insult.

132

meClical

DNA Replication and Repair

1. Nucleotide excision-repair mechanism of UV-damaged DNA. Ultraviolet light induces the formation of dimers between adjacent pyrimidines in DNA Cusually between adjacent thymines). The formation of pyrimidine dimers prevents DNA replication and normal gene expression. As shown in Figure II-3-3, four steps are involved in repairing this damage.

a. Incision. A UV-specific endonuclease recognizes the damaged DNA and makes a nick in the phospho diester backbone several bases away on each side of the dimer.

In a Nutshell Nucleotide excision repair removes a UV dimer and replaces it with normal nucleotides.

b. Removal. A helicase in the same enzyme complex removes the oligonucleotide. c. Polymerization. DNA polymerase fills in the gap by adding nucleotides, starting with the addition of the first nucleotide to the free 3' OR group created by the incision and moving in a 5' to 3' direction.

d. Ligation. DNA ligase forms a phosphodiester bond between the new DNA segment and the original DNA strand.

Thymine dimer~

I I II I I I I I I I rlu'.Jhl'l"'1MIIMI....I "-11 ....1'1..-1

I

t

Incision step Nicking by UV-specific endonuclease

fCl>\

~1'1"'1T"1~IIMI'I"'IT"I~llnl""nlIMI'I"'IT"I~IIMI'I"'IT"lrl

t

Removal by associated helicase

~ 5'-3' . . ,.1"T"1T"111....1'1T"1MII"""I"'IMil, " .1111111111111

I

DNA polymerase I

~t p synthesizes new DNA

1111111111111, t, ,1111111111111 Ligation by DNA ligase

I I 111111 111111 II 111111 111111 II Figure 11-3-3. Nucleotide excision-repair mechanism for UV-damaged DNA.

2. Base excision repair. The most common base transition is the deamination of cytosine to uracil in the DNA duplex. The deamination leaves an improper base pair between the two strands CU -G instead of C-G). The repair mechanism for this type of damage involves the following steps. a. Removal of the uracil base. A uracil-DNA glycosidase recognizes the mismatch and hydrolyzes the N-glycosidic bond between uracil and the deoxyribose moiety, leaving an apyrimidinic site CAP site) in the DNA strand. b. Cleavage of phosphodiester. A specific endonuclease recognizes the damage and nicks the phospho diester bonds adjacent to the missing base, thus releasing dexoyribose and

metlical

133

Molecular Biology

creating a gap. Endonucleases that cleave at apyrimidinic sites (or apurinic sites) belong to a family known as AP nucleases. c. Insertion of new cytosine nucleotide and ligation. DNA polymerase fills the gap, and the nick is sealed by DNA ligase.

In a Nutshell • An autosomal recessive deficiency in nucleotide excison repair results in xeroderma pigmentosa. • An autosomal recessive deficiency in base excision repair results in ataxia telangiectasia.

114

iiieilical

C. Diseases associated with DNA repair. Because repair mechanisms play such an important role in mutation surveillance and prevention, inherited defects that alter the activities of the repair enzymes can lead to dramatic increases in frequency of mutations. Several autosomal recessive diseases, such as xeroderma pigmentosa, ataxia telangiectasia, Bloom syndrome, and Fanconi anemia, are associated with either documented or suspected defects in DNA repair mechanisms. All of these diseases occur with high frequency and are associated with a predisposition to malignancy. 1. Xeroderma pigmentosa is an autosomal recessive trait characterized by extreme sensitivity to sunlight, skin changes, and a predisposition to malignancy. The disease results from a defective excision-repair mechanism for UV-damaged DNA. 2. Ataxia telangiectasia is an autosomal recessive disorder, characterized by hypersensitivity to ionizing radiation, degenerative ataxia, dilated blood vessels, chromosome aberrations, and lymphomas. The disease results from a defect in AP endonuclease.

Transcription The process of making an RNA copy of DNA is called transcription and is catalyzed by the enzyme RNA polymerase. In any particular region of the DNA double helix, only one of the two DNA strands is transcribed into RNA. The RNA strand is always synthesized in a 5' to 3' direction, but from an antiparallel and complementary DNA template strand. The completed RNA is therefore antiparallel and complementary to the template or "anti-sense" DNA strand. The RNA is, however, parallel and identical to the "sense" strand of the DNA duplex (except that it uses uracil instead of thymine). Cells contain three types of RNA, each having a different function. Messenger RNA (mRNA) carries the information specifying the amino acid sequence of proteins. Transfer RNA (tRNA) and ribosomal RNA (rRNA) play important roles in protein synthesis. This chapter reviews the key structural features of the genes coding for the three different types of RNA. the transcriptional machinery, and the steps involved in the process of transcription.

STRUCTURE AND TRANSCRIPTION OF PROKARYOTIC GENES In prokaryotes, both the gene structure and the machinery for transcription are considerably simpler than in eukaryotic systems. A. DNA elements. Specific sequences in the DNA act as start and stop signals for transcription.

1. Bacterial promoter region. The region of DNA that binds RNA polymerase and allows transcription to be initiated is known as the promoter region. As shown in Figure 11-4-1, the promoter region contains a starting point at which transcription is initiated and two consensus sequences that are recognized by RNA polymerase. The sequences at which RNA polymerase bind are located at positions -lO and -35. By convention, the starting point is designated as +1. (Negative numbers are not transcribed and indicate that the nucleotide is upstream from the initiation site, i.e., on the 5' side of the starting site). a. Pribnow box. The Pribnow box is an A-T rich sequence (TATATT) located 10 base pairs upstream from the site at which transcription begins. This sequence is present in almost all promoters and is involved in the initial unwinding of DNA by RNA polymerase. b. Hexamer at -35 position. Another consensus sequence of six nucleotides is located 35 base pairs upstream from the start site for transcription. This TTGACA sequence is involved in the initial recognition of the promoter by RNA polymerase.

In a Nutshell Most E coli promoters contain two recognition sequences for RNA polymerase: • Pribnow sequence at -10 • -35 sequence

c. Start site. The first nucleotide transcribed into RNA is usually a purine (Pu), either A or G. The next two nucleotides (designated NN) can be any of the four nucleotides found in RNA (A, G, C, or U).

KAPLAlf _ I meillea

135

Molecular Biology

Promoter

Coding region

II -35

-10

+1

5' -",,'-..rTTmGU\AC:CA~-..ffjITAiTI RNA polymerase

Initiation of transcription

binding sites

Figure 11-4-1. Bacterial promoter.

2. Bacterial terminators. Termination of transcription involves the release of both the DNA template and the newly synthesized RNA from RNA polymerase. Bacterial RNA polymerase has two methods of termination. a. Rho-independent termination occurs when the newly synthesized RNA folds back on itself and forms a hairpin loop that is stabilized by hydrogen bonding between complementary bases. The hairpin loop must be followed by a run of 6-8 U residues that form weak bonds with the complementary run of 6-8 A residues in the DNA template (Figure II-4-2). These two structural features of the newly synthesized RNA promote dissociation of the RNA from the DNA template.

u C U

C G

G-C C-G C-G G-C G-C A A In a Nutshell Rho-independent termination requires a specific 2° helical stem to form in the newly transcribed RNA; rhodependent termination requires rho protein to move along the newly transcribed RNA and to "catch" the RNA polymerase. 136

meClical

5'-GCACCGCA

UUUUUUU-OH 3'

Figure 11-4-2. Structural features required for rho-independent termination.

b. Rho-dependent termination. Rho factor is a protein that terminates transcription. It binds to the newly formed RNA and moves toward the RNA polymerase that has paused at a termination site. Rho then displaces the RNA polymerase from the 3' -OH end of the RNA. Rho factor has ATPase activity that is RNA dependent, and it requires a polyribonucleotide that is greater than 50 bases long.

Transcription

B. Transcriptional machinery. In prokaryotic systems, all forms of RNA are synthesized by a

single RNA polymerase. 1. Structure of RNA polymerase. This multisubunit enzyme exists in two forms: a core enzyme and a holoenzyme. The core enzyme has four subunits (a2~W). It is capable of polymerizing ribonucleotides, but it does not recognize promoter regions in the DNA. The holoenzyme has an additional sigma subunit that allows the enzyme to recognize promoter sequences. 2. Requirements for RNA synthesis. The synthesis of RNA requires all four ribonucleotides (ATP, GTP, CTP, and UTP), a divalent cation (either Mg2+ or Mn2+), and a template. Double-stranded DNA is the preferred template, but single-stranded DNA can also be used. Synthesis occurs in the direction of 5' to 3', and each time a nucleotide is added to the 3'-OR end, pyrophosphate is released. RNA polymerase does not require a primer and cannot proofread and correct mistakes. 3. Function of RNA polymerase subunits. The a subunits are involved in binding to the consensus sequences in the DNA promoter region. The ~ subunit contains the active site that binds nucleotide triphosphates and forms the phosphodiester bond of the polyribonucleotide chain. The Wsubunit plays a role in the attachment of the enzyme to the DNA template. Sigma factor recognizes promoter sequences and provides the enzyme with specificity.

In a Nutshell RNA Synthesis • Requires an antiparaliel and complementary template • Occurs 5' to 3' • Is not proofread • Can be initiated de novo (no primer is required)

C. Steps involved in transcription. Transcription in prokaryotes involves three steps: initia-

tion, elongation, and termination. 1. Initiation. The sigma subunit of the holoenzyme recognizes consensus sequences in the

promoter, binds to DNA, and helps unwind the DNA double helix so that one strand can serve as a template. The initiating ribonucleotide triphosphate is usually a purine (either A or G). After the first few phospho diester bonds are formed, the sigma subunit dissociates from the holoenzyme, and the core enzyme begins elongation. 2. Elongation. The core enzyme moves along the template extending the RNA chain, and the region of local unwinding moves with it. As the enzyme leaves a region, the DNA duplex reforms, and RNA is displaced as a growing polynucleotide chain. Growth of the chain is always in the 5' to 3' direction. RNA polymerase moves along the DNA template strand in the 3' to 5' direction.

Note In prokaryotes, the processes of transcription and translation are coupled. Translation of mRNA starts before the completion of transcription. In these systems, almost all gene regulation occurs at the transcriptional level.

3. Termination. The DNA template contains stop signals for transcription, as described in paragraph A.2. D. Inhibitors of prokaryotic transcription. Many antibiotics exert their action by interfering with bacterial RNA or protein synthesis while having no effect on eukaryotic RNA and protein synthesis. 1. Rifampin inhibits initiation of RNA synthesis. 2. Actinomycin D binds to DNA and inhibits RNA synthesis by blocking movement of RNA polymerase along the template. 3. Streptolydigin binds to the

~

subunit of RNA polymerase and blocks elongation.

metlical

137

Molecular Biology

STRUCTURE AND TRANSCRIPTION OF EUKARYOTIC GENES Both the structure of eukaryotic genes and the mechanisms for transcription are more complex than in prokaryotic systems. A. Transcriptional machinery: three RNA polymerases. Each type of RNA is transcribed by a different RNA polymerase. The requirements of each of these enzymes are the same as for prokaryotic RNA polymerase. None of the RNA polymerases have the proofreading activities that DNA polymerases have. 1. RNA polymerase I is localized in the nucleolus and transcribes the genes for three types of ribosomal RNA: 28S, 18S, and 5.8S rRNA. rRNA is the most abundant RNA in the cell.

2. RNA polymerase II is found in the nucleoplasm, where it transcribes genes coding for proteins. The primary transcripts synthesized by RNA polymerase II are known as heterogeneous nuclear RNA (hnRNA), which are precursors for mRNA and snRNA (small nuclear RNAs). The snRNAs (UI-U5) are a part of the "spliceosome;' a complex involved in removing intron sequences from precursor RNA molecules. 3. RNA polymerase III is located in the nucleoplasm and transcribes the genes for transfer RNA (tRNA) and 5S rRNA. B. Structure and transcription of ribosomal RNA genes. The genes coding for rRNA are transcribed by RNA polymerase I in the nucleolus. The nucleolus contains large loops of DNA from several chromosomes, each containing a cluster of genes for rRNA. As shown in Figure 11-4-3, multiple copies of the genes for rRNA are tandemly arranged so that each gene is separated from the next by a spacer. The primary RNA transcript of each of these genes is a 45S RNA that serves as a precursor for 18S, 28S, and 5.8S rRNA. The primary transcript also contains spacer regions between each of the rRNAs. The spacer regions are removed by a series of endonucleolytic cleavages. Processing of the precursor rRNA results in equimolar amounts of each of three types of rRNA. After the processing shown in Figure 11-4-3, the rRNA species are modified by methylation of specific 2'-OH groups. This reaction is believed to confer stability on the rRNA molecules and protect them from endonucleolytic cleavage after incorporation into ribosomes. C. Genes coding for protein. RNA polymerase II is found in the nucleoplasm, where it transcribes the genes whose RNAs will be translated into proteins. RNA polymerase II cannot initiate transcription itself and is absolutely dependent on additional proteins, known as transcriptional factors, that act at promoter sites. The essential features of genes that are transcribed by RNA polymerase II are shown in Figure II -4-4 and are briefly discussed. 1. Start site. The initial nucleotide in position + 1 of the RNA transcript is usually A, flanked by pyrimidines.

2. Promoters. There are two common sequences in promoters that are used by RNA polymerase II.

138

iiieClical --------------

-------

Transcription

45S rRNA precursor

CI)

co

CI)

CI)

to

C\I

co

45S rRNA precursor

CI)

co

co

CI)

CI)

to

C\I

co

co

45S rRNA precursor

S'p

L...L~~

OH3 '

___. L . - _

CI)

CI)

CI)

to

C\I

cq

co

CD

co

o


Q.

Processing of the 45S rRNA precursor

(/J

Mature rRNA CI)

CI)

CI)

tri

C\I

co

co

co

Figure 11-4-3. Structure and transcription of ribosomal RNA genes.

Poly- A+ addition site

>c

~o

0·-

O"l

~
O"l


a::

-70

-25

~ ..c

..c

t;(

«

0

Exon 1

Intron 1

t

Exon 2

x

0

~ ~

1+1 c 0 '5>

AGGTAAGT

NCAGG

t

t


"0
til

U5 C

~

c

::> Ln

11 AATAAA

c 0 '5>

(ij

"0
+

~

til

U5 c ~

C O"l

'iii

«

:>. ~

C ::> M

Figure 11-4-4. Structure of a typical gene transcribed by RNA polymerase II.

a. TATA box (Hogness box). This sequence of base pairs, which is important in the initiation of transcription, is found in all eukaryotes. It is located approximately 25 base pairs upstream (-25) from the start point and is almost identical to the Pribnow box found in bacterial systems. TFIID, the critical transcription factor for RNA polymerase II, binds here. b. CAAT box. This sequence is usually found between positions -75 and -80, but it can KAPLAN"dme leaI

139

Molecular Biology

function at distances that vary considerably from the start point. Binding of transcription factors at this site may influence the formation of initiation complexes at other sites.

In a Nutshell RNA polymerase II initiation requires: • Specific transcription factors bound to enhancer sequences • General transcription factors bound to promotor sequences • Association of RNA polymerase II with transcription factors to form initiation complex

3. Regulatory regions. Sequences that either increase or decrease the rate at which transcription is initiated by RNA polymerase II exist at various places in the gene. Although they are usually located upstream from the start site, they may also be internal to the gene or downstream from the gene. Enhancer sequences bind transcription factors that increase the rate of transcription, whereas silencer sequences bind factors that decrease the rate of transcription. 4. Exon and introns. The segment(s) of the gene that are maintained in the mature mRNA and code for protein are known as exollS. Introns are the portions of the primary RNA transcript that are removed by splicing together the exons (Figure 11-4-5). The splicing usually occurs at a consensus sequence (GU ... AG) found at the boundaries between introns and exons (discussed in more detail below).

prima7rahnn:c~; Mature mRNA

5'

--l 5'

Exon 1

---1

H

Exon 1

Exon 2

H

Exon 3

H

I Exon 2 I Exon 3 I Exon

Exon 4

r--

3'

4}-- 3'

Figure 11-4-5. hnRNA and mature mRNA.

In a Nutshell RNA polymerase III initiation requires: • Transcription factors bound at intemal promotor sequence and at start site • Association of RNA polymerase III with transcription factors at start site to form initiation complex

Note In eukaryotes, because transcription and translation are not coupled, the primary transcript (hnRNA) is extensively modified in the nucleus, yielding mature mRNA that is then transported to the cytoplasm for translation into protein.

140

ineClical

D. Genes coding for tRNA and 5S rRNA. These genes are transcribed in the nucleoplasm by RNA polymerase III. The promoters for both the 5S rRNA and the tRNA genes are internal and are found downstream from the start site of transcription. l. 55 rRNA genes. The genes for this small rRNA are tandemly repeated in a single cluster

located far away from the genes for tRNA. These are the only rRNA genes that are transcribed outside the nucleolus. 2. tRNA genes. The genes for tRNA are clustered and are transcribed as larger precursor RNA molecules, which are processed by endonucleases. Internal promoters are found in two regions downstream from the start site. tRNA is subject to a number of posttranscriptional modifications, including modification of specific bases and the addition of the sequence -CCA to the 3' -OH of the tRNA.

PROCESSING OF EUKARYOTIC RNA The heterogeneous nuclear RNAs (hnRNAs), synthesized by RNA polymerase II, leave the nucleus as messenger RNAs (mRNAs). The processing of the hnRNA starts while transcription is still occurring and involves covalent modification of both the 5' and 3' ends, followed by cutting and splicing to eliminate the intervening sequences that separate the coding regions. A. 5' Capping. The 5' end of the RNA is "capped" shortly after the initiation of RNA synthesis. This process involves the addition of an "inverted" methylated guanosine molecule to the first nucleotide in the RNA transcript. The 7-methyl-guanosine is linked through a 5'-5' triphosphate linkage. The 5' cap plays an important role in the initiation of protein synthesis and protecting the mRNA chain from degradation. B. Polyadenylation of the 3'-OH end. Mature mRNA molecules have a poly-A tail that is

between 20 and 250 nucleotides long. The tail is added to hnRNA by the enzyme poly-A polymerase. A consensus sequence near the end of the gene provides a signal that initiates

Transcription

polyadenylation. Polyadenylation is believed to increase the stability of hmRNA. Not all mRNA molecules are polyadenylated: histone mRNAs, for example, have no poly-A tails. C. RNA splicing. hnRNA contains coding sequences (exons) that are separated from one another by intervening sequences (intrans). In the conversion ofhnRNA to mature mRNA, the introns are removed and the exons are spliced together. This process is illustrated in Figure 11-4-6. During the excision of introns, the 5' cap and the poly-A tail are not removed. The base sequence at the beginning of an intron is GU ... and at the end of an intron is ... AG. This consensus sequence defines the sites at which cutting and splicing occur. The intron is excised as a loop (lariat) of RNA that is degraded. After excision, ligation of the exons occurs. These reactions occur in the nucleus. STEP 1.

-0 Q)

'til

-0

co

!:;c

Ui c

~'rn

!:;c

Q)

Ui c

'til

co

co

Co

Q) l!)lo..

t

~'rn Q) C')lo..

Intron 1

Exon 1

Exon 2 AAUAAA

5' Cap AGGUAAGU

t

t

AGG

t

["'il

AAAn

HnRNA

t

2

STEP 2.

'(jj

+ «'co >'c Exon 1

0-0 a.. co

a..'(jj

~.... AAUAAA

.......""'"""'........ AG

« .Q b:g

DOl

Exon 2

5' Cap

+ c

[ii,1

AAAn

Formation of lariat

G

STEP 3. Exon 1

Exon 2

====;.;.1 AAUAAA leII AAAn

5'Cap

G

AG STEP 4.

Excision of lariat

Exon 1 Exon 2

5' Cap

........··.··, ••. ·\.. 1 AAUAAA

II

(mRNA) AAAn

Ligation of exons

AGG

plus

Lariat (degraded in nucleus) Figure 11-4-6. Processing of hnRNA. KAPLAtf _ I med lea

141

Protein Synthesis

Translation is the process by which the base sequence in mRNA is decoded into an amino acid sequence. All three types of RNA play different and essential roles in translation. The genetic code is defined as the relationship between the sequence of bases in DNA (or its RNA transcript) and the sequence of amino acids in proteins. mRNA is the template for protein synthesis and acts as a "working copy" of the gene in which the code words for each amino acid (codons) have been transcribed from DNA to mRNA. The tRNA has a three-base anticodon that hydrogen bonds with the complementary codon in mRNA thus aligning amino acids in the appropriate sequence prior to peptide bond formation. Ribosomes are complexes of protein and rRNA that serve as the molecular machines, coordinating the interactions between mRNA, tRNA enzymes, and protein factors required for protein synthesis. This chapter reviews the structure and function of the three types of RNA, the genetic code, the structure and function of ribosomes, and the events involved in protein synthesis. Commonly used antibiotics that inhibit protein synthesis are also reviewed.

ROLE OF RNA IN PROTEIN SYNTHESIS Each of the three major types of RNA plays an essential role in protein synthesis. A. Messenger RNA (mRNA) acts as the "working copy" of the gene coding for a protein. The mRNA carries the information from the genome in the nucleus to the cytosol where protein synthesis occurs. Messenger RNA is synthesized as an hnRNA precursor and is processed to mature mRNA in the nucleus. The mature mRNAs are capped at their 5' end with 7-methylguanosine attached through a triphosphate linkage to the first nucleotide in the mRNA. Most mRNAs also contain a poly-A tail attached to the 3' end. Messenger RNA constitutes approximately 5% of the total cellular RNA and has a shorter half-life than other types of RNA. The length of the mRNA is related to the size of the gene. The key structural features of mRNA are shown in Figure 11-5-1.

KAPLAN"iIme leaI

143

Molecular Biology

5' Untranslated region

Coding region or open reading frame

Poly-A+ signal

Poly-A+ tail

UGA 5' Cap - - AUG - - - - - - UAA - - - AAUAAA--- AAAn 3' UAG Start codon Stop codon (Methionine)

t

t

3' Untranslated region

Figure 11-5-1. Structure of eukaryotic mRNA.

B. Ribosomal RNA (rRNA). Ribosomal RNA is the most abundant form of RNA, comprising approximately 80% of the cellular RNA. Ribosomes, the machines for synthesizing protein, are complexes containing protein and rRNA. In prokaryotic systems, there are three forms of rRNA: 23S, 16S, and 5S rRNA, which vary in length from 120 to 3,700 nucleotides. Eukaryotic rRNA has four forms of rRNA: 28S, 18S, 5.8S, and 5S. In eukaryotic systems, all of the forms of rRNA except 5S rRNA are synthesized in the nucleolus. The precise function of rRNA is unclear, but it is necessary for the organization of a functional ribosome.

Note The 5' end of an RNA that has been cut out of a larger precursor RNA is a monophosphate; the 5' end of a precursor or unprocessed RNA is a triphosphate.

C. Transfer RNAs (tRNAs) are the adaptor molecules in protein synthesis. They have a threebase region (anticodon region) that recognizes and hydrogen bonds to the complementary codon in mRNA, which specifies a particular amino acid. The tRNAs react with amino acids at their 3' ends. Transfer RNAs are small, containing approximately 80 nucleotides. The key structural features of tRNA are shown in Figure 11-5-2 and are described briefly below. 3' Amino acid OH - - attachment site A

C C

Phosphorylated 5' terminus - - 5' P

A G···· C

nvC loop

"" ______________"Extra arm" (variable)

Figure 11-5-2. Structure of tRNA.

144

mectical ---

._---_.----- - - - - - -

Protein Synthesis

1. The 5' end of tRNA is a monophosphate rather than a triphosphate, and the base is usually G. 2. The 3' end of tRNA has the sequence CCA. The "activated amino acid" is covalently attached to the 3' OH of the terminal adenosine. The bond linking the amino acid to tRNA is a high-energy bond that provides energy for peptide bond formation, an endergonic reaction.

Note The CCA amino acid attachment site of tRNA is encoded in prokaryotes but is added posttranscriptionally in eukaryotes.

3. Unique structural features of tRNA include three loops and an "extra arm" created by internal hydrogen bond formation. The anticodon loop contains three bases that are antiparallel and complementary to the bases in the codon of mRNA. 4. A high degree of secondary structure is found in tRNA due to the bending back of the molecule on itself with the formation of internal base pairs that stabilize the secondary structure. About half of the nucleotides in tRNA are base paired to form double helices. 5. A large number of unusual bases are found in tRNA that are not found in other types of RNA. These are believed to be important in maintaining the characteristic secondary structure. Inosine, pseudouridine, dihydrouridine, ribothymidine, and methylated guanosine are found exclusively in tRNA.

THE GENETIC CODE The genetic code is the relationship between the sequence of bases in DNA (or its working RNA transcript) and the sequence of amino acids in proteins.The genetic code is read in triplets. The code words are almost (but not absolutely) universal. Human mitochondria have a few differences in codons. A. A codon is a group of three nitrogenous bases (base triplet) that represents an amino acid or signals the initiation or termination of protein synthesis. The codons in mRNA are antiparallel and complementary to the anticodons (recognition sites) found in tRNA. The codons in mRNA are shown in Figure II-5-3. There are 64 possible combinations of the four bases in mRNA, and thus the genetic code contains 64 codons. B. Amino acid codons. There are 61 triplets that specify 20 amino acids. (The remaining three codons are "stop" codons.) Because more than one codon can specify the same amino acid, the genetic code is said to be redundant or degenerate. Different co dons for the same amino acid usually differ in the third base. C. Stop codons. There are only three codons that do not specify an amino acid. These codons (UAA, UAG, UGA) act as termination signals during protein synthesis.

D. Start codon. AUG (which codes for methionine) and sometimes GUG are signals for the initiation of translation. In prokaryotic systems, polypeptide chains start with a modified amino acid, formylmethionine (fMet). Prokaryotes have a specific tRNA that carries fMet and recognizes the initiating AUG codon in mRNA. In eukaryotic systems, the AUG closest to the 5' end of the mRNA is the start signal for protein synthesis. Eukaryotes also have a specific initiating tRNA that carries fMet.

meilical

145

Molecular Biology

8econd base U U

C

UCUj

UUU} UUC Phe UCC UCA UUA} UUG Leu UCG

CUUj CUC

C CUA CUG

Ser

CCUj CCC Leu

CCA CCG

Pro

ACUj

AUU} lie AUC ACC A AUA ACA AUG fJ~1 ACG

Val

GCA GCG

G

UAU} UAC Tyr UAA Stop UAG Stop

UGU} UGC Cys UGA Stop UGG Trp

U C A G

CAU}H' CAC IS

CGUj CGC

U C A G

CGA CAA} Gin CAG CGG

Arg

Thr

U AAU} AGU} AAC Asn AGC Ser C A AGA} ~}LYS AGG Arg G

Ala

GAU} GAC Asp GGC GGA GAA} GAG Glu GGG

GUUj GCUj GCC

GUC G GUA GUG

A

GGUj Gly

U C A G

Figure 11-5-3. The genetic code.

PROKARYOTIC AND EUKARYOTIC RIBOSOME STRUCTURE The structure and function of ribosomes are very similar in prokaryotes and eukaryotes. A. Common features. E. coli and eukaryotic ribosomes share the following features. 1. Function. Ribosomes are the sites at which protein synthesis occurs.

2. Components. Protein and rRNA are the building blocks for ribosomes. Each of the forms of rRNA and most of the proteins are present in only one copy per ribosome.

Note In general. the small subunit is necessary for initiating protein synthesis; the large subunit catalyzes the elongation steps.

3. Subunits. As shown in Figure 11-5-4, ribosomes have two subunits, one large and one small. (Note that the Svedberg units [S] are not additive.)

8mall subunit

Prokaryotes: 308: 168 rRNA + 20 proteins --- Eukaryotes: 408: 188 rRNA + 30 proteins Prokaryotes: 508: 238 and 58 rRNA + 34 proteins ---Eukaryotes: 608: 288,5.88, and 58 rRNA + 40 proteins

Ribosome

Prokaryote ribosome: 308 + 508 = 708 Eukaryote ribosome: 408 + 608 = 808 Figure 11-5-4. Composition of ribosomes.

B. Differences in prokaryotic and eukaryotic ribosomes. As seen in Figure 11-5-4, eukaryotic ribosomes are larger than prokaryotic ribosomes. In prokaryotes, the 70S ribosome is made up of a large subunit (50S) and a small subunit (30S), whereas eukaryotes have an 80S ribosome containing a large subunit (60S) and a small subunit (40S). Eukaryotic ribosomes contain four forms of rRNA, whereas prokaryotes have only three forms. Other minor differences are found in the number of proteins found in both the large and small subunits of eukaryotic and prokaryotic systems.

146

meCtical

---~~~~~-

Protein Synthesis

C. Eukaryotic ribosome assembly. In eukaryotes, the large ribosomal subunit (60S) is assembled in the nucleolus, whereas the small subunit (40S) is assembled in the nucleus. The ribosomal proteins are synthesized in the cytoplasm and are transported into the nucleus, where they combine with the appropriate rRNA species to form the large and small ribosomal subunits. After assembly, the ribosome moves through the nuclear pores into the cytoplasm by an unknown mechanism. Because prokaryotes have no defined nucleus, all of these processes occur in the cytoplasm.

In a Nutshell

ACTIVATION OF AMINO ACIDS AND ATTACHMENT TO tRNA The formation of peptide bonds between amino acids is an endergonic process and therefore requires a source of energy. The energy is derived from ATP and is transferred to the bond that links the amino acid to the 3'-OH group of the tRNA molecule. Enzymes that "activate" the carboxyl group of amino acids are known as aminoacyl-tRNA synthetases. These enzymes are highly specific for both the amino acid and the tRNA. There is a specific enzyme for each amino acid. As shown in Figure 11-5-5, the attachment of the amino acid to the tRNA occurs in a twostep process. R I

Step I

I

0

II

NH2-CH-C-AMP+tRNA R I

Sum

I

0

II

ATP+NH 2 -CH-COOH - - - - - NH2-CH-C-AMP+PPj R

Step 2

R

Each tRNA must be "charged" with the correct amino acid by a separate tRNA synthetase.

R I

0

II

- NH 2-CH-C-tRNA+AMP R I

0

II

NH 2-CH-COOH+ATP+tRNA - - NH 2-CH-C-tRNA+AMP+PPj

Figure 11-5-5. Formation of aminoacyl-tRNA.

The AGO for this reaction is close to zero, but in the cell, the overall reaction is highly exergonic and essentially irreversible because the high-energy bond in the pyrophosphate product is rapidly hydrolyzed by pyrophosphatases.

STEPS INVOLVED IN TRANSLATION The process of translating the genetic code found in mRNA into a specific sequence of amino acids in a protein involves three steps: initiation, elongation, and termination. Protein synthesis occurs in the direction of amino terminal to carboxy terminal, and the mRNA is read from the 5' end to the 3' end. A. Initiation of protein synthesis. Initiation of protein synthesis requires special tRNA molecules and the formation of an initiation complex. 1. Initiating tRNA molecules. The codon AUG in mRNA usually signals the beginning of protein synthesis. AUG is the codon for methionine. a. Prokaryotes. The initial methionine residue has a formyl group attached to it (fMet). The initiating tRNA is known as tRNAfMet. The tRNA that carries methionine to any other position is known as tRNAMet. b. Eukaryotes. The initiating methionine has no formyl group. The initiating tRNA is known as tRNAiMet, whereas the tRNA involved in elongation of the protein is known simply as tRNAMet. KAPLAN" . ._

me lea

I

147

Molecular Biology

2. Formation of initiation complex. The assembly of the 70S initiation complex found in prokaryotes is a stepwise process involving the formation of an intermediate 30S complex.

In a Nutshell In prokaryotes, the initiating AUG is defined by its position near a Shine-Oalgarno sequence. Therefore, prokaryotic mRNAs may contain multiple genes ("polycistronic"). Because each gene in the mRNA begins with a Shine-Oalgarno + AUG sequence, each will be independently translated by ribosomes. In eukaryotes, the ribosomal small subunit (with tRNAMet + e1F-2 + GTP bound) must begin each time at the 5' cap, then scan the mRNA for the first AUG to initiate, where it is joined by the large subunit. Eukaryotic mRNAs are typically monocistronic (one gene per mRNA).

a. Assembly of the 30S complex. Formation of the 30S complex requires mRNA, the 30S ribosomal subunit, three initiation factors (IF-I, IF-2, and IF-3), GTP, and tRNAtMet. The mRNA contains the start codon (AUG or GUG) that recognizes tRNAtMet. There is a special sequence of bases upstream from the AUG codon called the ShineDalgarno sequence. This sequence of bases binds with the 16S rRNA in the 30S ribosomal subunit. The binding of the mRNA to the 30S ribosomal subunit requires the transient help of IF-3. After mRNA binds to the 30S ribosomal subunit, IF-3 dissociates. The complex between mRNA and the 30S ribosomal subunit then binds IF-2, GTP, and tRNAtMet to produce an active 30S initiation complex. This reaction requires IF-I. b. Assembly of the 70S initiation complex occurs by the interaction of the 50S ribosomal subunit with the 30S complex. The formation of the 70S complex results in the hydrolysis of GTP to GDP and Pi and the dissociation of both IF-l and IF-2. The structure of the 70S initiation complex is shown in Figure 11-5-6. The association of the ribosomal subunits creates two important sites for protein synthesis within the ribosome, the A site and the P site. (1) The aminoacyl site (A site) binds the incoming tRNA molecule carrying an activated amino acid. (2) The peptidyl site (P site) is the site on the ribosome at which tRNAtMet initially binds. After formation of the first peptide bond, the P site is occupied by the growing peptide chain. The tRNAtMet recognizes two sites: the P site on the ribosome and the start site (AUG) on the mRNA. B. Elongation of the protein. This process is a three-step cycle that is illustrated in Figure II-5-6.

Each step is described below.

----,TT"'"""Trl~,.,.---

----,TT"'"""Trl.,..,.,.---3'

3'

Delivery of activated tRNAArg

P site

•

A site

P site

A site

70S Initiation complex Peptide bond formation by peptidyl transferase

-rl...--

mRNA 5' --r..,-".c"....

P site

..

tI

----,TT"'"""Trl.,..,.,.--- 3' Translocation

r

Empty A site

r

UAC

P site

Uncharged tRNA

Figure 11-5-6. Elongation phase of protein synthesis in bacteria.

148

mellical

A site

Protein Synthesis

1. Binding of aminoacyl-tRNA to the A site. The A site will always contain the next amino acid to be added to the peptide chain. The specific aminoacyl-tRNA that comes into the A site is determined by the mRNA codon that is positioned above the A site. Thus, in Figure II-5-6, tRNAArg is delivered to the A site because CGC codes for Arg (see Figure 11-5-3). The delivery of the aminoacyl-tRNA to the A site requires elongation factor EF-Tu and the hydrolysis of GTP to GDP and Pi. GDP remains associated with EF-Tu until it is displaced by another elongation factor, EF-Ts. The new Tu-Ts complex is dissociated by the binding of a second GTP to give GTP-Tu complex.

2. Peptide bond formation. Formation of a peptide bond between the amino acid (or peptide) in the P site and the amino acid in the A site is catalyzed by peptidyl transferase, which is an integral part of the 50S ribosomal subunit. This reaction results in the release of the amino acid from the tRNA in the P site. The resulting peptide is bound to the tRNA in the A site. 3. Translocation. In order for elongation to continue, translocation has to occur. This involves three movements. First, the uncharged tRNA leaves the P site. Next, the peptidyl tRNA moves from the A site to the P site. Finally, the mRNA moves a distance of three nucleotides to bring a new codon into a position above the empty A site. The movement of mRNA requires an elongation factor known as translocase (EF-G). Hydrolysis of GTP is required to release EF-G from the ribosome. At some point during elongation of the peptide chain, the formyl group is removed from the initial methionine residue. C. Termination of protein synthesis. When anyone of the three stop codons is encountered,

elongation is terminated. Cells do not have tRNAs with anticodons that are complementary to stop codons. Release of the peptide from the ribosome requires a protein known as a release factor (RF) that binds to the stop codon. Hydrolysis of the peptidyl-tRNA bond requires an enzyme, peptidyl-tRNA hydrolase, and GTP, which is hydrolyzed to GDP and Pi' The polypeptide leaves the ribosome, the ribosome dissociates into its 30S and 50S subunits, and the mRNA is released.

Note During peptide bond formation, the amino group of the amino acid bound to the A site tRNA is bound to the a-carboxy group of the amino acid bound to the Psite tRNA.

In a Nutshell Elongation is catalyzed by the large subunit: • Charged tRNA binds to the A site (requires GTP hydrolysis) • Peptide bond formation • Translocation (requires GTP hydrolysis)

INHIBITORS OF PROTEIN SYNTHESIS Several antibiotics recognize differences between prokaryotic and eukaryotic protein synthesis and can be used to inhibit one independent of the other. Additionally, some bacterial toxins inhibit protein synthesis in animal cells. Table II -5-1 lists several inhibitors of protein synthesis.

Table II-5-l. Inhibitors of protein synthesis. Inhibitor

Prokaryote or Eukaryote Step/Site of Action

Tetracycline

Prokaryotes

Streptomycin Erythromycin Chloramphenicol Puromycin

Prokaryotes Prokaryotes Prokaryotes Both

Cycloheximide Sparsomycin

Eukaryotes Eukaryotes

Ricin Eukaryotes Diphtheria toxin Eukaryotes Pseudomonas toxin Eukaryotes

Initiation; prevents binding of aminoacyl-tRNA to ribosome Initiation; causes misreading of code Translocation Ribosomal peptidyl transferase Elongation; binds to A site and prematurely terminates chain growthl Ribosomal peptidyl transferase . Initiation; inhibits formation of initiation complex Initiation; binds to 60S subunit Translocation; inactivation ofEF-2 Translocation; inactivation ofEF-2

KAPLI~. I meulca

149

Regulation of Ciene Expression

Regulation of gene expression is an essential feature in maintaining the functional integrity of a cell. Regulation of a gene may occur in a variety of ways; some are positive, whereas others are negative. Regulation of gene expression in prokaryotes almost always involves either initiation or termination of transcription. In eUkaryotes, transcription is more complicated than in prokaryotes, and, therefore, there are more possible sites for regulation. In both prokaryotes and eukaryotes, transcriptional regulation is usually achieved by the interaction of proteins with specific sequences in the DNA, resulting in either an increase or a decrease in the rate of transcription. All of the cells in eukaryotic organisms contain the same genome, yet different types of cells contain unique complements of mRNAs and proteins. Therefore, mechanisms for specifying cell-specific transcription exists in eukaryotes but not in prokaryotes. In this chapter, the basic mechanisms for regulating transcription in both prokaryotes and eukaryotes are summarized. A few selected examples of gene regulation are discussed that illustrate different mechanisms of transcriptional control.

REGULATION OF TRANSCRIPTION IN PROKARYOTES The regulation of gene expression in prokaryotes is usually achieved by regulating the rate at which transcription is initiated. The examples that will be considered are the positive and negative control of the lactose (lac) operon in E. coli. A. Operon concept. In bacteria, a set of structural genes and a regulatory region constitute an operon. The structural genes code for a group of proteins required for a particular metabolic function. The regulatory region is upstream (to the 5' side) of the structural genes. The structural genes in an operon are coordinately regulated, i.e., the expression of all the structural genes is controlled by the same regulatory region in the DNA. B. Description of the lac operon. The arrangement of the structural and regulatory genes in the lac operon of E. coli is shown in Figure 11-6-1. The regulatory gene (i) codes for a repressor that can interact with the operator sequence (0). The operator sequence is situated adjacent to the three structural genes (Z, Y, and A) that code for three enzymes concerned with lactose metabolism (B-galactosidase, permease, and transacetylase). The expression of these three genes is regulated by the operator sequence. RNA polymerase binds at the promoter site (P).

1. Negative regulation of the lac operon. In the absence of lactose, the repressor protein binds to the operator sequence and blocks the movement of RNA polymerase along the template, thus inhibiting transcription (Figure II -6-1A). When lactose is present, it induces transcription by the following mechanism: Lactose is converted to 1,6-allolactose, a molecule that binds to the repressor and changes its conformation so that it no longer binds to the operator region. Thus, the repressor is released and the three structural genes are transcribed as a single mRNA that codes for all three proteins (Figure II-6-1B).

Note Each structural gene in the polycistronic lac mRNA has a Shine-Dalgarno sequence to define its initiating AUG.

Note The lac repressor is made constitutively, i.e., whether or not lactose is present.

meclical

151

Molecular Biology

Messenger RNA coding for more than one protein is known as a polycistronic mRNA and is found only in prokaryotic systems. In this model of negative regulation of an operon, a protein acts as the repressor and lactose (or l,6-allolactose) acts as the inducer of transcription.

In a Nutshell Full transcription of the lac operon requires: • Absence of the repressor (negative regulator) • Presence of the activator (positive regulator)

2. Positive regulation of the lac operon. The function of ~-galactosidase in lactose metabolism is to cleave lactose to glucose and galactose. The galactose is ultimately converted to glucose by other enzymes. Therefore, if the bacteria has both glucose and lactose in the growth medium, there is no reason to activate the lac operon. When glucose is low, the lac operon is activated, and the bacteria begin to use lactose to generate glucose. The effects of glucose are mediated by cAMP. When glucose is low, cAMP concentration is high and vice versa. When cAMP increases, it binds to a protein known as CAP (catabolite activator protein). The CAP-cAMP complex activates transcription of the lac operon by binding to the promoter region and allowing RNA polymerase to initiate transcription. Thus, the CAP-cAMP complex is a positive regulator of the lac operon.

A. In the absence of lactose Regulatory region

Control region

Structural genes jrj----------------------------~

p

01

z

y

A

! Repressor mRNA

t

Repressor binds to 0 and structural genes cannot be transcribed

Repressor

B. In the presence of lactose Regulatory region

Control region

p

! t

Structural genes

jrj-----------------------------,

o

z

y

A

!

.-

Repressor mRNA

Beta-galactosidase, permease, transacetylase polycistronic mRNA

Irll

Repressor-Inducer complex

Repressor + Inducer

The repressor cannot bind to 0 because it is bound to the inducer and so structural genes are transcribed.

Figure 11-6-1. Negative control of the lac operon.

152

iiieClical

Regulation of Gene Expression

PACKAGING OF EUKARYOTIC DNA The DNA molecule in eukaryotes is associated with histones. The DNA-histone complex is known as chromatin and is organized into discrete units of higher structure in the eukaryotic chromosome. A. Eukaryotic chromosomes contain two specialized structures that are required for replication and segregation during mitosis. 1. Telomeres are G-rich, block-like structures located at the end of eukaryotic chromosome

arms. They are essential for the completion of DNA replication. 2. Centromeres are the chromosomal sites where the two daughter chromatids attach during mitosis. Centromeres are essential for aligning the DNA molecule on the mitotic spindle during mitosis. They are required for segregation and stability of the mitotic apparatus. The centromeres attach to the spindle fibers and are responsible for movement during both mitosis and meiosis.

Note Telomeres protect the ends of the linear DNA molecules from being degraded by exonucleases and are needed to complete replication.

B. Chromatin composition. Chromatin is composed oflinear double-stranded DNA and five types of histones (HI, H2A, H2B, H3, and H4). Histones have a high density of positive charge that interacts with negatively charged phosphate groups of DNA. Histones contain a large number of side chains that are modified by methylation, acetylation, or phosphorylation after the protein is translated. C. Chromatin organization. There are various levels of organization that chromatin undergoes

during packaging.

Note

1. Nudeosomes. Electron micrographs of chromosomes show long fibers that are 10 nm

Highly transcribed regions of DNA contain fewer nucleosomes than less transcribed regions.

in diameter and contain discrete bead-like structures equally spaced along the lO-nm fiber. The bead-like particles are DNA-histone complexes known as nucleosomes, which are connected to one another by linear regions of DNA. The core of the nucleosome contains approximately 140 base pairs of DNA wrapped twice around eight histones (two each of H2A, H2B, H3, and H4). Approximately 160 base pairs of linear DNA connect adjacent nucleosomes. Nucleosomes constitute the first level of packaging of DNA. 2. Solenoid fibers. Histone HI binds to the linear DNA between nucleosomes. Chromatin that contains HI forms solenoid-like fibers, with six nucleosomes per turn of the helix. The solenoid fibers constitute the second level of packaging of eukaryotic DNA. A still higher level of organization occurs when the solenoid fibers are organized into loops of greater than 20 kilobases. D. Chromatin and gene regulation. Chromatin can be organized into two classes based on their genetic activity. 1. Heterochromatin contains DNA that is genetically inactive and highly condensed. The

regions of DNA that condense in early prophase are known as heterochromatin. Most of these regions are located near the centromere, and, although they are not transcribed, they playa role in pairing and segregation of homologous chromosomes. Heterochromatin is found in the 30-nm solenoid fibers folded into looped domains. 2. Euchromatin makes up most of the genome, and about 10% of euchromatin contains genes that are actively transcribed. This active euchromatin is made up of lO-nm fibers of nucleosomes connected to one another by linear DNA.

I KAPLAlf medlea

153

Molecular Biology

In a Nutshell

REGULATION OF EUKARYOTIC GENE EXPRESSION

The major eukaryotic gene regulation mechanisms occur in the nucleus, including:

Eukaryotic gene expression is much more complex than that of prokaryotes and can occur at several levels. Although regulation of mRNA transcription initiation appears to be the most common type of gene regulation in eukaryotes, several other levels of control also exist. Alternative processing of mRNA primary transcripts into different mature mRNAs provides an important mechanism for cell-type specific gene expression. Within the cytoplasm, both the stability of the mRNA and the initiation of translation can be regulated. Other examples are also mentioned.

• Regulation of transcription initiation • Alternative mRNA processing • DNA modifications or rearrangements

Note Regulatory proteins (transcription factors) exert their effects in trans, i.e., they can diffuse through the nucleoplasm to affect any and all genes that contain their recognition sequences (enhancers, silencers).

A. Initiation of eukaryotic mRNA transcription 1. Description. As with prokaryotes, the rate of mRNA transcription is regulated by the interaction of regulatory proteins that bind to DNA sequences near the promoter and modulate the rate at which the initiation complex can form on the promoter. In eukaryotes, general transcription factors (including TFIID and TFIIB) must bind to the promoter to allow RNA polymerase II to bind and form the initiation complex. Specific transcription factors bind to regulatory DNA sequences, often called enhancer or silencer sequences, and modulate the formation of the initiation complex, thus regulating the rate of transcription. 2. Transcription factors. Transcription factors are proteins that bind to regulatory DNA sequences and to other transcription factors or to RNA polymerase II in order to regulate initiation of transcription. Typically, transcription factors contain at least two recognizable domains, a DNA-binding domain and an activation domain. a. The DNA-binding domain binds to a specific sequence in the promoter, enhancer, or silencer. Several types of DNA-binding domain motifs have been characterized and have been used to define certain families of transcription factors. Families based on DNA-binding domains include "zinc fingers" (seen on steroid hormone receptors), and "homeodomain" (seen on some transcription factors involved in development). b. The activation domain allows the transcription factor to bind to another transcription factor or to RNA polymerase II to stabilize the formation of the initiation complex. These are less well characterized. c. Some transcription factors must dimerize in order to form functional DNA-binding domains and functional activation domains. These include the "helix-loop-helix" family and "leucine zipper" family. Within each family, partners may form heterodimers or homodimers, creating dimeric combinations with a variety of DNA sequence specificity and a variety of activating or inhibiting abilities. Because different cells can express different partners within each family, this greatly increases the possible range of gene regulation. The genes that code for fos and jun, two transcription factors important in regulating cell proliferation, are members of the "leucine zipper" family. When expressed abnormally (or overexpressed), these two genes can contribute to transformation of the cell; therefore, fos and jun are both proto-oncogenes.

Note DNA sequences (promoters, enhancers, silencers) exert their effects in cis, i.e., they can affect only the gene to which they are attached.

3. Enhancer and silencer sequences. These are DNA sequences to which the specific transcription factors bind to regulate transcription. Enhancer sequences contain binding sites for specific transcription factors that increase transcription. Enhancers are functional both upstream and downstream of the promoter and in either orientation. Silencers contain binding sites for specific transcription factors that inhibit transcription. B. Regulation by specific transcription factors. Each gene contains a variety of enhancer and

silencer sequences in its regulatory region. Each enhancer or silencer sequence may be recognized by a different combination of specific transcription factors. The exact combination

154

iile&ical

Regulation of Gene Expression

of specific transcription factors available (and active) in a particular cell at a particular time determines which genes will be transcribed at what rates. 1. Regulating transcription factor function. Because specific transcription factors are pro-

teins, their expression can be cell-type specific. Additionally, hormones can regulate the activity of specific transcription factors. 2. Examples

Note

a. Thyroid receptor (T3R) is localized in the nucleus. Besides its DNA-binding and activation domains, it contains a thyroid-binding domain at its C-terminus. This receptor is found in many tissues, but it is expressed in very high levels in the anterior pituitary, where it increases transcription of the growth hormone gene. The effects of thyroid hormone on the anterior pituitary are shown in Figure 11-6-2. Thyroid hormone activates growth hormone transcription. T3 (or T 4) enters the cell and moves into the nucleus, where it binds to its receptor. The T3R complex undergoes a conformational change that allows it to bind to specific enhancer sequences located upstream from the growth hormone gene. In the nucleus, the rate of transcription is regulated by RNA polymerase II, resulting in increased mRNA for growth hormone. Translation of mRNA in the cytosol results in increased growth hormone synthesis. The steroid hormones exert their effects by a similar mechanism. The DNA-binding domain of thyroid hormones and steroid hormones contains "zinc fingers;' a structural motif that binds to DNA.

_....,t""-_AAAn HnRNA

Steroid and thyroid hormone receptors are hormoneactivated specific transcription factors that allow these hormones to change gene expression.

•

Processing

1

Growth 5'Cap - - - AAAn hormone mRNA Transport to cytoplasm

Cytoplasm

Growth 5'Cap --:---- AAAn hormone mRNA Translation •

•

!

Growth hormones

Secreted growth hormone

Figure 11-6-2. Activation of growth hormone transcription by thyroid hormone. KAPLA~. I meulca

155

Molecular Biology

b. CREB is a specific transcription factor found in many cell types that is active only when cAMP levels rise in the cell. The cAMP-dependent protein kinase phosphorylates CREB, enabling it to bind to its enhancer sequence in various genes and activate their transcription. In hepatocytes, CREB affects the transcription of some of the key enzymes in gluconeogenesis. c. AP-l is a family of transcription factors. Members include the fos-jun complex consisting of one molecule of fos bound to one molecule of jun. Fos and jun are proteins encoded by proto-oncogenes; mutations in these genes have been associated with a wide variety of tumors. A range of cellular stimuli, including growth factors, phorbol esters, and neurotransmitters, regulate the activity of the fos-jun complex. The dimerization of these proteins is highly specific and occurs through a domain on the proteins known as the "leucine zipper." This domain consists of repeating leucine residues occurring at every seven amino acids in a region of a-helical structure.

Note

C. Alternative mRNA processing

Alternative mRNA processing allows different proteins to be synthesized from the same gene and pre-mRNA.

1. mRNA processing. Eukaryotic mRNA primary transcripts are extensively processed before the mature mRNA is transported into the cytoplasm. On each mRNA the 5' end is capped, the 3' end is cleaved to allow addition of polyadenosine, and the introns are spliced out. The presence of the sequence AAUAAA in the mRNA signals a 3' cleavage/polyadenosine addition site, and splice donor (GU) and splice acceptor (AG) sequences define splicing patterns.

2. Cell-type specific gene regulation. Alternative use of such signals allows the formation of different mRNAs from transcripts of the same gene. The alternative mRNAs are translated into different proteins. This is often seen in the production of different proteins in different cell types produced from the same gene. 3. Example. The calcitonin gene contains exons for both calcitonin, a hormone that regulates blood calcium and phosphate levels, and CGRP, a protein that regulates neural receptor expression. In thyroid C cells (stromal cells located between the follicles in the thyroid gland), this primary gene transcript is cleaved in front of the CGRP exon and spliced to form an mRNA containing the calcitonin exon. In neural cells, a cleavage signal after the CGRP exon is used and the calcitonin exon is spliced out, leaving an mRNA containing the CGRP exon. D. Regulation of initiation of translation

Note Regulation of gene expression can also occur in the cytoplasm via: • Regulation of translation initiation • Regulation of mRNA stability

1. Initiation of eukaryotic translation requires the small ribosomal subunit, charged initiator tRNA (tRNAiMet), GTP, and several initiation factors. The two important initiation factors are eIF-2 and eIF-4.

a. eIF-2, GTP, and tRNAiMet form a complex and bind to the small (40S) ribosomal subunit, forming a pre-initiation complex. b. The cap-binding protein subunit of eIF-4 recognizes the 7-methyl-guanosine "cap" at the 5' end of eukaryotic mRNA and allows the pre-initiation complex to bind. c. The pre-initiation complex scans down the mRNA until it encounters the first AUG in the appropriate sequence; GTP is hydrolyzed to GTP, the large ribosomal subunit joins the complex, and the initiation factors leave. Translation continues much as in prokaryotes. 2. Some viruses, such as polio virus, ensure preferential translation of viral mRNAs by cleaving the eIF-4 cap-binding subunit but providing a cap-independent alternative recognition site for the pre-initiation complex (called a "ribosomal landing pad").

156

meClical

Regulation of Gene Expression

3. Initiation of translation can also be regulated by proteins that bind to sequences on the mRNA. a. Ferritin is a cytoplasmic Fe2+-binding protein expressed in hepatocytes only when cytoplasmic Fe2+ levels are high. Ferritin sequesters the Fe2+, preventing it from being toxic to the cell. b. Ferritin mRNA contains three "IRE" sequences between the 5' cap and the first AUG. The hepatocyte also makes an IRE-binding protein that binds to the IRE sequence and prevents the pre-initiation complex from scanning from the cap to the AUG; this inhibits the translation of ferritin.

Note Certain sequences found near the 3' end of some mRNAs decrease mRNA stability. Regulatory proteins can reversibly mask these destabilizing sequences, increasing protein synthesis.

c. As Fe z+ levels rise in the cell, iron binds the IRE-binding protein and changes its con-

formation. The Fe2+ -IRE-binding protein complex cannot bind to the IRE, so ferritin is translated. E. The level of packing of the DNA can also affect transcription. 1. Condensed DNA is not accessible to transcription factors or RNA polymerases and so is not transcribed. The level of packing of the DNA can be regulated by the frequency of methylation of cytosines found in the sequence 5' CG 3'. Such methylation inhibits the binding of specific transcription factors to their enhancer sequences and promotes higher levels of packing of the DNA. Different methylation patterns are found in different cells; the regulation of these patterns is not clear. 2. Transient modification of histones, such as acetylation or binding of ubiquitin (which may target them for degradation by proteases), is associated with unpackaging the DNA before transcription. F. Regulation of 5S rRNA gene (Xenopus). Regulation of rRNA transcription is much less common than the regulation of mRNA transcription but does occur in Xenopus oocytes. The

5S rRNA genes are tandemly repeated and organized in clusters. These genes have an internal promoter and are transcribed by RNA polymerase III. 1. Xenopus has two types of gene clusters for 5S rRNA: One gene cluster code for the somat-

ic-type 5S rRNA and the other cluster codes for the oocyte 5S rRNA. Both types of genes are present in all cells. Although the gene types differ from each other at only six positions in the 120 base-pair coding region, they are transcribed at very different rates. The somatic gene population constitutes 2% of 5S rRNA genes and accounts for more than 95% of the 5S rRNA synthesized in somatic cells. In the oocyte, almost all of the 5S rRNA is transcribed from the oocyte gene cluster. 2. The differential transcription of the two 5S rRNA genes can be accounted for by different interactions between the promoter and a transcription factor, TFIIIA. TFIIIA binds to the internal promoter, signaling RNA polymerase to initiate transcription. TFIIIA has a greater affinity for somatic 5S rRNA genes than for the oocyte 5S rRNA genes. In fact, the binding of TFIIIA to oocyte 5S rRNA genes is prevented in regions of the chromosome that contain histone HI. Early in oogenesis, there are very high levels of TFIIIA (101Z molecules/oocyte), and under these conditions all internal promoters of the 5S rRNA genes (both somatic and oocyte) bind to TFIIIA, activating transcription. As the 5S rRNA gene is expressed and the level of 5S rRNA increases in the nucleus, the 5S rRNA begins to bind TFIIIA and deplete the transcription factor, thus decreasing transcription. Therefore, the 5S rRNA regulates its own synthesis.

meClical

157

Genetic Engineering

Genetic engineering, also known as recombinant DNA technology, provides a means of analyzing and altering genes and proteins. Additionally, this technology can provide a source of specific proteins in almost unlimited quantities and has many applications in clinical medicine. Genetic engineering depends on having an array of tools for analyzing genes and proteins, including enzymes that can cut, join, and replicate genes. This chapter reviews the procedures and related techniques of genetic engineering and indicates how these procedures can be applied to clinical medicine.

TOOLS USED IN RECOMBINANT DNA TECHNOLOGY A. Enzymes that modify nucleic acids. Many enzymes that alter the structure of nucleic acids are used in genetic engineering. 1. Nucleases constitute a family of enzymes that hydrolyze phospho diester bonds. They are classified either as ribonucleases or deoxyribonucleases, depending on whether they hydrolyze RNA or DNA. Both ribonucleases and deoxyribonucleases can be further classified as endonucleases (if they cleave internal phosphodiester bonds) or as exonucleases (if they cleave terminal phosphodiester bonds, either at the 5' or 3' end of the chain). 2. Restriction endonucleases are enzymes that recognize specific double-stranded sequences in DNA and cleave the DNA at or near the recognition or "restriction" site. These enzymes are powerful tools in recombinant DNA technology, allowing specific genes to be excised from the genome. Many restriction endonucleases have been isolated from bacterial sources. Isochizomers are restriction endonucleases from two different sources that recognize the same DNA sequence. Most restriction nucleases recognize sites consisting of four to eight base pairs. The sequences in the two strands of the recognition site are palindromes, having the same 5' to 3' sequence in both strands. Two examples of restriction endonucleases are shown in Figure 11-7-1.

!

t

5'-GGCG-3' 3'-CCGG-5'

t

!

feoRI

5'-G + 3'-CTTAA-5'

!

Haelll

5'-MTTC-3' G-5'

The location of restriction sites along a DNA helix depends upon its nucleotide sequence: • Use of a restriction enzyme on mUltiple samples of the same sequence will always generate the same pattern of fragment sizes.

!

5'-GMTTC-3' 3'-CTTMG-5'

Note

5'-GG-3' + 5'-CC-3' 3'-CC-5' 3'-GG-5'

Figure 11-7-1. Examples of restriction endonucleases.

• A mutation or difference in the DNA sequence can change the presence or absence of a restriction site, thereby changing the sizes of the resulting fragments. KAPLA~.

meulCa

I

159

Molecular Biology

a. EcoRI recognizes a specific six-base-pair sequence in double-stranded DNA and cleaves one site in each strand, as indicated by the arrows in Figure II-7-1, producing staggered cuts that leave short single-stranded tails at the two ends of each fragment. Ends of this type are known as sticky ends because each tail can form complementary base pairs with the tail of any other fragment produced by EeoR!. b. HaeIII recognizes a specific four-base-pair sequence in double-stranded DNA and cleaves both strands at positions opposite one another, producing blunt ends with no unpaired tails. 3. Reverse transcriptase is an enzyme found in retroviruses whose genome is composed of RNA rather than DNA. This enzyme is an RNA-dependent DNA polymerase that can synthesize a single strand of DNA using a complementary RNA strand as a template. It is used in recombinant DNA technology to construct synthetic DNA molecules known as cDNA (complementary DNA) by copying the corresponding mRNA. 4. DNA and RNA polymerases are used to synthesize a particular DNA or RNA, respectively. Unlike RNA polymerase, DNA polymerase requires a primer (a 3' -OH group at the end of an oligonucleotide chain) to initiate synthesis. 5. DNA ligase forms a phosphodiester bond between two DNA fragments. This is an essential step in creating recombinant DNA molecules. 6. SI nuclease acts exclusively on single-stranded polynucleotides or single-stranded regions of double-stranded polynucleotides. Double-stranded regions are resistant to its action. 7. Terminal deoxynucleotidyl transferase catalyzes the addition of deoxynucleotides to the 3'-OH end of the DNA molecule. It does not require a template (Figure II-7-2). Most restriction endonucleases produce complementary "sticky" ends. However, DNA fragments that have "blunt" ends can be joined by using terminal deoxynucleotide transferase to add complementary homopolymer tails. Thus, two molecules can be joined if poly( dA) tails are put on one molecule and poly( dT) tails are put on a second molecule.

5 ' - - - - - - - - 3' 3' 5'

tI

Terminal deoxynucleotidyl transferase dTTPP

5'-------TTTT3' 3'TTTT 5'

In a Nutshell

Figure 11-7-2. Terminal deoxynucleotide transferase reaction.

To radioactively label the 5' end of a nucleic acid:

8. Alkaline phosphatase removes the S'-phosphate group from either single-stranded or double-stranded DNA or RNA to give a 5' -OH group.

• Remove existing 5' phosphate with alkaline phosphatase.

9. Polynucleotide kinase catalyzes the transfer of the terminal phosphate group of ATP to a 5' -OH group in either DNA or RNA. The physiologic function of this enzyme is unknown. However, it is useful in radiolabeling nucleic acids, a procedure used in determining the base sequence of polynucleotides. Because naturally occurring nucleic acids usually contain 5' phosphate rather than 5' OH, labeling with polynucleotides can be accomplished only after the S'-phosphate group is removed with alkaline phosphatase.

• Use radioactive ATP and poly-nucleotide kinase to replace it with radioactive phosphate. 160

meClical

10. Eco RI methylase catalyzes the methylation of the adenine (A) residue in the EeoR! recognition sequence, thereby protecting the DNA sequence from cleavage by BeoR!.

Genetic Engineering

B. Oligonucleotides. Short DNA molecules, usually 16 to 40 base pairs, can be chemically synthesized from nucleotide building blocks. Two specific uses of these oligonucleotides are discussed below. 1. Hybridization screening probes. Radiolabeled oligonucleotides can be used in

hybridization experiments to screen cells for specific DNA clones. Oligonucleotides with base sequences complementary to different parts of the gene of interest are synthesized. Colonies of cells that hybridize with two different oligonucleotide probes are considered to be strong candidates for containing the desired gene. 2. Detection of specific mutations in genes. The knowledge of either the protein sequence or the DNA sequence of a particular gene allows synthesis of specific oligonucleotides that will hybridize with specific regions in the gene. A mismatch of a single base can result in the formation of unstable hybrids. The shorter the oligonucleotide, the more sensitive the probe to base changes in the gene.

TECHNIQUES AND APPLICATIONS IN MOLECULAR BIOLOGY A. Gel electrophoresis is very useful in recombinant DNA technology for separating fragments resulting from cleaving DNA with restriction endonucleases. Electrophoresis permits the separation of DNA (or RNA) molecules by measuring the rate at which they move in an electric field. DNA molecules have a negative charge because of the phosphate groups. In an electric field, these molecules move toward the positive pole at a rate that is dependent upon their size. Smaller DNA molecules move more rapidly than do larger molecules. 1. Types of gels. Two types of gels are typically used for separating DNA.

a. Polyacrylamide gels are used to separate small single-stranded DNA fragments ranging from about 10 to 500 nucleotides.

In a Nutshell Probes (in a single-stranded form) will hybridize with any homologous sequence in the (denatured) genomic DNA. • Long probes can be made from known genes or cDNAs. Minor sequence differences, such as allelic differences, or sometimes even the difference between a mouse and a human version of a gene, will not prevent hybrid formation. • Short oligonucleotide probes are more sensitive to differences in nucleotide sequence.

b. Agarose gels are used to separate double-stranded DNA fragments ranging from approximately 300 to 20,000 base pairs in length. 2. Applications a. Separation of DNA on the basis of size. In the experiment shown in Figure II -7 -3, a 5-kilobase (kb) DNA molecule was digested with two different restriction endonucleases (RE). Digestion with RE-A results in two fragments of the same size (2.5 kb), whereas digestion with RE-B results in three fragments of different sizes (2.5 kb, 1.5 kb, and 1 kb). After digestion, the fragments are separated by electrophoresis on an agarose gel. Molecular weight markers (MW) of different sizes ranging from 0.7 kb to 5 kb and a sample of undigested DNA (5 kb) are also submitted to electrophoresis. The position of DNA fragments in the gel is detected by staining the gel with ethidium bromide, a dye that binds to DNA and can be visualized as a fluorescent band when the gel is illuminated with UV light. Note that the pattern of electrophoresis of the DNA fragments differs between RE-A and RE-B. With RE-A, only one band is detected because the enzyme cuts the 5-kb DNA into two pieces of equal size.

meclical

161

Molecular Biology

7

(5 kb)

Sample DNA

Res'dct;on Enzyme

,,\es.,;cllon Enzyme B

~(2.5kb)

~(2.5kb)

~(2.5kb)

~

(1.5kb) (1 kb)

2 DNA fragments

3 DNA fragments

MW UC RE-A RE-B kb 5

- pole

2.5 2

Electrical field

0.7

+ pole

UC = uncut DNA

Figure 11-7-3. Restriction enzyme digestion and agarose gel electrophoresis.

b. Separation of DNA molecules on the basis of shape. Gel electrophoresis can distinguish between different configurations of a DNA molecule. Supercoiled DNA is more compact and migrates faster than open circular DNA and linear DNA of the same mass.

B. Hybridization. In molecular biology, hybridization is defined as the complementary pairing of an RNA and a DNA strand, or of two different DNA strands, to form a stable doublestranded molecule. 1. Definitions and principles of hybridization are illustrated in Figure II -7 -4. The breaking of hydrogen bonds between two complementary nucleotide strands is defined as denaturation. Conditions promoting denaturation are high temperatures or treatment of DNA with high salt, alkali, urea, or formamide. The temperature at which the double-stranded DNA molecule denatures into single-stranded DNA is defined as the melting temperature (Tm). In addition to solution conditions of ionic strength, pH, etc., the Tm is dependent upon the relative number of A-T (or A-U) and G-C base pairs in a given DNA or DNA-RNA hybrid. Because G-C base pairs have three hydrogen bonds, they are more stable than the A-T and A-U base pairs with only two hydrogen bonds. Thus, the higher the number of G-C base pairs in a given DNA molecule, the higher the Tm. The Tmis also dependent upon the number of mismatched base pairs: The greater the number of mismatches, the less stable the hybrid and the lower the Tm. Renaturation or annealing between two separated complementary strands occurs upon the cooling of the heatdenatured DNA or by the removal of agents that promote denaturation.

162

meclical ------------------

Genetic Engineering

~ ~ ~

Double-stranded DNA

~aturat;o" ~ ~

~ ~ ~ ~

Initial base pairing

Single-stranded DNA

Figure 11-7-4. Denaturation and renaturation.

2. Applications. The principles of hybridization are used in numerous techniques, including Southern blotting, Northern blotting, and in situ hybridization. a. Southern blotting is used to detect DNA fragments in an agarose gel by hybridization with a specific radiolabeled oligonucleotide probe. The technique is illustrated in Figure II-7-5 and involves the following steps:

A.

B.

* Isolate genomic DNA

* Restriction

C.

* Agarose gel

electrophoresis

digestion

kb MW 27

Long DNA fragments

DNA fragments 0.5 Genomic DNA

Short DNA fragments

DNA fragments Agarose gel

D.

E.

* Denature gel in alkali * Blot-transfer onto

* Hybridize with

F.

* Autoradiography

radioactive probe * Wash blot

nitrocellulose * Bake nitrocellulose

Singlestranded -+-----+- DNA fragments ...

Nitrocellulose filter

Washed nitrocellulose filter

Figure 11-7-5. Southern blotting.

-

Photographic film

I KAPLAlf me d lea

163

Molecular Biology

Note A Southern blot of two samples of DNA exposed to the same restriction enzyme and probe may reveal different fragments (in size and/or number). Such a difference is due to a sequence difference that has affected the position of a restriction site and is called a restriction fragment length polymorphism (RFLP).

In a Nutshell Northern blots are often used to detect which cell types express mRNA from a particular gene.

Bridge to Physiology In situ hybridization can detect the chromosomal location of a gene, or it can detect the cells in an intact tissue that express that gene.

164

meClical

(1) Cleavage of genomic DNA by treatment with a specific endonuclease. The fragments are separated according to size by agarose gel electrophoresis. Because the sample used in the figure was genomic DNA, thousands of fragments were produced. When submitted to electrophoresis, the digested DNA appears as a continuous smear of fragments from the top to the bottom of the gel. (2) Denaturation of the DNA fragments is achieved by treatment with alkali. The gel is then placed on a buffer-saturated filter paper, covered with a sheet of nitrocellulose, and overlaid with dry filter paper. (3) Immobilization on nitrocellulose. The filter paper draws buffer out of the gel. The buffer carries single-stranded DNA with it, and when the DNA comes in contact with the nitrocellulose filter, it binds to the nitrocellulose and is immobilized. (4) Detection of a specific DNA fragment by hybridization. The filter is dried and incubated with a solution containing a radioactive DNA probe that is complementary to the sequence of the DNA of interest. The radioactive probe and the DNA on the filter are allowed to renature under conditions that promote hybridization. Under these conditions, only the DNA that is complementary to the radioactive oligonucleotide probe will be recognized by the probe. The filter is washed and dried, and the region of hybridization is visualized by autoradiography. Southern blotting is a very sensitive method and can detect DNA sequences present in a single copy per genome. b. Northern blotting is a technique similar to Southern blotting, but it is used to determine the size and abundance of mRNA for a specific gene in a sample of total cellular RNA. Figure II -7 -6 uses the total RNA from liver to test for the mRNA expression for albumin. The total RNA includes rRNA, tRNA, and mRNA. Because mRNA contains poly-A tails, it can be isolated from the other types of RNA by hybridizing it to an oligo d(T) column. The mRNA is removed from the column by heat denaturation and is run on a denaturing agarose gel containing formamide. The mRNA is then transferred onto nitrocellulose filter paper. The filter is baked and hybridized with a singlestranded radioactive probe. The filter is then washed, and the position of the mRNA for albumin is identified by autoradiography. By use of marker RNA molecules of known size, the size of the specific mRNA can be determined. c. In situ hybridization can be used to detect the distribution of a specific mRNA in tissue sections. The radioactive nucleic acid probe is incubated directly with the tissue. After hybridization, it is possible to determine exactly which population of cells in the tissue expresses the specific mRNA of interest. This technique can also be used to detect the position of a specific DNA sequence on a chromosome. C. Cloning vectors. Cloning vectors are DNA molecules that have inserts of foreign DNA incorporated into their genome. There are two important features of cloning vectors: They possess sites at which the foreign DNA can be inserted without disrupting its function, and they replicate in the host cell independently of the host cell chromosome. Two of the most commonly used cloning vectors are plasmids, which are routinely used for cloning short DNA fragments (fewer than 5-10 kilobases), and bacteriophages, which are used for cloning larger DNA fragments (up to 20 kilobases).

Genetic Engineering

A.

* Prepare liver total RNA

B.

* Isolate poly-A+ RNA

using oligo d(T) column • mRNA with poly· (A) tail

IIIIIII

C.

* Agarose-formaldehyde

gel electrophoresis rRNA poly A+

An

IIIIIII An • rRNA (288, 188)

I II III

288

-

188

-

• tRNA

+

D.

+ Agarose-formaldehyde gel

rRNA poly A+

* Blot-transfer onto

nitrocellulose * Bake nitrocellulose * Hybridize with

radioactive probe (albumin cDNA or oligonucleotide) * Wash blot * Autoradiography

288 -

c::=I

188 -

c::=I

-

....~- Albumin mRNA

Photographic film Figure 11-7-6. Northern blotting.

1. Plasmids

a. Plasmids are small, circular, double-stranded DNA molecules (2-10 kilobases) that replicate in bacteria. Multiple copies of plasmid DNA can be found in bacterial cells. The important features of a plasmid cloning vector are shown in Figure 11-7-7. They contain an origin of replication (ori) that permits autonomous replication, one or more genes that confer specific antibiotic resistance, and sites for restriction endonucleases that can be used for inserting specific DNA fragments. b. Polylinker DNA, containing many overlapping sites for restriction endonucleases, can also be inserted into the plasmid (Figure 11-7-8). The advantage of adding polylinker DNA is that it can provide unique sites that can be used for inserting other foreign pieces of DNA. In principle, there is no limit on the size of foreign DNA that can be inserted into the plasmid, but larger molecules do not multiply as well as smaller ones.

KAPLAlf I meillea

165

Molecular Biology

Pvull

~ .....

Q)

I:: Q)

III

0>

(')

'S.

Q)

EcoRI

I::

ell

U5

Pvull

s·

u

'iii ~

§

'u

!

!

'6.

(!l

CD (/) iii'

Neal

iii ~

(') (!l

E

«

EcoRI

Bgnl

San Figure 11-7-7. Important features of a plasmid cloning vector.

BamHI

I

Hind/II

I

I

I

S'GGATCCCGGGAAGCTI3' 3'CCTAGGGCCCAAGCTIS'

I

Smal

I

Figure 11-7-8. Polylinker DNA.

Note Transformation has two definitions. It can refer to the introduction of exogenous DNA into a bacterium, or it can refer to the process by which an animal cell becomes a cancer cell.

166

meCfical

c. The cloning of a DNA fragment into a plasmid involves cleaving the plasmid with a restriction endonuclease (such as EeoR!) that results in sticky ends. If the DNA fragment to be cloned also has EeoR! sticky ends, it can be joined to the plasmid by DNA ligase.

d. The plasmid, containing foreign DNA, is introduced into the host bacterium by a process called transformation. Bacterial membranes are made permeable to plasmids by treatment with CaCho The efficiency of transformation is low (0.001 %). Transformed bacteria can be selected by growing them on a medium containing antibiotic. Only the bacteria that contain plasmids with the antibiotic resistance gene will grow under these conditions.

--------~--~----

Genetic Engineering

2. Bacteriophages (phages) a. Phages are bacterial viruses with a relatively large and linear DNA molecule (40-50 kilobases) enclosed in a shell of protein. The phage genome codes for the coat proteins and a number of other specific viral proteins. The structure of the protein coat imposes a physical limitation on the size of the DNA that can be enclosed in the virus (about 50 kilobases). Consequently, there is a limit to the size of foreign DNA that can be inserted into this type of cloning vector. Genetic engineering has tried to overcome this problem by replacing parts of the viral DNA that do not contain essential viral proteins with foreign DNA. b. Lambda phages that replicate in E. coli are most commonly used as cloning vectors. They have a genome of approximately 50 kilobases. However, they can be reduced to 30 kilobases by elimination of nonessential genes, thus providing room for insertion of up to 20 kilobases of foreign DNA. If the recombinant DNA is too small, the phage is typically not viable. Infection of E. coli is initiated by adsorption of lambda onto the surface of E. coli. The phage DNA enters the bacterium and converts it into a "phagesynthesizing factory" that eventually lyses and releases approximately 106 phages that can infect other bacteria. Not all phages are lytic; however, only lytic phages are used as cloning vectors.

Note Plasmid or bacteriophage vectors can be used to express human genes in bacteria; this is a source of human insulin used to treat diabetics.

D. Host cell for cloning vectors. Cloning vectors are usually transformed into a prokaryotic host cell, with E. coli usually being the host of choice. E. coli grows quickly, with a dividing time of approximately 20 minutes. This makes E. coli an ideal choice for a cloning vector. The growth curve of E. coli has three distinct phases. In the lag phase, the bacteria are adjusting to their environment and are activating their replication machinery. In the log phase, exponential growth is occurring. With each cell division per generation, the number of bacteria is doubled. Transformations are performed at this stage of growth. In the stationary phase, the nutrients in the medium become scarce, and the replication machinery slows down.

CONSTRUCTION OF DNA LIBRARIES The purpose of cloning is to isolate a particular gene in large quantities. The most common approach to this problem is to construct a set of recombinant DNA clones from a source that contains the gene or DNA sequence of interest. A collection of clones containing all of the sequences found in the original source is known as a DNA library. There are two approaches to constructing a library. One approach starts with the total population of mRNA of a given cell type, synthesizes complementary DNA (cDNA) copies of the mRNA, and inserts the cDNA into cloning vectors, thereby producing a cDNA library. The other approach is to start with digested genomic DNA and insert the fragments into bacteriophage cloning vectors, producing a genomic library. A. cDNA libraries. A cDNA library is a collection of recombinant vectors that together contain a complete copy of all of the messenger RNAs in a particular cell. There are five steps involved in constructing a cDNA library: isolation of the total mRNA population from a cell, synthesis of cDNA using mRNA as a template, introducing the cDNA library into a cloning vector, transforming the cDNA library into E. coli, and screening the cDNA library for specific cDNA clones. The chief advantage to a cDNA library versus a genomic library is that the cDNA clones do not have introns or other noncoding sequences found in genomic DNA.

In a Nutshell A cDNA library represents the genes expressed in a particular cell type; a genomic library contains all of the genes present in the DNA.

1. Preparation of mRNA. The cell or tissue type used as a source of mRNA must contain the

specific mRNA of interest. For example, reticulocytes are a good source for mRNA that codes for (1,- and l3-globin proteins. Once a cell type has been identified, the total RNA population (rRNA, mRNA, and tRNA) is isolated from the cells. mRNA usually constitutes <5% of the total RNA population. Because most mRNA molecules contain poly-A+ KAPLAN· . I medlea 167

Molecular Biology

tails, the total RNA can be passed through a column coated with oligo d(T). The poly d(T) column will retain the mRNA on the column while allowing the other RNA populations to pass through the column. The mRNA can then be removed from the column using a low-salt solution. 2. Synthesis of cDNA from mRNA. The ability to make a DNA copy from an RNA template relies on reverse transcriptase and a primer, oligo d(T), which anneals with the poly-A+ tail of mRNA (Figure II-7-9). In the presence of all four deoxyribonucleotides, reverse transcriptase synthesizes a strand of DNA that is complementary to the mRNA. The resulting RNA-DNA heteroduplex is treated with NaOH to hydrolyze the RNA strand. The DNA strand is then used as a template for DNA polymerase I to synthesize doublestranded DNA. The primer for synthesis of the second strand is provided by reverse transcriptase, which makes a hairpin loop ending with a 3' -OH group. The double-stranded DNA is treated with Sl nuclease, which removes the hairpin loop and single-stranded staggered ends, to create a blunt-ended cDNA duplex. 3. Introduction of the cDNA library into a cloning vector. In order to form recombinant DNA between the cDNA and the plasmid (or phage) DNA, both must have "compatible" restriction endonuclease ends. For example, if BeaRI is used to create sticky ends, the cDNA must have BeaRI sites only at its ends. As shown in Figure II-7-9, internal BeaR! sites are methylated, making them insensitive to cleavage. To ensure that BeaR! sites are present at the ends of the cDNA, linker DNA containing restriction endonuclease sites are added to each end. Both the plasmid cloning vector and cDNA are treated with BeaR! to generate "compatible" sticky ends that, when mixed, will anneal with one another and can be covalently linked by the action of DNA ligase to generate the cDNA library. 4. Transforming the cDNA library into B. coli. The cDNA library is introduced into competent B. coli that have been made partially permeable. These bacteria are grown in a medium containing an antibiotic. Only those bacteria with plasmids will grow in the presence of antibiotics. 5. Screening the cDNA library for specific cDNA clones. Frequently, the most difficult part of gene cloning is the identification of the bacterial colonies that contain a specific DNA fragment of interest. Two approaches are used to screen for specific clones. In one method, in situ hybridization with a DNA probe identifies bacterial colonies containing a particular cDNA. In the second method, antibodies can be used to identify the ability of a clone to express a specific protein.

Note In situ hybridization identifies colonies containing DNA fragments complementary to the probe; these are not necessarily complete genes.

168

meclical

a. In situ colony hybridization screening is illustrated in Figure II-7-10. The bacterial colonies to be screened are blotted with a piece of nitrocellulose filter paper. Some of the bacteria in each colony will adhere to the filter, producing a replica. The petri dish still contains the original colonies and serves as a master reference plate. The filter is treated with alkali to lyse the cells and to separate the two strands of DNA. After neutralization and treatment with protease to remove protein, the filter is incubated with a radiolabeled DNA probe containing part of the sequence of the gene of interest. The colony that gives a positive autoradiographic signal can be picked from the master reference plate. If some sequence information is available on the protein encoded by the DNA of interest, it is possible to deduce a sequence that can be used for constructing an oligonucleotide probe. Due to redundancy in the third position of the genetic code, there are a number of possible mRNA sequences that code for the same

----------

Genetic Engineering

3 1AAAAA _ _ _ _ _ _ _ _ _ _ 51

!

3 0H 1

51 TTTTT 3 1AAAM

~~ 51

Hairpin loop first strand of cDNA

Treatment with NaOH to remove mRNA template

! 5 1 TTTTT

mRNA

Oligo d(T) primer Reverse transcriptase +4 dNTPs + Mg2+

3 0H 1

tI

~

First strand of cDNA synthesized

DNA polymerase I

+4 dNTPs + Mg2+ Second strand of cDNA synthesized

5 1 TTTTT

~ S1 nuclease Blunt-ended cDNA

!

~._.CORI Methylase

Il2ill EcoRI site

CH3

CH3

I

I•

t

I

Methylation of EcoRI sites within the cDNA

EcoRI linker

CH3

CH3

LI

•

Linker DNA ligated to cDNA

Cut linkers with EcoRI and ligate the cDNA with EcoRI cut plasmid cloning vector

Figure 11-7-9. Construction of cDNA library.

peptide, and there are several oligonucleotides that can be constructed as probes. This is illustrated in Figure II -7 -11, where five of the six amino acids in the peptide shown have degenerate genetic codes in the third base of the codon. The oligonucleotide probes can be made radioactive by using polynucleotide kinase and y_ 32 p_ATP.

KAPLA~. I meulCa 169

Molecular Biology

COIO~ Master reference plate

W

1. Transform competent bacteria with cDNA library and grow on agar plates with antibiotic

2. Replica plate onto nitrocellulose filter

Nitrocellulose filter replica 3. Lyse bacteria with NaOH Neutralize Proteinase K treatment Wash and bake filter 4. Hybridize filter with radioactive probe Wash and autoradiograph 5. Pick up positive colony from master reference plate Autoradiograph Figure 11-7-10. Colony hybridization screening.

Gin -

Pro -

Met -

His -

Lys - Lys

A

A C C A A CAG-CCG-AUG-CAU-AAG-AAG U Figure 11-7-11. Sequence of degenerate oligonucleotides based on amino acid sequence.

Note Expression vectors must be used if production of the actual protein is desired.

170

meclical

b. Screening by expression of a specific protein. If an antibody is available that recognizes the protein encoded by a particular gene, it can be labeled and used to find the clone that is producing the protein. In this method of screening, the cDNA is inserted in a special type of cloning vector called an expression vector (Figure 11-7-12). The expression vector has a special promoter, the lac Z promoter, and a small fragment of the l3-galactosidase cloned into it.

Genetic Engineering

lacZ

Fragment of /3-galactosidase gene cDNA insert

Transcription

Figure 11-7-12. Lac Z expression vector.

Bacteria are transformed with this vector that contains the cDNA library and are plated. Under certain conditions, the lac Z promoter is activated, and the small fragment of the ~-galactosidase gene and the entire insert of cDNA is transcribed into "fusion" mRNA, which codes for a "fusion" protein. To find the desired fusion protein, replicas are made on nitrocellulose fIlters. The bacterial colonies on the fIlters are lysed with chloroform. The filter is incubated with antibody to the protein of interest. The fIlter is washed to remove unbound antibody and is incubated with 125I-Iabeled protein A, which binds to the antibody. After autoradiography, the positive colony can be isolated from the master reference plate. B. Genomic libraries. A genomic library is a large collection of recombinant vectors that have been constructed in such a way that, collectively, they contain a complete collection of all the DNA sequences in the entire genome. In contrast to cDNA libraries, genomic libraries contain both introns and exollS. 1. Construction of genomic libraries. Genomic DNA is isolated from a cell and fragmented with a restriction endonuclease into managable sizes (ranging from 500 bases to 18 kilobas-

es). The size of fragments is critical because in making a library, it is desirable to obtain the entire gene coding for a particular protein in a single restriction fragment. The 18- to 20kilobase restriction fragments are isolated from the rest of the fragments by centrifugation on a sucrose density gradient or by gel electrophoresis. These large fragments are methylated with EcoRI methylase to prevent EcoRI sites within the fragments from being cleaved. Linker DNA with sites for EcoRI are then ligated to the ends of the 18- to 20-kilobase fragments. These fragments are cut with EcoRI, yielding sticky ends that anneal with compatible cohesive EcoRI ends of lambda phage cloning vector. The cloning vector is joined to the genomic inserts by DNA ligase. The phage is then packaged, and bacteria are infected with phage that replicates lytically in the bacteria. 2. Screening genomic libraries. The best way to find a particular gene is to screen bacterial clones by using a single-stranded radioactive cDNA probe. The technique is similar to in situ colony hybridization screening.

KAPLA~.

meulCa

I 171

Molecular Biology

DNA SEQUENCING DNA sequencing allows a rapid and reliable determination of the order of nucleotides in isolated DNA fragments by reducing the DNA to four sets oflabeled fragments. The reactions that generate the fragments are base specific, so the length of the fragment corresponds to the position in the DNA where the specific base occurs. A. Sanger single-stranded DNA sequencing is based on the in vitro synthesis of DNA carried out in the presence of chain-terminating nucleotides (dideoxynucleotides). The steps involved can be described in three phases: the preparation of single-stranded DNA, the enzymatic synthesis of four sets of base-specific fragments, and the analysis of the fragments by gel electrophoresis. 1. Preparation of single-stranded DNA. For sequencing purposes, it is desirable to have

large quantities of single-stranded DNA. The use of M13 bacteriophage as a cloning vector makes this possible. The M13 phage has a circular single-stranded genome that has a double-stranded replicative form. Double-stranded DNA is inserted into the doublestranded replicative form of M13 DNA using the standard techniques for recombinant DNA. Host bacteria are then transformed with the double-stranded recombinant DNA, and the new phages produced in the bacteria will contain only one of the complementary strands. 2. Enzymatic synthesis of DNA in the presence of terminating nucleotides. The singlestranded DNA is used as a template for directing the synthesis of the complementary strand by DNA polymerase 1. DNA pol I also requires the four deoxynucleotides and a primer to which nucleotides are added. The synthesis is carried out in four separate tubes, each containing a single-stranded DNA template, a primer, all four deoxynucleotides, and a small amount of a dideoxy analog of one of the nucleotides. When DNA pol I incorporates the radiolabeled dideoxy analog (instead of the corresponding deoxynucleotide), chain elongation stops. For example, one tube will contain dideoxy ATP, another dideoxy CTP, etc. Thus, four sets of fragments are generated that correspond to the positions of these nucleotides in the DNA.

Note The smallest fragment is the 5' nucleotide. The next largest contains the first two nucleotides, etc. So the sequence is read from the bottom of the gel up.

In a Nutshell peR is a very powerful amplification method that uses repeated cycles of DNA synthesis.

172

meClical

3. Analysis of radiolabeled fragments by gel electrophoresis. The fragments are heated to 100°C to separate the strands and are run on a denaturing polyacrylamide gel. The four sets of fragments are run side by side on the gel, and the positions of the radiolabeled fragments are identified by radioautography. Because small fragments migrate faster in the gel, the sequence is reconstructed by reading the gel from bottom to top.

OTHER METHODS A. Polymerase chain reaction (PCR). This is a technique in which a short sequence of either DNA or RNA can be amplified by more than 10 6 times within a few hours. PCR allows very small amounts of DNA or RNA to be analyzed without cloning and without the need for Southern or Northern blotting. The procedure is illustrated in Figure II-7-13. The DNA to be amplified is flanked by two short oligonucleotide primers that hybridize to opposite strands of the target sequence. Synthesis of the DNA target sequence is achieved by the addition of Taq polymerase, which is heat stable, and the four deoxynucleotides. The primers are oriented in such a way that at the end of each cycle, the two new strands of DNA are complementary, and together they constitute a new copy of the target sequence. Repeated cycles of heat denaturation, hybridization with primers, and polymerization by Taq polymerase results in the exponential amplification of the target sequence. Within a few hours, millions of copies of the starting sequence can be generated.

Genetic Engineering

fill:UfI.(:I,I;IUll

t

Heat to 94°C

III i 1111111111 11111111111111

!

Add primers Anneal at 50°C

11111111111111 I III III i 111111

!

dNTPs and Taq Polymerase Polymerization at 72°C

11111111111111

~ ~

11111111111111

~

Completion of cycle 1 .

l!if;!IHli';iHf!m:II:I!I!!liil +

III:li:t!,'iI2Iilli:1111!i~li;1

t

Repeat cycle again Figure 11-7-13. Polymerase chain reaction.

B. Western blotting. This procedure is used to identify a specific protein that may be present

in very small concentrations in a complex mixture of proteins. As shown in Figure 11-7-14, the protein mixture is separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins are immobilized by transferring them to a nitrocellulose filter. The filters are incubated with antibody to the protein of interest (the antigen). Excess antibody is removed by washing the filter, and the antibody-protein complex is identified by incubating the filters with radiolabeled Protein A, which binds to the antibody. The presence of the protein of interest is identified by autoradiography.

Clinical Correlate Western blots are used to confirm the presence of anti-HIV antibodies in the serum of infected patients.

C. Shuttle vectors. The unique feature of a shuttle vector is that it can replicate in two different

organisms. These vectors, carrying a specific gene, are replicated in one organism (usually E. coli) and are then transfected into eukaryotic cells, where synthesis of protein encoded by

the specific gene occurs. Shuttle vectors are engineered to consist of the following components: a bacterial origin of replication (ori), a mammalian ori, a poly-A+ tail, antibiotic resistance genes, and a mammalian promoter.

meClical

173

Molecular Biology

A.

* Prepare cellular protein

B.

extracts

C.

* SOS-polyacrylamide gel

electrophoresis (S OS-PAG E)

* Blot-transfer onto

nitrocellulose * Block nitrocellulose

with milk * Incubate with

antibody against protein of interest

Ab-Ag complex

D. * Wash nitrocellulose * Incubate with

E.

* Wash nitrocellulose * Autoradiography

1251-Protein A sepharose kO MW Extract

200

1 -

~Protein

of interest

14

Photographic film Figure 11-7-14. Western blotting.

174

meclical

--~

SECTION III

Cell Biology

- - - -

------------

Overview of Cell Types

Cells are separated from their environment by a plasma membrane (and sometimes a cell wall). The function of the plasma membrane is to control the passage of molecules into and out of the cell. There are two basic categories of cells: prokaryotes and eukaryotes. Prokaryotic cells (bacteria) are relatively small and lack a nucleus, whereas eukaryotic cells (fungi, animal, and plant cells) are larger and have a well defined nucleus separated from the cytoplasm by a double membrane structure. This chapter reviews the major structural components of prokaryotic and eukaryotic cells, the properties of the plasma membrane that surrounds all cells, and the mechanisms for transporting molecules across the plasma membrane.

STRUCTURAL FEATURES OF PROKARYOTIC CELLS Prokaryotic cells (bacteria) are relatively small (1-10 J..lm long) and consist of a plasma membrane that surrounds a simple internal cytoplasmic compartment containing DNA, RNA, and all of the enzymes and small molecules required for cell maintenance and reproduction. The plasma membrane is usually surrounded by a rigid protective cell wall. In contrast to eukaryotic cells, prokaryotes do not contain subcellular membrane-bound organelles. A. Cell wall. The thick rigid structure that surrounds the plasma membrane may range in thickness from 15 to 100 nm and is composed oflipids, proteins, and polysaccharides. The cell may be coated with a capsule, which is typically a polysaccharide consisting of repeating units of two or three monosaccharides and is usually attached to phospholipids in the membrane. B. Plasma membrane. The plasma membrane controls the movement of materials in and out

of cells. All naturally occurring membranes consist of an amphipathic bilayer composed of lipids and membrane proteins. C. Nucleoid. The DNA of prokaryotic cells is found in a region of the cytosol known as the nucleoid. This region is equivalent to the nucleus in eukaryotic cells, but it is not separated from the cytoplasm by a membrane. D. Cytoplasm. The total internal compartment of prokaryotic cells is defined as the cytoplasm.

E. Flagella. The outer surface of many bacteria is coated with flagella, which are thread-like structures that allow the bacteria to move rapidly.

STRUCTURAL FEATURES OF EUKARYOTIC CELLS Eukaryotic cells are considerably larger than prokaryotes and have subcellular compartments that are defined by networks of internal membrane structure. The linear dimensions of eukaryotic cells generally range from 10-100 /lm. The structure of a typical eukaryotic cell is shown in Figure III-I-I. KAPLAlf _ I meillea

177

Cell Biology

--~---------------------------------------------------------------------------------

Nucleolus

Lysosome Golgi apparatus

-+-=---"'----!t-- Nucleus

Rough endoplasmic reticulum (RER)

Smooth endoplasmic reticulum (SER)

Figure 111-1-1. Eukaryotic cell structure.

A. Plasma membrane. As in prokaryotes, the plasma membrane separates the cell from its extracellular environment and is primarily concerned with controlling the movement of molecules into and out of the cell.

Note Topologically, the lumen of any of the endomembranous system organelles or vesicles is equivalent to the outside of the cell.

B. Endomembranous system. In eukaryotes, an extensive membrane system exists, including the plasma membrane, the smooth and rough endoplasmic reticula, Golgi apparatus, endosomes, lysosomes, and secretory vesicles. The endomembranous system transfers macromolecules and membrane components from one region of the cell to another and transfers macromolecules to and from the cell's extracellular environment. The specific functions for the various members of the endomembranous system are discussed in Chapter 4, Subcellular Organelles, in this section. C. Mitochondria. Most of the energy metabolism in eukaryotic cells occurs in mitochondria.

They contain two separate membrane systems and are found in varying numbers in different cell types. D. Nucleus. The nucleus contains two major components.

Note Molecules less than 40 kD in size can diffuse freely between the nucleoplasm and the cytoplasm through the nuclear pores.

178

meClical

1. The nuclear envelope consists of a double membrane structure with nuclear pores that allow molecules to move between the cytoplasm and the nucleoplasm. The outer membrane is continuous with the rough endoplasmic reticulum.

2. The nucleolus is a dense region in the nucleus with a high content of RNA. It is the site of ribosomal RNA synthesis and ribosomal assembly.

Overview of Cell Types

E. The cytoplasm (cytomatrix) is a colloidal substance within the plasma membrane of the cell but outside of the nucleus. The organelles contained within the cytoplasm are discussed in Chapter 4, Subcellular Organelles, of this section.

STRUCTURE AND FUNCTION OF THE PLASMA MEMBRANE Biologic membranes are bilayers that are approximately 7.5 nm in width and are assembled from a mixture of lipids and proteins. The ratio of lipid to protein is different in various membranes of the cell. The content of protein is proportional to the number of functions performed by the membrane, with the lowest content being found in myelin, which functions as a simple insulator ofaxons. The inner mitochondrial membrane, which is very active in electron transport and oxidative phosphorylation, has the highest protein content. A. Composition of plasma membrane. The plasma membrane of cells is composed of approximately equal proportions of lipid and protein. 1. Lipids. The lipids found in the plasma membrane are phospholipids, unesterified choles-

terol, and glycolipids. All of the lipid components are amphipathic, having polar head groups that interact with the aqueous environment. The hydrophobic tails interact with one another to form the interior of the bilayer. The fatty acids of the phospholipids, sphingolipids, and glycolipids form the hydrophobic tails. a. Phospholipids are the major building blocks of all biologic membranes, comprising between 50 and 60% of the total membrane lipid. The phospholipids found in animal plasma membranes include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and sphingomyelin (also a glycolipid-see below). b. Cholesterol is a major component of plasma membranes, comprising between 15 and 25% of the total lipid. The cholesterol is always in the unesterified form. e. Glycolipids make up the remainder of the plasma membrane lipids. Although glyco-

lipids usually make up less than 100/0 of the total lipid in the plasma membrane, they are very important in cell-cell and cell matrix interactions. 2. Proteins. The dynamic functions of the membrane are carried out by protein. Plasma membrane proteins, which are frequently glycoproteins, include hormone receptors, transport proteins, ion channels, and enzymes. There are two basic types of membrane proteins: integral and peripheral proteins. a. Integral membrane proteins are embedded in the lipid bilayer and cannot be removed without disrupting the bilayer. They span the lipid bilayer one or more times, and their association with the membrane is stabilized by hydrophobic interactions. The protein domain that spans the lipid bilayer is usually an a-helix composed of approximately 20 amino acids. Detergents are required to solubilize integral membrane proteins. b. Peripheral membrane proteins are loosely associated with the membrane and can be removed by alterations in the pH, by an increase in ionic strength, or by other conditions that interfere with ionic interactions. They are bound to the membrane by interaction with the polar head groups of phospholipids or with exposed regions of other membrane proteins. B. Properties of the lipid bilayer. The driving force for formation of lipid bilayers is the hydrophobic interaction between the fatty acid chains of the lipid components.

Note A subset of peripheral membrane proteins contains fatty acids or prenyl groups covalently bound to an amino acid. These "tails" anchor the protein to the membrane. Examples include the src tyrosine kinase and the ras G protein. KAPLA~. I meulca

179

Cell Biology

1. Structure. The bilayer consists of two leaflets held together by hydrophobic interactions

between the fatty acids in each leaflet (Figure III-I-2). The polar head groups of the outer leaflet interact with the aqueous environment outside the cell, whereas those of the inner leaflet interact with the cytosol.

Intrinsic

t- Hydrophilic e~ Phospholipid Ir- Hydrophobic ~

Figure 111-1-2. The structure of biologic membranes.

2. Asymmetry. Lipids, proteins, and carbohydrates are all asymmetrically distributed across the lipid bilayer. The asymmetry of the two sides of the membrane allows each side to be functionally distinct. a. Phospholipid asymmetry. The choline-containing lipids (phosphatidylcholine and sphingomyelin) are enriched in the outer leaflet, whereas amine-containing phospholipids (phosphatidylserine and phosphatidylethanolamine) are found primarily in the inner leaflet.

In a Nutshell All membrane carbohydrates are attached to lipids or proteins and are exposed to the "exterior" of the cell.

Bridge to Physiology Increasing the proportion of unsaturated fatty acids or, to a lesser degree, shorter-chain fatty acids increases membrane fluidity. Cholesterol decreases fluidity somewhat but prevents actual freezing; it always increases mechanical stability.

180

iiieClical

b. Carbohydrate asymmetry. The carbohydrate portions of both glycoproteins and glycolipids are found on the exterior of the plasma membrane, where they may function in holding adjacent cells together. c. Protein asymmetry. Proteins are not randomly inserted in membranes. Some proteins have their N-terminus outside the cell, whereas others have their N-terminus on the cytosolic side of the membrane. However all molecules of a specific protein are always inserted in the membrane with the same orientation. 3. Fluidity. The fluidity of the plasma membrane is influenced by the content of cholesterol and by the nature of the fatty acids found in the phospholipids. The higher the cholesterol content, the more tightly the phospholipids of the bilayer are packed, resulting in a membrane that has high rigidity and low fluidity. Note, however, that cholesterol prevents actual freezing of the membrane. Rigidity is also increased by long-chain saturated fatty acids that pack more tightly than unsaturated fatty acids or short-chain fatty acids. The effect of fatty acid composition on rigidity is demonstrated by the fact that the melting temperature (Tm) of membranes is lowered by increasing the proportion of either unsaturated fatty acids or shortchain fatty acids. 4. Mobility. Phospholipids move laterally within the membrane at very high rates. Some integral membrane proteins can also move laterally within the plane of the membrane, whereas the movement of others is restricted by interaction with other proteins either in the cytosol or outside the cell.

- - -

----------------------------

Overview of Cell Types

TRANSPORT ACROSS THE PLASMA MEMBRANE The plasma membrane is a selective barrier that maintains distinct internal and external environments by controlling the passage of molecules into and out of the cell. Several mechanisms are used for the transport of molecules across the membrane. A. Osmosis. Water moves across a cell membrane from a compartment with a lower solute concentration to a compartment with a higher solute concentration. The movement of water requires no expenditure of energy. Osmotic pressure is defined as the force that would be required to prevent the movement of water between compartments.

B. Passive transport. The movement of molecules across a membrane from a region of higher concentration to a region of lower concentration is termed "passive transport" because it requires no external source of energy. There are two types of passive transport: simple diffusion and facilitated diffusion. 1. Simple diffusion. Small uncharged molecules such as oxygen and carbon dioxide can pass through the lipid bilayer by simple diffusion from a region of higher concentration to a region of lower concentration; ions can move through open ion-selective protein channels via simple diffusion.

2. Facilitated diffusion. Some molecules have very low permeability because of their size or polar nature, and they require protein carriers to move from a region of higher concentration to a region of lower concentration. The carrier-mediated movement down a concentration gradient is termed "facilitated diffusion:' All of the transport proteins are integral membrane proteins that span the membrane multiple times. For example, the glucose transporters contain 12 membrane-spanning domains. C. Active transport. The movement of molecules across a membrane from a region oflow concentration to a region of high concentration requires both a carrier protein and the expenditure of energy. The hydrolysis of ATP by an ATPase is directly coupled to this transport, and the free energy released in hydrolysis is used to move ions up an electrical or concentration gradient. This process is called "active transport:' and it is fully reversible. Three examples of proteins in active transport are the Na+K+ -ATPases (which pump Na+ out and K+ into the cell), Ca2+-ATPases (which pump Ca2 + out of cells, or in muscle, from cytosol into the sarcoplasmic reticulum), and the H+ -ATPases (which acidify lysosomes). 1. Cotransport. Cotransport is a form of active transport in which cells import molecules such as glucose or amino acids against a Na+ or H+ gradient. The movement of the ion across a membrane and down a concentration gradient is coupled to transport of the other molecule up a gradient. Ifboth molecules move in the same direction, this is known as symport. For example, glucose and Na+ move from the intestinal lumen across the apical surface of brush border cells via the glucose-Na+ symport protein. If the movement is in opposite directions, it is called antiport. For example, cardiac muscle cells decrease the frequency of contractions by exporting Ca2+ and importing Na+. D. Signal transduction. Some macromolecules do not need to cross the plasma membrane to affect the cell's biologic functions. The extracellular binding to the appropriate membrane receptor triggers a cascade of intracellular events. The binding of an agonist such as a hormone to its receptor causes a conformational change in the receptor. The receptor then interacts with a protein known as a stimulatory G protein (Gs ). Gs is composed of three subunits, (alpha, beta, and gamma) and the alpha subunit contains a molecule of GDP. The agonist-receptor complex triggers an exchange of bound GDP for GTP on Gs' and this dissociates alpha-GTP from beta and gamma. The alpha-GTP activates an intracellular effector protein such as adenylate cyclase. This transduces the signal created by the binding of the agonist to its membrane receptor. The GTP bound to alpha is hydrolyzed back to GDP, which deactivates the adenylate cyclase and returns the G protein to its unstimulated state.

In a Nutshell Simple diffusion is not saturable. Facilitated transport requires specific binding sites for the substrate and therefore is saturable and competitively inhibited.

Clinical Correlate Cystic fibrosis is caused by a defect in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, a cAMP-activated chloride channel. This results in altered chloride permeability in epithelial cells.

Note There is a family of receptors that interact with the G protein. They are all glycosylated integral membrane proteins that have seven transmembrane segments. An example of this class of protei n is the betaadrenergic receptor for the hormone epinephrine. KAPUlf I meillea

181

Cell Biology

VESICULAR TRAFFIC AND THE PLASMA MEMBRANE A. Endocytosis. Movement of macromolecules from the outside of the cell into the cell by the active invagination of the plasma membrane is known as endocytosis. Endocytosis has been divided into three categories: receptor-mediated endocytosis, fluid-phase endocytosis (pinocytosis), and phagocytosis. All three classes require energy. 1. Receptor-mediated endocytosis. The molecule being internalized binds to a receptor on

the surface of the cell. The receptor-ligand complexes concentrate at clathrin-coated pits on the plasma membrane. These coated pits bud to form clathrin-coated vesicles. The vesicles lose their clathrin coats and fuse with early endosomes. The pH of the early endosomes is slightly acidic due to an ATP-driven proton pump, and this lowering of pH causes the dissociation of the ligands from the receptors. The dissociated ligands fuse with late endosomes or lysosomes for degradation, and the receptor vesicles (recycling vesicles) return to the plasma membrane. 2. Fluid-phase endocytosis. This process is also known as pinocytosis ("cellular drinking"), and it refers to the uptake of molecules that are in solution. Receptors are not involved and it is not a concentrating process.

Note Constitutive secretion is the "default" pathway for proteins entering the endomembranous system (by cotranslational import into the RER mediated by SRP). All other destinations (retention in RER or Golgi, delivery to Iysosomes or regulated secretory vesicles) require additional signals. In the absence of such signals, the protein will reach the plasma membrane or be secreted from the cell.

182

meClical

3. Phagocytosis. This process is known as "cellular eating" and is carried out by macrophages and neutrophils. B. Exocytosis. Movement of macromolecules out of the cell is achieved by exocytosis. There are

two types of exocytosis: regulated exocytosis and constitutive secretion. In both cases, the molecules that leave the cell are packaged into secretory vesicles. 1. Regulated exocytosis. The secretion of hormones requires a signal before the secretory

vesicle will fuse with the plasma membrane. The vesicles are coated with clathrin. 2. Constitutive secretion. Secretory vesicles that are not coated with clathrin are continuously fusing with the plasma membrane. These vesicles contain membrane proteins, membrane lipids, and plasma proteins.

Nucleus and Nucleolus

The nucleus of the cell contains DNA, RNA, and nuclear proteins. The nucleus is spherical in shape and is separated from the cytoplasm by a nuclear envelope consisting of two distinct lipid bilayers. The outer nuclear membrane is continuous with the endoplasmic membrane. The inner nuclear membrane is associated with the nuclear lamina, a network of proteins involved in the structural organization of the nucleus. The inner nuclear membrane also has contact sites for interacting with the chromatin and with newly synthesized RNA. The transport of molecules into and out of the nucleus is facilitated by pores in the nuclear envelope. The nucleolus is a dense organelle in the nucleus where ribosomal RNA synthesis and ribosome assembly occur. The functions of the nucleus are to synthesize DNA and repair mistakes made during synthesis and to synthesize RNA and to process it before transport to the cytosol. This chapter reviews the structure of the nucleus and the packaging of DNA into chromatin and the structure and function of the nucleolus.

NUCLEUS Eukaryotic cells have their DNA sequestered in the nucleus, a membrane-bound organelle that can be seen by light microscopy. The subnuclear components can be visualized by the electron microscope. A. Function. The nucleus is involved in both mitosis and meiosis. Several events required for cell division occur in the nucleus, including DNA replication and transcription of DNA into precursor RNA molecules (Figure 1II-2-l). All of the enzymes required for replication and repair of newly synthesized DNA, as well as for transcription and processing of precursor RNA molecules, are found in the nucleus. The synthesis and assembly of ribosomes occurs in a region of the nucleus known as the nucleolus. A detailed review of these processes is discussed in the first five chapters of Molecular Biology (Section II).

KAPLAN'dme leaI

183

Cell Biology

---

./

--

-

"" I

Nuclear pore

-+~-i

/

/

rRNA

/

•

•

DNA Replication and Repair

\

\ \

,,

\

~

I

(

·1 ,Ribosome

Ribosomal assembly

/ 0(

,,

\ \

,

mRNA

RNA Transcription and Processing

mRNA

+ + I

/

tRNA

DNA

.~

DNA

•

tRNA

/

/ /

Nucleus

"-

""

-..

- - ---

Cytoplasm

./ ."""

Figure 111-2-1. Functions of the nucleus.

B. Structure. The major structural features of the nucleus are the nuclear envelope, the nuclear lamina, the nuclear pores, and the nucleolus. 1. Nuclear envelope. The periphery of the nucleus is defined by a double membrane known as the nuclear envelope. Each of the nuclear membranes is composed of a distinct lipid bilayer that is approximately 6 nm thick. Thus, the nuclear envelope is thicker than the plasma membrane. The outer nuclear membrane is continuous with the endoplasmic reticulum. It is associated with ribosomes that are actively synthesizing proteins. The inner nuclear membrane is coated with a meshwork of fibrous proteins known as the nuclear lamina. The double membrane structure contains pores or channels through which most macromolecules must pass to enter or leave the nucleus.

Note Phosphorylation of the lamina during prophase of mitosis initiates nuclear disassembly into small vesicles.

2. Nuclear lamina. The nuclear lamina is a lattice-like network of proteins that play an important role in the structural organization of the nucleus. The lamina is composed of intermediate fllaments having a diameter of approximately 10 nm and containing three types of proteins known as lamins A, B, and C. The lamins contain specific sites that allow the chromatin to be connected to the inner membrane of the nuclear envelope. They are essential for the breakdown and reformation of the nuclear envelope that occurs during the cell cycle. 3. Nuclear pores. The presence of pores in the nuclear envelope provides a mechanism of communication between the cytosol and nucleus. Molecules such as water, ions, amino acids, and small proteins (e.g., ribosomal proteins) pass through the pores. The transport through pores is highly selective because nearly all of the proteins made in the cytosol are

184

mellical

Nucleus and Nucleolus

excluded from the nuclear compartment. The inner and outer membranes of the nuclear envelope are fused to form openings that are approximately 90 nm in diameter. The periphery of the pore is defined by a complex of proteins arranged in an octagonal pattern. 4. Nucleolus. The region in the nucleus where rRNA synthesis occurs and ribosomes are assembled is known as the nucleolus. With electron microscopy, three morphologically distinct zones can be observed. A granular zone found at the periphery of the nucleolus consists of ribosomal precursor particles in various stages of assembly. A centrally located fibrillar zone contains ribonuclear protein fibrils, and a pale-staining fibrillar center contains DNA that is not being transcribed. The size and shape of the nucleolus is dependent upon its activity. In cells synthesizing large amounts of protein, the nucleolus may occupy 20-25% of the total nuclear volume, whereas in resting cells the nucleolus may be almost invisible. The difference in the size of the nucleolus is primarily due to a difference in the size of the granular zone.

In a Nutshell The nucleolus is the site of RNA polymerase I-mediated transcription of rRNA and the site of large subunit ribosome assembly. rRNA is the most abundant RNA in the cell.

CHROMATIN Chromatin is a complex of DNA and proteins. The proteins can be classified as either histone proteins or nonhistone proteins. A. DNA (deoxyribonucleic acid) is a double-stranded helical molecule that carries the genetic information of the cell. 1. Structure. DNA is composed of four nitrogenous bases linked together by a deoxyribose

phosphate backbone. The four nitrogenous bases consist of two purines (adenosine and guanine) and two pyrimidines (cytosine and thymine). The nucleotides are linked via 3'-5' phosphodiester linkages. DNA exists in three conformations: B DNA, Z DNA, and A DNA. The differences in these three types of DNA are discussed in the first chapter of Molecular Biology (Section 11). The most common type is B DNA, a classic Watson-Crick double helix with major and minor grooves that serve as sites for DNA-protein interactions. 2. Histone proteins. The histones are positively charged proteins enriched with lysine and arginine residues. They have no enzyme activity but are involved in DNA packaging. There are five types of histones: HI, H2A, H2B, H3, and H4. The histones are modified post-translationally by phosphorylation, acetylation, and methylation. The histones are important in forming two types of structures in chromatin: nucleosomes and solenoid fibers (Figure III-2-2). a. Nucleosomes. Each nucleosome contains two copies each of histones H2A, H2B, H3, and H4 and approximately 150 base pairs of DNA. The histones form an octamer, which has DNA wrapped around it to give chromatin the appearance of "beads on a string" when examined by electron microscopy. The nucleosomes are the basic repeating units of the chromatin fiber, having a diameter of approximately 10 nm. Adjacent nucleosomes are linked to one another by approximately 60 base pairs of DNA. b. Solenoid fibers. Histone HI is involved in packaging long strings of nucleosomes into a helical, secondary chromatin structure known as a solenoid, which appears under the electron microscope as a thick 30-nm fiber. Each turn of the solenoid contains six nucleosomes (Figure 1II-2-2).

Note Some transcription factors increase RNA polymerase II-mediated mRNA transcription by displacing the nucleosomes in the gene's DNA.

3. Nonhistone proteins. The enzymes involved in nuclear functions such as replication, transcription, DNA repair, and regulation of chromatin function are known as the nonhistone proteins. They are acidic or neutral proteins. B. Chromatin organization. During the cell cycle, chromosomes go through orderly stages of

condensation and decondensation. Various stages in the condensation of DNA are shown in KAPLA~. I meulca

185

Cell Biology

Figure III-2-2. After formation of the nucleosomes and solenoid fibers, the solenoids are further packed into loops, each containing approximately 100,000 base pairs of DNA. The loops are the beginning of the knob-like thickenings called chromomeres that are observable under the microscope in early prophase as mitosis begins. The chromomeres further condense, as shown in Figure III-2-2, to eventually form the chromosome.

c

DNA

A. 2 nm

[

B. 10 nm

[

String of nucleosomes

C. 30 nm

[

6 Nucleosomes/turn solenoid fiber

o ~ E

1111111111111111111

(;

LL

c

~

D. 300 nm

Loops

E

e

x

..c

o

E. 700 nm

[QRV I I

F. 1,400 nm [

Chromatin

I

===_==

Chromosome

Figure 111-2-2. Chromatin organization.

Note

C. Types of chromatin. Chromatin inside a eukaryotic cell nucleus can be classified into two

major types based on its state of condensation: heterochromatin and euchromatin.

Almost the entire inactive X chromosome in each somatic cell in a woman is condensed into heterochromatin.

1. Heterochromatin is highly condensed (30-nm solenoid fibers or higher states of condensation) and is transcriptionally inactive. The amount of heterochromatin in a cell is related to the transcriptional activity of the cell. In a typical eukaryotic cell, approximately 10% of the chromatin is heterochromatin.

2. Euchromatin is a more extended form of DNA and is the potentially transcriptionally active form of chromatin. In a typical cell, euchromatin accounts for approximately 90% of the total chromatin, although only about 10% is in the 10-nm fiber of nucleosomes and is being actively transcribed.

186

iiie&ical

Subcellular Organelles Eukaryotic cells, when viewed with the electron microscope, contain a number of well defined subcellular compartments or organelles, each enclosed by a membrane system that separates them from the cytosol. The organelles of eukaryotic cells include the nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, Iysosomes, and peroxisomes. Additionally, a part of the endoplasmic reticulum is associated with ribosomes, which are actively engaged in protein synthesis. This chapter reviews the structure and function of each of these subcellular organelles as well as some clinical conditions arising from a specific enzyme or organelle deficiency.

RIBOSOMES Ribosomes are cytoplasmic organelles composed of ribosomal RNA and protein. They contain binding sites for all of the interacting molecules involved in protein synthesis. A. Structure. The mature 80S ribosome is a multienzyme complex of rRNA and protein consisting of a large (60S) and a small (40S) subunit. The ribosomes are assembled in the nucleus and are transported to the cytoplasm through the nuclear pores. 1. The large subunit contains three species of rRNA (28S, 5.8S, and 5S) and a set of approx-

imately 50 proteins. 2. The small subunit contains a set of approximately 30 specific proteins that are associated with an 18S species of rRNA. B. Assembly. The large ribosomal subunit is synthesized in the nuceolus, whereas the small subunit is synthesized in the nucleus. Each subunit is assembled from rRNA and ribosomal proteins from the cytoplasm. After assembly of the ribosomal subunits, the subunits pass through the nuclear pore into the cytoplasm where they combine to form a ribosome. C. Function. The ribosomes are the cellular machines used for protein synthesis. They can exist

either as free polysomes in the cytoplasm or as membrane-associated polysomes, which are attached to the rough endoplasmic reticulum. Polysomes consist of a single mRNA that is being translated by several ribosomes at the same time, all moving on the mRNA from the 5' end toward the 3' end (Figure III -3-1). The two ribosomal subunits associate on the mRNA, with the small subunit binding first. Subsequent binding of the large subunit is necessary before protein synthesis can begin. Individual ribosomes are spaced approximately 80 nucleotides apart along the mRNA molecule. Each ribosome is independently synthesizing a single polypeptide. 1. Free polysomes are the sites of synthesis for proteins destined for the nucleus, peroxi-

somes, or mitochondria. 2. Membrane-associated polysomes are the sites of synthesis of secretory and membrane proteins.

meClical

187

Cell Biology

@


+ 6 Start codon

t

Nascent polypeptide chain

!N _

~
~

N Mature polypeptide

Figure 111-3-1. Structure of a polysome.

ENDOPLASMIC RETICULUM More than 50% of the total membrane in eukaryotic cells is made up of endoplasmic reticulum. There are two types: rough endoplasmic reticulum and smooth endoplasmic reticulum.

Note Protein synthesis for cytosolic, mitochondrial, nuclear, and peroxisomal proteins is completed in the cytosol.

Note Proteins destined for the RER, SER, Goigi apparatus, Iysosomes, plasma membrane, or for secretion from the cell must finish translation on RERbound ribosomes.

188

meClical

A. Rough endoplasmic reticulum (RER). The RER is a single lipid bilayer continuous with the outer nuclear membrane. It is organized into stacks of large flattened sacs called cisternae that are studded with ribosomes on its cytoplasmic side, giving the organelle its rough appearance. The internal space of the cisternae is known as the luminal space. Sacs and tubules form an interconnected branched network that covers a large part of the cell. Three functions are associated with the RER: protein synthesis, translocation of membrane proteins, and cotranslational modification of polypeptides. Proteins that are destined for secretion, the plasma membrane, the Golgi apparatus, and lysosomes, are synthesized by the rough endoplasmic reticulum. The RER is very prominent in cells that are specialized in the synthesis of proteins destined for secretion (e.g., pancreatic acinar cells). 1. Protein synthesis (Figure III-3-2). All protein synthesis begins on free polysomes. The association of the two ribosomal subunits (Step A) occurs at the 5' end of the mRNA, with the small ribosomal subunit binding first. After binding of the large subunit, protein synthesis begins (Step B). The association of ribosomes with the RER requires signal peptides, signal recognition particles, and signal recognition particle receptors. The mRNAs of proteins synthesized by RER-associated polysomes contain nucleotide sequences coding for approximately 30 hydrophobic amino acids. As the signal peptide is released by the ribosome (Step B), it is recognized by dle signal recognition particle (Step C). The signal recognition particle (SRP) contains six protein subunits and a 7S RNA. The SRP binds to both the ribosome and the signal sequence of the peptide being synthesized, causing a brief pause in protein translation. During the pause, the SRP binds to the SRP receptor (SRPR or docking protein), an integral membrane protein of the endoplasmic reticulum (Step D). The

Subcellular Organelles

G

6)

(A)

~ 5'

(C)

(8)

5'

3'

~

8

N

5'

3'

Signal sequence

3'

SRP

(E)

(0)

5'

3'

3'

SSR RR

RER membrane SRPR SSR

3'

RR

RER luman

•• ~ • IP (H)

(I)

5'

-~r.....-...--I..----

N

5' - - - - - - - 3'

G

3'

I~I

I I

RR

C

V)

,;,.

ASn\3e. N Dolichol pyrophosphate lipid carrier

RR

0 0

RER lumen

N.J'C)_Asn~

Figure 111-3-2. Protein synthesis and the rough endoplasmic reticulum.

binding of SRP to its receptor leads to its displacement from the ribosome and to the insertion of the signal sequence into the RER membrane, events that allow protein synthesis to continue. The ribosome receptor (RR) is made up of two proteins of the RER membrane, ribophorins I and II (Step E). The signal sequence and the newly synthesized polypeptide chain enter the RER membrane where the signal sequence interacts with a protein known as the signal sequence receptor (SSR). 2. Cotranslational modifications. Several modifications occur as the protein is being synthesized. Signal peptidases (SP) located in the RER membrane remove the signal peptide and are degraded by intraluminal proteases (Step F). N-linked glycosylation occurs by the transfer of preformed mannose-rich oligosaccharides from a dolichollipid carrier to specific asparagine side chains within the nacent polypeptide (Steps G and H). Intramolecular disulfide bonds are formed by protein disulfide exchange enzyme. After completion of polypeptide synthesis, the ribosome disassembles and detaches from the RER membrane (Step I).

Note The initial step of N-linked glycosylation occurs during translation. All O-linked glycosylation steps are posttransiational.

3. Insertion of membrane proteins into the endoplasmic reticulum membrane. Many proteins are completely released into the RER after synthesis (Figure III-3-2, Step I). These include proteins that function in the lumen of the endoplasmic reticulum, proteins that are to be secreted, and proteins destined to other organelles such as the Golgi apparatus

meClical

189

Cell Biology

and lysosomes. However, some proteins are destined to be inserted and retained in the membrane. The retention of such proteins is facilitated by two types of signals, the insertion signal (or the start-transfer signal) and the halt transfer signal (also known as the stop-transfer signal). How these signals are used depends on the number of times a protein crosses the membrane and on the orientation of the protein within the membrane (Figure III-3-3).

Extracellular side

Type I

Type II

Type III

Intracellular side Figure 111-3-3. Types of integral membrane proteins.

a. Type I proteins cross the membrane one time and are oriented so that the N-terminus faces the luminal surface and the C-terminus faces the cytoplasmic surface. For these proteins, the N-terminal start-transfer peptide is followed by a hydrophobic stoptransfer sequence that terminates the insertion. The start-transfer N-terminal signal peptide is then cleaved, leaving behind a single-pass integral membrane protein with its C-terminus in the cytoplasm and its N-terminus in the lumen of the ER.

In a Nutshell N-Linked Glycosylation • Initial step occurs during translation. • Initial step transfers preformed oligosaccharide from dolicholphosphate onto Asn in Asn-X-Ser sequence. • Preformed oligosaccharide is trimmed in RER. • New sugars (in a pattern specific to each protein) are added in the Golgi.

190

meClical

b. Type II proteins also cross the membrane one time, but their orientation is such that the N-terminus is in the cytoplasm. For these proteins, the start-transfer peptide sequence (insertion peptide) is in the interior of the protein and does not undergo cleavage. The N-terminus is synthesized first and is exposed to the cytoplasmic surface of the membrane. The C-terminus is then looped out of the membrane and into the lumen. c. Type III proteins cross the membrane several times. They contain a series of alternating insertion (start-transfer) sequences and halt (stop-transfer) sequences, thereby allowing the protein to cross the membrane multiple times. The start- and stop-transfer sequences are hydrophobic runs of 20-30 amino acids that form an a-helix in the lipid bilayer. 4. Cotranslational modification of polypeptides. Most proteins synthesized on the endoplasmic reticulum become glycosylated upon entry into the lumen of the ER. On the other hand, proteins that are synthesized on free polysomes are usually not glycosylated. In animal cells, oligosaccharides are attached to the glycoprotein through glycosidic bonds that are either N-linked (attached to the amide group in an asparagine side chain) or 0linked (attached to the hydroxyl group of a serine, threonine, or hydroxylysine side chain). The early stages of N -linked glycoprotein synthesis are summarized below. a. Synthesis of preformed oligosaccharide. A mannose-rich oligosaccharide is assembled on a dolichol pyrophosphate lipid carrier located in the membrane of the ER. h. Transfer of oligosaccharide to protein. The enzyme oligosaccharide transferase trans-

Subcellular Organelles

fers the carbohydrate moiety from the dolichol carrier to an asparagine residue with the sequence Asn-X-Ser/Thr (where X can be any amino acid except proline or aspartate). The enzyme is located on the luminal side of the RER membrane. Before polypeptide synthesis is completed, one or two mannose residues and three glucose residues are trimmed from the oligosaccharide by specific glucosidases, all enzymes of the ER. e. Classes of N-linked oligosaccharides. In mature glycoproteins, three classes of

oligosaccharides can be found (Figure 1II-3-4). All three classes share a core region (Man3GlcNAc2), but they differ in the composition of the branches. The core region is added to the protein in the RER, whereas the unique terminal monosaccharides are added in the Golgi. (1) High-mannose type oligosaccharides retain 2-6 mannose residues linked to the core region. Proteins with high-mannose type carbohydrate chains remain in the RER and are not transferred to the Golgi.

(2) Complex oligosaccharides contain sialic acid, galactose, and glucosamine in the branches coming off the core region. These terminal monosaccharides are added in the Golgi.

(3) Hybrid oligosaccharides consist of a structure in which one branch retains some of the mannose residues and the other branch contains galactose and gIucosamine. d. Pathway of glycosylation. The exact pattern of glycosylation of a newly synthesized protein can be used to trace the pathway of a protein through the intracellular compartments. For example, the ~-subunit of the Na/K ATPase is glycosylated. The nascent ~-subunit polypeptide (38 kD) acquires three core oligosaccharides of the high-mannose type in the endoplasmic reticulum, increasing its molecular weight to 45 kD' As the 45-kD protein moves through the Golgi, the molecular weight increases to 55-60 kD and the post-translational modification is completed, yielding the mature ~-subunit.

SA Man

Man

Man

I

I

I

Man

Man

Man

'-./

I

Man

Man

"-Man /

SA

I

I

Gal

Gal

I

I

GlcNAc

GlcNAc

I

I

Man

Man +/-GlcNAc -

"-Man /

Gal

I GlcNAc Man

I

Man

Man +/-GlcNAc -

'\.

Man

/

I

I

I

GlcNAc

GlcNAc

GlcNAc

I

I

GlcNAc

I

I

Asn

+/-Fuc - - GlcNAc

I

High Mannose

L4iJ Complex

Man

"-/

I GlcNAc

I

I

Asn

I

Hybrid

Figure 111-3-4. Classification of N-linked oligosaccharides.

B. Smooth endoplasmic reticulum (SER). The SER is a network of membranous sacs, vesicles, and tubules continuous with the RER but free of ribosomes. The membrane contains enzymes involved in the biosynthesis of phospholipids, triglycerides, and sterols. Some of the functions of the SER are listed below. KAPLA~. I meulCa

191

Cell Biology

1. Detoxification reactions. Many compounds are extremely water insoluble and, when present in sufficient quantities, are toxic. One of the approaches to detoxification is to make these compounds (e.g., steroid hormones, alcohol, drugs, insecticides) more water soluble so that they can be excreted. Two types of reactions that are most important in increasing the solubility are hydroxylation and conjugation reactions.

Note Phase I hydroxylations can result in toxic intermediates, which can be harmful unless modified by a phase II conjugation.

a. Hydroxylation reactions. The addition of hydroxyl groups to compounds is achieved by a multienzyme complex found in the smooth endoplasmic reticulum. The complex requires molecular oxygen, NADPH, and a minature electron transport system for using the reducing equivalents in NADPH to reduce oxygen to a hydroxyl group. These hydroxylase complexes contain cytochrome P 450, a flavoprotein, and a nonheme iron protein, all components of the SER. b. Conjugation reactions. Other enzymes found in the SER can transfer polar groups (sulfate, glucuronic acid) from activated carriers (PAPS and UDP-glucuronic acid) to the toxic water-insoluble molecule. 2. Glycogen degradation and gluconeogenesis. Glycogen is degraded to glucose-lphosphate, which is then isomerized to glucose-6-phosphate. Glucose-6-phosphate is also the last cellular intermediate in the pathway of gluconeogenesis. For glucose to leave the cell, the phosphate group has to be removed by the enzyme glucose-6-phosphatase, an integral membrane protein of the SER. 3. Reactions in lipid metabolism. Lipolysis begins in the SER with the release of a fatty acid from triglyceride. The SER is also the site where lipoprotein particles are assembled. 4. The sarcoplasmic reticulum. The SER in striated (skeletal and cardiac) muscle is known as the sarcoplasmic reticulum, an organelle that is active in the sequesteration and release of calcium ions.

GOLGI APPARATUS The Golgi consists of disc-shaped smooth cisternae that are assembled in stacks (dictyosomes) having a diameter of approximately 1 !im (Figure III-3-5). Numerous small membrane-bound vesicles are associated with the edges of the stacks. The Golgi has two distinct faces. The cis (forming) face is associated with the RER. The trans (maturing) face is oriented toward the plasma membrane. The trans-most region is a network of tubular structures known as the trans-Golgi network (TGN). The major functions of the Golgi apparatus include post-translational modification and the sorting of newly synthesized proteins and lipids. A. Transfer of proteins from the RER to the Golgi. The carbohydrate component of glycoproteins are processed to give complex-type oligo saccharides (see Figure III-3-4) in the Golgi. Regions of the RER that are smooth in appearance are known as transitional elements. These regions bud off to form vesicles that contain proteins destined for the Golgi. The vesicles go to the cis face of the Golgi and fuse with the Golgi membrane, allowing secretory proteins and membrane proteins to enter the Golgi. The transfer of proteins from the RER to the Golgi is an energy-dependent process and can be blocked by inhibitors of oxidative phosphorylation. B. N-Hnked oligosaccharides are attached to an asparagine residue of proteins during the course of polypeptide synthesis. Further modification of the carbohydrate moiety occurs in the Golgi and is required to produce complex and hybrid oligosaccharide chains (see Figure III-3-4). The Golgi stack contains biochemically distinct compartments. For example, the cis sac contains a-mannosidase I, N-Ac-glucosamine phosphotransferase, and N-Ac-glucosaminidase; the

192

meClical

Subcellular Organelles

medial sac contains cx-mannosidase II, transferase I, transferase II, and fucosyl transferase; and the trans-most sac contains galactosyl transferase. Membrane vesicles transport the proteins across the Golgi from the cis face to the trans face. Some proteins remain in the Golgi; others will reach the trans-Golgi network and become secretory proteins, a part of the plasma membrane, or lysosomal proteins.

Membrane ---:,r---t->JI(,i vesicles

Rough Endoplasmic Reticulum

Figure 111-3-5. Goigi apparatus.

C. O-linked oligosaccharides are attached to the hydroxy group on the side chain of serine,

threonine, or hydroxylysine residues in proteins. In contrast to N-linked oligosaccharides, they are not assembled prior to transfer and there is no dolichollipid intermediate involved. The O-linked oligosaccharides are built up one monosaccharide at a time on the protein. D. Packing, concentration, and storage of secretory granules occurs in the trans-Golgi network. Secretory proteins are transferred as dilute solutions to condensing vacuoles located on the trans face of the Golgi, where they become concentrated into membrane-bound secretory granules or vesicles. The vesicles bud off the TGN and release their contents into the extracellular space via exocytosis. E. Modification of glycoproteins

In a Nutshell O-linked Glycosylation • Occurs in Golgi apparatus after translation is complete • Adds sugars to Ser or Thr • Adds sugars one at a time • Tends to be less complex than N-linked glycosylation

1. Phosphorylation of mannose in glycoproteins is a signal that targets proteins to the lyso-

somes. Lysosomes have receptors that recognize and bind proteins with mannose-6phosphates prior to uptake. The phosphate is added in a two-step sequence of reactions that are catalyzed by N-Ac-glucosamine phosphotransferase and N-Ac-glucosaminidase. The first enzyme transfers N-Ac-glucosamine-l-P from UDP-N-Ac-glucosamine to specific mannose residues in the oligosaccharide chain. The second enzyme removes N-Ac-glucosamine, leaving phosphate attached to the 6-position of mannose. 2. Sulfation of oligosaccharide chains of proteoglycans occurs in the Golgi.

meclical

193

Cell Biology

3. Limited proteolysis of precursor proteins, e.g., pro insulin is cleaved to insulin and C-peptide in the Golgi. F. Diseases associated with Golgi apparatus include I-cell disease and hyperproinsulinemia.

Note I-cell disease patients lack the enzyme necessary to create mannose-6-phosphate on lysosomal enzymes. Therefore, these enzymes follow the default (no signals needed) pathway of constitutive exocytosis and are secreted from the cell.

1. I-cell disease is a disease in which a whole family of enzymes is sent to the wrong destination. The cause of the disease is a deficiency in N-Ac-glucosamine phosphotransferase, which is one of the two enzymes required to convert specific mannose residues to mannose-6-phosphate in glycoproteins (hydrolases) destined for lysosomes. Individuals with this disease are characterized by having huge "inclusion bodies" in cells due to the accumulation of undegraded glycoconjugates in lysosomes. The lysosomes are missing the hydro lases that normally degrade these macromolecules. The missing enzymes are found in the plasma and other body fluids, where they have normal levels of activity. The absence of the mannose-6-phosphate on the hydrolases results in their secretion rather than their incorporation into lysosomes.

2. Hyperproinsulinemia is characterized by elevated levels of proinsulin in the serum resulting from a deficiency in the peptidase associated with the secretory granules that contain proinsulin. The clinical manifestations are similar to those seen in patients with n oninsulin -dependent diabetes.

LYSOSOMES The lysosomes are spherical membrane-enclosed organelles that are approximately 0.5 diameter and contain enzymes required for intracellular digestion.

~m

in

A. Types of lysosomes. Lysosomes are a heterogeneous population of organelles, whose contents depend upon the degradation stage of the material. 1. Primary lysosomes have not yet acquired the materials to be digested. They are formed by budding from the trans side of the Golgi. 2. Secondary lysosomes are formed by the fusion of the primary lysosome with the substrate to be degraded and have contents that are in various stages of degradation.

Clinical Correlate Lysosomal storage diseases are caused by a lack of lysosomal enzymes or transporters, resulting in an accumulation of toxic materials.

B. Hydrolytic enzymes. Approximately 60 hydrolytic enzymes have been identified in lysosomes. These include nucleases for degrading DNA and RNA, lipases for degrading lipids, glycosidases for degrading glycoconjugates (glycoproteins, proteoglycans, and glycolipids), proteases and peptidases for degrading protein, and a variety of phosphatases. The specific complement of enzymes varies significantly between different lysosomes. All of the lysosomal enzymes are acid hydrolases, with optimal activity at a pH of approximately 5.0. The requirement for an acidic environment prevents this class of enzymes from causing damage if accidentially released into the cytoplasm. The synthesis of the lysosomal hydrolases occurs in the RER; the hydrolases are transferred to the Golgi, where they are modified and packaged into lysosomes. The key modification reaction that targets these hydrolytic enzymes to lysosomes is the addition of phosphate to the 6-position of mannose residues of their oligosaccharide chains. C. Functions. The major fuction of lysosomes is to degrade macromolecules to their building

blocks. The material to be degraded may be either extracellular or intracellular. The degradation of intracellular materials such as membrane or organelles is known as autophagy. Extracellular materials are taken up by endocytosis. 1. Fluid-phase endocytosis. Many large molecules enter the cell by the active invagination of the plasma membrane, resulting in an endocytic vesicle that fuses with a lysosome.

194

meClical

Subcellular Organelles

2. Receptor-mediated endocytosis. Some extracellular molecules recognize receptors that are widely dispersed over the plasma membrane. The binding of a molecule (ligand) to its receptor causes the receptors to aggregate in special regions called clathrin-coated pits, which invaginate, allowing the receptor and its ligand to enter the cell as a coated vesicle. For example, receptor-mediated endocytosis is the process by which cells take up LDLcholesterol. The vesicle loses its coat and then fuses with another vesicle to form an endosome. Within the endosome, the LDL receptor becomes incorporated into a membrane vesicle that buds off and returns to the cell surface. The remaining part of the endosome fuses with a primary lysosome, forming a secondary lysosome. In the secondary lysosome, the cholesterol esters are hydrolyzed to form free cholesterol.

Note The decreased pH of the endosome typically causes the ligand (e.g., LDL) to dissociate from its receptor, allowing receptor recycling.

PEROXISOMES The peroxisomes are a heterogeneous group of small, spherical organelles with a single membrane and a diameter that ranges from approximately 0.15 to 0.5 ~m. They contain a number of enzymes that transfer hydrogen atoms from organic substrates (urate, D-amino acids, very long chain fatty acids) to molecular oxygen with the formation of hydrogen peroxide. Catalase, the major peroxisomal protein, degrades the hydrogen peroxide to water and oxygen. A. Functions. In addition to the synthesis and degradation of hydrogen peroxide, peroxisomes are important in a number of other metabolic activities. 1.

~-oxidation of very long chain fatty acids (>C24 ) starts in the peroxisome and proceeds until the carbon chain has been reduced to a length of approximately C lO . Oxidation of the residual C lO is completed in the mitochondria.

2. Phospholipid exchange reactions occur in the peroxisome. Phospholipids are synthesized by the endoplasmic reticulum and are imported into the peroxisome, where enzymes convert phosphatidylserine and phosphatidylethanolamine.

Note Proteins destined for import into peroxisomes are fully translated in the cytosol and contain a (-terminal amino acid sequence that directs them to the peroxisome import machinery.

3. Bile acid synthesis occurs in peroxisomes. B. Synthesis of peroxisomal enzymes occurs on polysomes. After translation, the enzymes are incorporated directly into peroxisomes. C. Peroxisomal deficiency. Several genetic diseases are associated with the impairment or absence of peroxisomes. These patients fail to oxidize very long chain fatty acids and accumulate bile acid precursors. The four most common disorders are Zellweger cerebro-hepato-renal syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and hyperpipecolic acidemia.

Clinical Correlate Zellweger syndrome is caused by a lack of the machinery (proteins) needed to recognize and import the various peroxisomal enzymes.

MITOCHONDRIA Mitochondria are similar to bacteria in size and shape. They have a characteristic cigar shape, with a width of 0.5 ~m and a length that can vary from 1-10 ~m. They are found scattered throughout the cell. Liver cells contain 700-1,000 mitochondria per cell, each with a half-life of approximately 10 days. Mitochondria contain their own double-stranded circular DNA, and they make some of their own proteins. Like bacteria, mitochondria reproduce by dividing. A. Mitochondrial compartments. As shown in Figure III-3-6, mitochondria are defined by a double membrane.

meCtical

195

Cell Biology

Cristae -l-_-Outer membrane Inner membrane Intermembrane compartment

Figure 111-3-6. Structure of the mitochondrion.

1. The outer membrane is smooth, continuous, and permeable. It contains an abundance of porin, an integral membrane protein that forms channels in the outer membrane through which molecules ofless than 10 kD can pass. 2. The inner membrane is impermeable to most small ions (Na+, K+, H+) and small

Note The electron transport chain pumps protons into the intermembrane space, creating a proton (electrochemical) gradient. The protons move back into the matrix through the Fa channel of ATP synthase, releasing energy for ATP synthesis.

molecules (ATP, ADP, pyruvate). The impermeability is likely related to the high content of the lipid cardiolipin. The inner membrane has numerous infoldings, called cristae, that greatly increase the total surface area. The enzymes for electron transport and oxidative phosphorylation are imbedded in the inner membrane. The number of mitochondria and the number of cristae per mitochondrion are proportional to the metabolic activity of the cells in which they reside. For example, both cardiac tissue and kidney tubule cells require large amounts of ATP for their normal function. These cell types therefore have an abundance of mitochondria with a large number of closely packed cristae. 3. The intermembrane compartment is the space between the inner and outer membranes. It contains enzymes that use ATP to phosphorylate other nucleotides (creatine phosphokinase and adenylate kinase). 4. The matrix is the region enclosed by the inner membrane, which contains several different components: a. Dehydrogenases oxidize many of the substrates in the cell (pyruvate, amino acids, fatty acids), generating NADH and FADH2 for use by the electron transport chain and energy generation. b. Double-stranded circular DNA genome and the enzyme machinery necessary for its replication and expression are all located in the matrix. The human mitochondrial nucleotide sequence encodes a few of the mitochondrial proteins. Mitochondrial DNA is always inherited from the mother, resulting in the maternal transmission of diseases of energy metabolism.

Note Most proteins destined for the mitochondria are completely translated in the cytosol but contain N-terminal amino acid sequences that direct them to the mitochondrial import machinery. Import requires unfolding, ATP, a membrane potential, and refolding. 196

meClical

c. RNA, proteins, and ribosomes allow mitochondria to carry out protein synthesis. However, most mitochondrial proteins are synthesized in the cytoplasm and are transferred into the mitochondria. d. Intramitochondrial granules containing calcium and magnesium are found in the matrix. Their function is not known, but it is believed that they may represent a storage site for calcium. B. Mitochondrial function. The major function of the mitochondria is to synthesize ATP

(Figure I1I-3-7). Cellular oxidation of glucose starts in the cytoplasm, where it is converted to pyruvate by glycolysis, yielding two molecules of ATP per glucose. Pyruvate is transported into the mitochondrial matrix, where pyruvate dehydrogenase oxidizes it to acetyl-CoA, the substrate for the TCA cycle.

Subcellular Organelles

glucose

Glycolysis cytosol

-~--,,--:-----+-.

pyruvate

inner membrane

NAD+

Intermembrane compartment

(proton pumps)

Figure 111-3-7. Energy generation by the mitochondria.

meCtical

197

Cytoskeleton

The cytoskeleton provides a supportive network of tubules and filaments in the cytoplasm of eukaryotic cells. The cytoskeleton is a dynamic structure responsible for cellular movement changes in cell shape, and contraction of muscle cells. It provides the machinery for intracellular movement of organelles. It is composed of three types of supportive stuctures: microtubules, intermediate filaments, and microfilaments. This chapter reviews the structure and function of each of the cytoskeletal components.

MICROTUBULES Microtubules are polymers of tubulin that undergo rapid assembly and disassembly. They are found in the cytoplasmic matrix of all eukaryotic cells. Microtubules have been implicated in a large number of cell functions, including mitotic and meiotic chromosomal movement, transport of vesicles from one site to another within the cell, and ciliary and flagellar motility. A. Structure. The major component of microtubules is tubulin, a protein dimer composed of two different polypeptides, a-tubulin and /3-tubulin. When viewed by electron microscopy, microtubules are hollow cylinders with a diameter of approximately 24 nm. A crosssectional view shows that the wall of the cylinder is made up of 13 protofilaments, each consisting of an alignment of tubulin dimers. The individual protoffiaments are arranged such that the a- and /3-tubulin subunits alternate along the length of the protoffiament. The tubules have a diameter of approximately 24 nm, and the wall is 5-6 nm thick. B. Assembly. Polymerization of tubulin to form microtubules is accomplished by microtubule organizing centers (MTOC) and two types of accessory proteins: tau proteins and microtubule-associated proteins (MAPs). Microtubules grow from the organizing centers. Both tau and MAPs, when purified, induce polymerization of tubulin and become bound to newly formed microtubules. Assembly can occur spontaneously in vitro in the presence of MAPs, GTP, and calmodulin. When the incubation is carried out at 37°C, self-assembly occurs with the hydrolysis of one molecule of GTP per microtubule. Lowering the temperature to 4°C results in depolymerization. As long as an adequate concentration of GTP is present in the reaction, it is possible to polymerize and depolymerize the microtubules indefinitely. Calcium can block or reverse polymerization, suggesting that calcium may also modulate microtubule formation in the cell. C. Polarity. Microtubules have polarity (a plus and a minus end). The minus ends are oriented

toward the inside of the cell and associated with the microtubule organizing center. All of the assembly and disassembly occurs at the plus ends of the microtubules, which are oriented toward the periphery of the cell.

In a Nutshell Microtubules are hollow cylinders formed by long protofilaments of tubulin dimers.

Note Typically, the negative end of microtubules is found at the centrosome (or basal body of cilia and flagella).

KAPLAlf I meillea

199

Cell Biology

D. Movement associated with microtubules. There are three major types of movement that involve microtubules. 1. Chromosomal movement during meiosis and mitosis depends on the activity of microtubules. Microtubule assembly is an important event in spindle formation. 2. Intracellular vesicle and organelle transport requires microtubules. Two specific microtubule-dependent ATPases, kinesin and dynein, are involved in the force generation, with the microtubules playing a more passive role in intracellular transport. 3. Ciliary movement is produced by the cilia beating in S-shaped waves and is important in clearing mucus from the respiratory tract. Movement is produced by the axoneme, a complex structure composed of micro tubules and a variety of other proteins that allow the beating action to occur. The structure of the axoneme is shown in Figure III -4-1. a. In axonemes, there are two central micro tubules surrounded by a circle of nine peripheral microtubule doublets. The peripheral doublets are fused so that they share a common tubule wall and form two subtubules (A and B). Subtubule A is a complete microtubule, but subtubule B is only a partial subtubule. The central singlets are connected by a bridge that runs between them and a sheath that surrounds them. Subtubule A of each peripheral doublet is connected by a spoke to the sheath. Adjacent doublets are connected to one another by nexin links.

Spoke - / + - - -

Central singlet

membrane

sheath

In a Nutshell Dynein moves toward to negative end; this powers axoneme bending in cilia and flagella and vesicle movement toward the nucleus (centrosome) or away from the nerve terminal in axons (retrograde).

200

ineilical

Bridge Figure 111-4-1. Structure of the axoneme.

b. A pair of dynein arms is attached to each A subtubule. The arms bind to ATP and rearrange themselves so that a binding site for the B subtubule in the tip of the arm is exposed. The B tubule interacts with the binding site, causing the arm to snap back and movement to occur. Each cycle of a single dynein arm slides adjacent doublets 10 nm past each other.

Cytoskeleton

E. Diseases associated with microtubules. Two disease states involve malfunction of microtubules: Chediak-Higashi syndrome and bronchiectasis. l. Chediak-Higashi syndrome is characterized by a defect in the ability of microtubules to polymerize within leukocytes. This leads to a delay in the fusion of phagosomes with lysosomes, thus preventing phagocytosis of bacteria.

2. Bronchiectasis is caused by immotile cilia in the respiratory epithelia, resulting in the inability of cilia to remove inhaled bacteria.

F. Drugs affecting tubulin polymerization may either promote or inhibit polymerization; both groups inhibit microtubule function. 1. Inhibitors include colchicine (which binds to tubulin and blocks polymerization), vinblastin, and vincristine. 2. Promoters are taxol (causes free tubulin to assemble) and D 20 (increases polymerization).

Bridge to Pharmacology Colchicine stops cell division during metaphase.

INTERMEDIATE FILAMENTS These components of the cytoskeleton, with a diameter of 10 nm, are intermediate in thickness between microtubules and microfilaments. These rope-like filaments function primarily in structural roles. Intermediate filaments are classified into three types: keratin filaments, vimentin-containing filaments, and neurofilaments. These tough and durable filaments are found predominantly in those parts of the cell that are subjected to mechanical stress, where they function to maintain cell shape and the positioning of organelles within the cell.

A. Keratin filaments are made up of a family of 20 closely related proteins. They are found in epithelial tissue, where they strengthen the epithelium. B. Vimentin-containing filaments are found in nonepithelial cells such as fibroblasts, chondrocytes, endothelial cells, and macrophages. Vimentin can copolymerize with other proteins, some of which are cell specific. For example, the intermediate filament in fibroblasts is made up solely of vimentin; those of glial cells and astrocytes contain both vimentin and glial fibrillary acidic protein; those of some muscle cells contain vimentin and desmin.

Note Keratin filaments form the tonofilaments that connect desmosomes in epithelial cells.

C. Neurofilaments are found in most neurons of the central and peripheral nervous system,

where they extend along the length of the axon. They are the major cytoskeletal component of nerve cells.

MICROFILAMENTS These filaments have a diameter of 6 nm and are composed of actin. Each actin filament (F-actin) consits of two strands of actin twisted into a helical pattern with 13.5 molecules of globular actin (G-actin) per turn of the helix. The globular monomer of actin is stabilized by one molecule each of calcium and ATP. The polymerization of G-actin to give F-actin is accompanied by the hydrolysis of ATP to ADP. Microfilament polymerization is believed to be under the control of small fluctuations in the concentration of calcium ions and cAMP.

In a Nutshell Microfilaments are doublestranded helices of polymerized actin.

A. Actin-binding drugs can interfere with the polymerization-depolymerization cycle of micro filaments. B. Movement associated with microfilaments. Two types of movement are dependent upon micro filaments. Local movement takes advantage of the polymerization and depolymerization properties of microfilaments. Sliding filament movement is generated by the interaction of actin filaments with myosin filaments.

meCtical

201

Cell Biology

1. Cytochalasin B inhibits the assembly of actin fllaments by binding to one end of the fll-

ament and preventing the addition of actin molecules to the fllament. Processes such as endocytosis, phagocytosis, cytokinesis, and cytoplasmic and amoeboid movements are all inhibited by cytochalasin B. 2. Phallodin binds along the length of the actin fllament, preventing depolymerization.

In a Nutshell • Myofibers are large multinucleated skeletal muscle cells. • Myofibrils are composed of tandem arrays of sarcomeres, or clusters of thin or thick filaments; these are the contractile elements. • Myofilaments are the thin (actin) and thick (myosin) filaments that slide past one another during contraction.

Note During contraction, the Z lines approach one another and the I bands get smaller, but the A band does not change size.

C. Microfilaments and muscle contraction. Skeletal (striated) muscle consists of long, cylin-

drical muscle fibers. These large fibers are multinucleated and arise from the fusion of a large number of precursor cells (myoblasts). The cytoplasm of these fibers is divided into discrete units (myofibrils), which extend the entire length of the muscle fiber. 1. Myofibrils are the contractile elements of muscle cells and are surrounded by a large

number of mitochondria that supply the ATP required for contraction. All other cellular components that are not involved in contraction are pushed aside and are tucked in between the outermost myofibril and beneath the sarcolemma (the plasma membrane of muscle cells). The ultrastructure of a myofibril is shown in Figure III-4-2. a. The sarcomere is the repeating contractile unit in the myofibril. Sarcomeres are separated from one another by the Z line. The A band spans the width of the myosin thick fllaments, including the region where they overlap with the thin fllaments. The actin thin fllaments are connected to the Z line and extend toward the center of the sarcomere, where they overlap with myosin fllaments. Each A band is flanked by I bands, the region containing actin fllaments only. b. Myosin molecules are long fibrous structures composed of six subunits: two heavy chains and four light chains. Each myosin heavy chain consists of a globular head and a fibrous tail. The two fibrous tails of the heavy chains are twisted into a double helical structure, forming regions where myosin molecules interact to form filaments. The globular heads of the myosin molecule contain ATPase activity as well as sites for binding to actin fllaments. c. Tropomyosin and troponin are involved in the regulation of actin and myosin interaction.

2. Tropomyosin is a fllamentous protein containing two subunits. It lies in the grooves on either side of the F-actin fllament and mediates access to the site on actin monomers that bind to the myosin head. Tropomyosin is found in actin fllaments of both muscle and nonmuscle cells. 3. Troponin is a complex of three subunits: T, I, and C. Troponin T binds to tropomyosin and, together with troponin I, inhibits the interaction of actin and myosin. Troponin C has a binding site for Ca2+, and when calcium is bound, actin and myosin interaction is promoted. Troponin is found only in skeletal muscle cells. Tropomyosin and troponin have a fixed relationship with F-actin: one molecule of tropomyosin and one molecule of troponin bind per every seven actin monomers.

202

meClical

Cytoskeleton

Myofibril

Sarcomere

F-Actin filament Myosin filament

C~;Jj.;~

C=:~ent

G-Actin molecules

CJIL--__

Heavy Light meromyosin meromyosin Figure 111-4-2. Ultrastructure of a myofibril.

a. Accessory proteins of the myofibril. As described below, several other proteins are associated with the sarcomere. (1) a-Actinin binds and anchors actin filaments to the Z line.

Note

(2) Myomesin cross-links adjacent myosin filaments at the M line (the center of the sarcomere).

Desmin, an intermediate filament, aligns adjacent myofibrils in the myocyte cytoplasm, giving it the classic "striated" appearance.

(3) Desmin is found at the Z line, where it holds myofibrils in place. (4) Titin is a large fibrous protein that extends over half of the sarcomere. It has elastic properties and keeps the myosin thick ftlaments centered in the sarcomere. (5) Nebulin is a large fibrous protein attached to the Z line and extends the length of the thin ftlament. It stabilizes the highly ordered structure of the myofibril by regulating the assembly of the actin filaments. D. Actin-myosin interaction in skeletal muscle. In relaxed muscle, the actin ftlaments extend

approximately halfway over the myosin ftlament (see Figure III-4-2). During contraction, the actin ftlaments slide toward each other, past the myosin ftlaments, resulting in the shortening of the sarcomere, the myofibril, and the length of the muscle. This is known as the sliding filament model of muscle contraction. There is no change in the length of either types of filaments. The movement of myosin along the actin ftlament is ATP dependent. The myosin head binds ATP,

Note Smooth muscle contains actin filaments attached to the plasma membrane as well as myosin filaments, but the fibrils are not aligned. When the myosin slides along adjacent filaments, the cell contracts in all directions. KAPLAff I me illea

20J

Cell Biology

which is hydrolyzed to ADP and Pi by myosin ATPase. ADP and Pi remain bound to the ATPase site. The myosin head moves to an adjacent thin filament, and as it makes contact with the thin filament, it undergoes a conformational change and releases ADP and Pi. A second molecule of ATP binds to the myosin head, causing it to be released from the thin filament. Hydrolysis of ATP results in the myosin head resuming its original conformation. The myosin head is then ready for another cycle.

Bridge to Physiology • In skeletal and cardiac muscle, increased Ca 2+ binds to troponin, which shifts tropomyosin, allowing actin to bind myosin, thereby activating myosin ATPase activity. • In smooth muscle, increased Ca 2+ binds myosin light chain kinase, which then phosphorylates the myosin (or its light chain), thereby activating its ATPase activity and initiating contraction.

204

meClical

E. Troponin and tropomyosin in muscle contraction. Control of muscle contraction is provided by troponin, tropomyosin, and a change in intracellular calcium concentrations. When the calcium concentration is low (10- 7 M), troponin I and troponin T are strongly bound to tropomyosin, holding it in the actin groove so that it blocks the myosin binding site on the actin monomers. Troponin C is either loosely associated with tropomyosin or is bound to troponin I and troponin T. When calcium concentrations increase above 10-7 M in the cell, troponin C binds calcium and undergoes a change in conformation, forcing troponin I and troponin T to move. The movement exposes the myosin binding site on the actin, permitting interaction between the two filaments to occur. This, however, does not necessarily result in contraction. Contraction can result only if ATP is present. F. Control of intracellular calcium concentrations. Small membranous folds, known as

T-tubules, extend from the sarcolemma and surround each myofibril at the Z line. The T-tubules act as a communication system for the depolarization of the plasma membrane that occurs when a nervous stimulation signals the muscle to contract. Depolarization of the sarcolemma is relayed to the sarcoplasmic reticulum (SR), a sheath of flattened vesicles that surrounds the myofibrils and stores large quantities of calcium. Normally, extracellular calcium concentrations are high (10- 3 M), whereas those in the cytosol are low (10- 7 M). Upon depolarization, the calcium levels rise from 10-7 to greater than 10-5 M during contraction. The increase is due primarily to the movement of calcium ions through Ca2 + release channels in the membrane of the sarcoplasmic reticulum. The change in calcium concentration occurs rapidly and relieves the inhibition of myosin binding to actin. Once the stimulation of the nerve ceases, calcium is pumped back into the sarcoplasmic reticulum by the action of a SR-specific ATPase. The calcium is bound in the SR by calsequesterin, an acidic protein with a high density of aspartate and glutamate residues.

Cellular Adhesion and Extracellular Matrix The relationship between the cell and its environment is controlled by specialized macromolecular complexes that bind cells together to form tissues and organs. The plasma membrane of mammalian cells has specialized junctional complexes that allow cells to bind to neighboring cells or to the extracellular matrix, which provides a structural framework for the association of cells. This chapter reviews the basic structure of the different types of cell junctions and the extracellular matrix and the way that they all function in relating the cell to its environment.

CELL JUNCTIONS Cell junctions anchor cells to each other, seal boundaries between cells, and form channels for direct transport and communication between cells. Each of the three types of junctional complexes has a characteristic ultrastructure (Figure III-5-l) designed to perform a specific function. A. Anchoring junctions are present in all cell types but are most numerous in tissues that are subject to extreme mechanical stress, such as the epithelial cells of skin, intestine, and uterus. These junctions couple neighboring cells to one another through interactions that are mediated by the cytoskeleton. Anchoring junctions are found in both vertebrates and invertebrates and are important in both adhesion and cellular movement. There are two major types of anchoring junctions, based on the type of cytoskeletal element involved: adherens junctions and desmosomes. Adherens junctions connect groups of actin filaments in adjacent cells, whereas desmosomes connect intermediate filaments of adjacent cells.

meCtical

205

Cell Biology

-tt--- Microvilli

Apical surface

Plasma membrane Tight [ junction Adhesion ,-------' belt

§~~~~~~ Intermediate I 'Alf=IIlAI--filaments (keratin)

. .~It-IIt---

Hemidesmosome

Basal lamina

Figure 111-5-1. Cell junctions.

1. Adherens junctions can couple cells with other cells or with a component of the extra-

cellular matrix. The junctional complexes are composed of two major proteins: intracel-

lular attachment proteins (lAP) and transmembrane linker glycoproteins (TLG). As shown in Figure III-5-2, lAP has binding sites for both TLG and actin filaments. TLG spans the plasma membrane and interacts with another TLG from a neighboring cell or with the extracellular matrix.

Transmembrane linker -t----t-t------+-+--, glycoprotein

-I-+----+.,.-

Intracellular attachment proteins

+-_ _ __+_ Cytoskeletal element

Plasma membrane Extracellular matrix

Transmembrane - - - - - linker glycoprotein

Figure 111-5-2. Adherens junctions.

206

ii1e&ical

---~----

Cellular Adhesion and Extracellular Matrix

a. Cell-cell adhesion junctions form an "adhesion belt" (also known as the zonula adherens) that encircles each of the interacting cells (see Figure III -5-1). These junctions are found between cells in epithelial sheets and are located in the apical region of the cells. The adhesion belt contains contractile actin filaments. Contraction of the adhesion belt is responsible for many of the morphologic changes that occur in embryonic development. One of the characteristic features of cardiac muscle is that adjacent cells are joined by intercalated discs, which are composed of adhesion junctions. The discs are L-shaped structures that allow cardiac muscle cells to interdigitate with one another, forming fibers. b. Cell-matrix junctions connect cells and their actin filaments to the extracellular matrix. These junctions are also known as adhesion plaques and are important in some types of cellular movement. 2. Desmosomes are button-like structures (see Figure III-5-1) that bind to intermediate filaments, anchoring them in place. The type of intermediate filament attached to the desmosome depends on the cell type. For example, epithelial cell desmosomes are attached to keratin filaments, brain desmosomes are attached to vimentin filaments, and cardiac muscle cell desmosomes are attached to desmin filaments. When viewed in the electron microscope, desmosomes appear as dense plaques. These plaques, known as desmoplakins, are composed of intracellular attachment proteins. The intracellular attachment proteins bind to transmembrane linker proteins, known as desmogleins, which hold adjacent cells together. These points of attachment between cells are known as "spot welds;' and the interaction is mediated by calcium. Hemidesmosomes anchor the basal surface of epithelial cells to the extracellular matrix. B. Tight junctions form a physical barrier between adjacent cells whose plasma membranes come in direct contact with one another. 1. Structure. Tight junctions are composed of a network of membrane proteins that

form a belt-like structure that encircles epithelial cells along their apical surface (Figure III-5-3). The junction between cells is so tight that no extracellular space can be seen. 2. Function. Tight junctions prevent the movement of small molecules between the two cells. They also block the exchange of lipid molecules between the outer region of the two plasma membranes that are in direct contact.

Clinical Correlate Pemphigus is a potentially fatal autoimmune disease that affects the skin. Individuals with this disease produce antibodies against transmembrane linker glycoproteins. The antibodies bind to these proteins and disrupt the desmosome structure, resulting in severe blistering and leakage of body fluids into the loosened epithelia. Only the desmosomes in skin are affected, suggesti ng that there are tissue-specific isoforms of desmosomes.

KAPLAlf I medlea 207

Cell Biology

Apical surface Microvilli Plasma membrane Intercellular space tMf--

Tight junction

Tight junction protein 14 nm

CeliA

Cell B

CeliC

Cell D

____ Basal membrane

Basal lamina

Figure 111-5-3. Tight junctions.

C. Gap junctions, also known as communication junctions, attach adjacent cells in a way that

allows transfer of chemical and electrical signals. 1. Structure. The key structural component of gap junctions is the connexon, a hollow cylinder made up of transmembrane proteins (Figure III-S-4). Connexons of adjacent cells interact with one another to form a continuous channel that is open to the interior of both cells. Although connexons of neighboring cells are in direct contact, the plasma membranes of the two cells are separated by a slight gap of 2-4 nm.

Bridge to Physiology Increased intracellular Ca 2+ will close gap junctions. This prevents the death of one cell from killing its coupled neighbors.

208

2. Function. Gap junctions permit communication between cells by allowing small ions or water-soluble molecules to pass between cells. The phenomenon of cell coupling via gap junctions provides a mechanism for coordinating the activities of individual cells. Cells may be coupled both chemically and electrically. Gap junctions are prominent in nerve and brain cells, cardiac muscle, smooth muscle, and liver cells. In nerve cells, gap junctions set up direct electrical connections that fuse the two cells into a single electrical unit. This communication is maintained by the concentration of Ca2+ and cAMP levels. An increase in the cytosolic Ca2 + concentration decreases the permeability of gap junctions, whereas an increase in cAMP increases the ability of the cells to communicate.

meclical ~---~~--~~----~----~---

Cellular Adhesion and Extracellular Matrix

Connexon

2-4nm

7nm Figure 111-5-4. Gap junctions.

EXTRACELLULAR MATRIX The extracellular matrix provides a structural framework for the organization of cells into tissues and organs. Three major types of macromolecules make up extracellular matrix: proteoglycans, structural proteins, and adhesive proteins. A. Proteoglycans are composed of approximately 90-95% carbohydrate and 5-10% protein. 1. Structure. The carbohydrate components of proteoglycans are known as glycosamino-

glycans (GAGs). They are long linear polysaccharide chains made up of repeating disaccharide units. The polysaccharide chain is linked to protein through a linker oligosaccharide that is common to all proteoglycans. 2. Function. Proteoglycans form a hydrated gel in which the fibrous proteins are embedded. These molecules serve as excellent shock absorbers and lubricants. B. Structural proteins. The two major structural proteins are collagen and elastin, both of

which are fibrous proteins. 1. Collagen is composed of three polypeptide chains wound around one another to form a

triple helical structure. They are highly enriched in glycine and proline (every third residue is glycine) and contain hydroxylysine and hydroxyproline. There are at least 30 different collagen peptide chains. Types I-IV are of primary significance. Collagens I-III are fibrous forms of collagen. Collagen IV is assembled into a sheet-like meshwork and is located exclusively in the basal lamina. The major function of collagen is to strengthen and organize the extracellular matrix. 2. Elastin is the main component of elastic fibers. These fibers are also rich in glycine and proline. After secretion into the extracellular space, they are cross-linked with each other via desmosine and isodesmosine residues to provide a mesh structure. Their major function is to provide elasticity to tissues such as lungs and to large arteries leaving the heart. C. Adhesive proteins. The two major adhesive proteins found in the extracellular matrix are

fibronectin and laminin. 1. Fibronectin is a large fibrous protein consisting of peptides that are similar but not iden-

tical. These polypeptides are held together by disulfide bonds. Fibronectin is synthesized by fibroblasts, monocytes, and endothelial cells. It binds to both the cell and to several

Bridge to Physiology Vitamin C is required for the formation of hydroxyproline in collagen and for the formation of interchain hydrogen bonds that stabilize the triple helix.

In a Nutshell Fibronectin is necessary for the formation of fibroblast adhesion plaques and for fibroblast movement.

meclical

209

Cell Biology

other matrix macromolecules, thereby providing a linkage between the cell and the extracellular matrix. Fibronectin is found in several locations. a. Cell surface fibronectin appears to be transiently present on the cell surface. b. Plasma fibronectin increases blood clotting and promotes wound healing. c. Extramatrix fibronectin is highly insoluble and stabilizes cells within the matrix. 2. Laminin is the major glycoprotein in the basal lamina, where it forms continuous mats of polypeptide underlying all epithelial cell sheets. It is secreted by epithelial cells and is composed of a complex of three polypeptide chains arranged in the shape of a cross. The chains are held together by disulfide bonds. Laminin has numerous functional domains, each binding a different protein such as collagen IV, heparan sulfate, etc. Laminin mediates the attachment of cells to connective tissue.

210

meClical

SECTION IV

Genetics

Mendelian Inheritance

The simplest inheritance patterns depend upon the genotype of an individual at a single locus. Traits inherited in this manner are said to follow a mendelian inheritance pattern. The mode of inheritance of a trait within a family can help suggest a diagnosis, which can be confirmed with diagnostic tests. It can also provide information to relatives regarding the probability of their being affected with the same condition.

TERMINOLOGY A. In a mendelian sense, a gene is an inherited factor that determines a given genetic characteristic of an organism. An individual has two copies of most genes, although the copies may not be identical. Alternative expressions of the same gene are called alleles. The locus of a gene refers to its physical location on a chromosome. B. The genotype is the genetic constitution of an individual at a given locus. The phenotype is

the observable expression of a particular gene in an organism due to the interaction of the gene with other genes and the environment. An individual is homozygous when he possesses two copies of a similar allele of a gene pair (e.g., AA or aa). An individual is heterozygous when he has two different alleles of the gene (e.g., Aa). Hemizygous refers to the special case in which an individual normally has only a single copy of a gene, as in the case of X-linked genes in males. C. An allele is said to be dominant if the phenotype associated with it is expressed in heterozygotes. An allele is recessive if it requires the homozygous condition to be expressed. A carrier

is an individual who carries a deleterious allele, but does not express the phenotype. Incomplete dominance describes the condition in which two different alleles produce a blended effect in the heterozygote. The clearest examples of incomplete dominance occur in plants. When certain plants are heterozygous for red and white flower pigments, pink flowers are produced. For dominant diseases, penetrance can be defined quantitatively by determining the proportion of obligate gene carriers who express the phenotype. This frequency is given as a percent (i.e., polydactyly has a penetrance of 95%). Expressivity is the degree to which a particular effect is expressed by individuals. Pleiotropy is the diverse effects of a single gene on several different organ systems and functions. The same mutant allele that causes pleiotropic effects will show variable expressivity in different individuals because of the effects of other genes modifying its expression or environmental interactions.

In a Nutshell Gene: an inherited factor that determines a given genetic characteristic

Allele: alternative expression of a gene Locus: the physical location of a gene on a chromosome Genotype: the genetic constitution of an individual at a single locus Phenotype: the observable expression of the interaction of a genotype with the environment Homozygous: having two similar alleles at a locus Heterozygous: having two different alleles at a locus Hemizygous: having one gene at a locus, characteristic of X-linked genes in males

KAPLA~.

meulca

I

213

Genetics

D. Pedigree analysis is a method of recording family information to trace the passage of a gene through generations. Figure IV-l-l summarizes basic pedigree nomenclature.

Generation

II III IV

2

D 0

0

•• C)[J

®

•

3

Male

00

Dead

Female

0-0

Mating

Unknown sex

0=0

Consanguineous or incestuous mating

Affected

DO

Sibship

DO

Dizygous twins

Carrier of an autosomal recessive (optional) Carrier of an X-linked recessive (optional) Stillborn or abortion

Figure IV-1-1. Pedigree nomenclature.

AUTOSOMAL DOMINANT TRAITS A. Characteristics 1. Of the over 5,000 catalogued traits that exhibit Mendelian inheritance, over 50% are inherited in an autosomal dominant manner. Figure IV-1-2 illustrates a typical autosomal dominant inheritance pattern.

In a Nutshell Autosomal dominants affect both male and female children and generally do not skip generations because only one copy of the mutant allele is needed for expression of the trait.

Figure IV-1-2. Autosomal dominant inheritance pattern.

2. The phenotype can be expressed if a dominant allele is present in only one dose. In other words, only one mutant allele need be present for the disease to be expressed. 3. Deleterious autosomal dominants occur at very low frequencies, so affected individuals are almost always heterozygous for the trait.

214

meClical

Mendelian Inheritance

4. Affected parents can have either affected or normal children. The characteristic does not "skip" generations, unless there is reduced penetrance. 5. Because the trait is carried on an autosome rather than a sex chromosome, both male and female progeny can be affected. B. Diseases

1. Familial hypercholesterolemia is a condition characterized by increased serum cholesterolleading to premature atherosclerotic disease and myocardial infarctions early in life. Homozygotes for this disorder lack functional LDL receptors and cannot internalize LDL or IDL via receptor-mediated phagocytosis. Their plasma LDL-cholesterollevels are three to five times that of normals. Heterozygotes have about half the normal number of functional LDL receptors and their LDL-cholesterollevels are twice that of normals. The incidence of homozygotes is approximately 111,000,000, and the incidence of heterozygotes is 11500. 2. Huntington disease is an autosomal dominant of late onset that leads to neurological degeneration, emotional disturbances, cognitive decline, and chorea. It is caused by the loss of neurons in the neostriatum and cortex. The gene responsible has been mapped to chromosome 4 (4pI6.3). It codes for a 348 kD polypeptide and contains a CAG trinucleotide repeat. In normal individuals, this repeat is present 11 to 34 times. In individuals with Huntington disease, the repeat has expanded and is present between 37 and 86 times. The larger the number of repeats, the earlier the age of onset of the disease. The incidence is approximately 8/100,000. 3. Neurofibromatosis type I, or von Recklinghausen disease, is an autosomal dominant with high penetrance, but variable expressivity, that maps to the long arm of chromosome 17 (l7ql1.2). The disease has three major features: multiple neural tumors anywhere on or in the body, numerous pigmented cutaneous lesions (cafe-au-lait spots) and pigmented iris hamartomas (Lisch nodules). EM studies show that the tumors are the result of the proliferation of fibroblasts or Schwann cells in peripheral nerves, possibly due to ras inactivation. There is no treatment except for surgical resection of symptomatic tumors. The incidence is approximately 113,000. 4. Marfan syndrome is due to a defect in the gene for fibrillin located at 15q21.1; the major clinical findings involve the skeleton, cardiovascular system, and the eye. Affected individuals tend to be tail with long extremities and long, tapering appendages. Many spinal deformities are seen including kyphosis and scoliosis, and the joints may be hyperextensible. The most characteristic ocular change is bilateral subluxation of the lens, or ectopia lentis. Mitral valve prolapse and dilation of the ascending aorta due to cystic medial degeneration are common. The latter is the more important defect because the loss of medial support leads to dilation of the aortic valve ring and the root of the aorta, giving rise to aortic incompetence. Weakening of the media predisposes the aorta to an intimal tear, which can produce a dissecting aneurysm. The incidence of Marfan syndrome is approximately 1115,000.

AUTOSOMAL RECESSIVE TRAITS A. Characteristics 1. Autosomal recessives account for approximately 1800 of the 5000 mendelian traits identified. Figure IV-1-3 illustrates a typical autosomal recessive inheritance pattern, in this case due to a consanguineous mating.

Flashback to Biochemistry For more information on lipoprotein metabolism, see Lipids (Chapter 5) in Biochemistry (Section I).

In a Nutshell Huntington disease is caused by expansion of a CAG trinucleotide repeat on chromosome 4. It is inherited as an autosomal dominant.

Clinical Correlate Type II neurofibromatosis is much rarer than type I and is characterized by acoustic neuromas and cafe-au-Iait spots, but Lisch nodules are usually absent. The gene has been localized to chromosome 22.

Clinical Correlate Many diseases affecting genes that code for structural proteins (e.g., collagen: Ehlers-Danlos syndrome, or fibrillin: Marfan syndrome) are inherited as autosomal dominants.

Note Other important autosomal dominant conditions include: Ehlers-Danlos syndrome, Gilbert syndrome, polycystic kidney disease, osteogenesis imperfecta, and chondrodystrophic dwarfism.

meClical

215

Genetics

In a Nutshell Autosomal recessive disorders require two copies of the mutant allele for expression. Therefore, these diseases often skip generations; unaffected parents can produce affected sons or daughters. Figure IV-1-3. Autosomal recessive inheritance pattern.

2. Autosomal recessive traits require two doses of the mutant allele (homozygosity) for the phenotype to be expressed. 3. The hallmark of autosomal recessive inheritance is that unaffected parents can produce affected children of either sex. 4. Many autosomal recessive diseases are due to enzyme deficiencies. B. Diseases

1. Cystic fibrosis is an autosomal recessive disease caused by point mutations or small deletions in an integral membrane protein that functions as a chloride transporter, called the cystic fibrosis transmembrane conductance regulator (CFTR). The gene coding for this protein is located at 7q31.2.

a. Phenotypically, cystic fibrosis often presents with meconium ileus and is also characterized by deficiencies of pancreatic enzymes, pulmonary obstruction and infection leading to progressive pulmonary damage, bronchiectasis, cor pulmonale, and respiratory failure. Other abnormalities include cirrhosis of the liver, infertility, and elevated sodium and chloride concentrations in sweat.

In a Nutshell The majority of cases of cystic fibrosis are due to a deletion of three nucleotides coding for phenylalanine at position 508 in the CFTR.

Bridge to General Principles Refer to the Pathology chapter of the General Principles book for more information about Tay-Sachs disease and phenylketonuria.

216

meClical

b. Cystic fibrosis occurs at an incidence of 112,000 births in the Caucasian population; the carrier frequency is estimated at 1 in 25. Although many mutations within the gene could cause the phenotype, it is not practical or cost effective to sequence the entire region to screen for carriers. However, in the Caucasian population, the most frequent mutant allele causing cystic fibrosis is due to a small deletion at phenylalanine 508 in exon 10. It accounts for up to 70% of mutant cystic fibrosis alleles in Caucasian populations. An amplification of this region using the polymerase chain reaction (PCR) can be done, and the PCR products sequenced and compared with the normal sequence for this region. If comparison reveals the deletion, the donor of that template DNA would be classified as a carrier. If the mutation is not present, the probability that the donor of that DNA is a carrier of cystic fibrosis is greatly reduced, but still exists. Most diagnostic laboratories use this method to screen for between 4 and 10 of the most common mutations. Note that if a person belongs to a different ethnic group, PCR must be used to amplify the exons that contain the majority of the CF mutations within that particular ethnic group. 2. Tay-Sachs disease is an autosomal recessive disease caused by a deficiency of hexosaminidase A, which leads to the accumulation of ganglioside GM2 in neurons, producing a degenerative neurologic disease. Children appear normal at birth, but then begin to suffer from diminished responsiveness, deafness, blindness, loss of neurologic function, and seizures. Death usually occurs by 4 to 5 years of age. There is no therapy. The incidence of Tay-Sachs disease is highest among Ashkenazi Jews, in whom it occurs in approximately

Mendelian Inheritance

113,600 births. The carrier frequency in this population is estimated to be 1 in 30. Screening for carriers is inexpensive and measures hexosaminidase A activity in the blood. Cells obtained from amniocentesis can also be screened for hexosaminidase A activity. 3. Phenylketonuria is caused by a deficiency in the enzyme phenylalanine hydroxylase. Without this enzyme, phenylalanine is not converted to tyrosine, but is made into phenylketones. These phenylketones are excreted in urine and lead to the characteristic musty smell of the disorder. If phenylalanine is not restricted in the diet, the deficiency is characterized by severe mental retardation and microcephaly. Treatment consists of a diet low in phenylalanine and supplemented with tyrosine, which becomes an essential amino acid in these individuals. The incidence in those of European descent is approximately 1110,000.

X-LINKED RECESSIVE TRAITS A. Characteristics 1. X-linked recessive traits account for less than 10% of the 5,000 mendelian traits identified. Figure IV-1-4 illustrates a typical X-linked recessive inheritance pattern.

Clinical Correlate Newborns are routinely screened for phenylketonuria using the Guthrie test that measures the amount of phenylalanine in the blood. If a child with phenylketonuria is identified early, a special diet that restricts phenylalanine can prevent significant mental retardation.

Note Other important autosomal recessive conditions include: albinism, alkaptonuria, Gaucher disease, Hartnup disease, homocystinuria, maple syrup urine disease, sickle-cell anemia, thalassemia, Wilson disease, and xeroderma pigmentosum.

In a Nutshell

Figure IV-1-4. X-linked recessive inheritance pattern.

2. Since males are hemizygous for the X chromosome, the phenotype is expressed in males with only one dose of the gene. Females require two doses for the phenotype to be expressed. 3. A male with an X-linked recessive allele will pass it to all of his daughters, but to none of his sons. A carrier female who is heterozygous for the trait, if mated to a normal male, can have affected sons or normal sons and carrier daughters or homozygous normal daughters. There is a 50% chance the female will transmit the gene to each son or daughter. B. Diseases 1. Duchenne muscular dystrophy (DMD) is due to a mutation in an X-linked gene located at Xp21.2. The gene encodes the protein dystrophin. a. Two thirds of DMD cases each generation are familial and the remaining one third arise sporadically. The gene is 2.5 Mb long and has 78 exons.1t codes for a 3687 amino acid protein that contains 24 similar repeats of 109 amino acids each. Because of this similarity, new mutations are thought to arise because of the incorrect alignment of the repeats at meiotic synapsis, followed by crossover events that lead to deletion or additions. If the illegitimate recombination event results in a frameshift mutation that truncates dystrophin, DMD results. Interestingly, if the recombination event preserves the reading frame, Becker muscular dystrophy results.

X-linked recessive diseases usually affect males, who inherit the mutant allele from heterozygous (carrier) mothers. A male with an X-linked recessive will pass it to all of his daughters, but none of his sons. If his mate is homozygous dominant, the daughters will be carriers. A carrier female will pass the disease to half her sons and will pass the carrier tra~ to half her daughters.

Bridge to Musculoskeletal System For more information on Duchenne-type muscular dystrophy, refer to the Musculoskeletal Pathology chapter in Organ Systems Book 2 (Volume IV).

I KAPLAlf medlea

217

Genetics

Flashback to Biochemistry Refer back to Nucleic Acids (Chapter 1) in Molecular Biology (Section II) in this book for more information about Lesch-Nyhan syndrome.

Bridge to Heme/Lymph Hemophilia is discussed in greater detail in the Hematologic/Lymphoreticular Pathology chapter of Organ Systems Book 1 (Volume III).

Note Other important X-linked recessive conditions include: hemophilia A, hemophilia B, glucose-6-phosphate dehydrogenase deficiency, and colorblind ness.

b. DMD is characterized by onset of weakness in early childhood. Most affected children are confined to wheelchairs by age 10 and die from respiratory and cardiac complications of the muscle degeneration by age 20. The incidence is approximately 1/3,500. 2. Lesch-Nyhan syndrome results from a severe deficiency of hypoxanthine-guanine phosphoribosyltransferase (HGPRT), a key enzyme in the purine salvage pathway. It results in excessive uric acid production, mental retardation, spasticity, self-mutilation, and aggressive, destructive behavior. 3. Hemophilia A is caused by a deficiency of factor VIII that leads to the inability to clot blood normally. Affected individuals have prolonged bleeding times and suffer from easy bruising and hemorrhage. Treatment consists of the replacement of factor VIII by means of a concentrate prepared from pooled blood or by using recombinant DNA techniques. The incidence of hemophilia A is approximately 1110,000 in Caucasian males. 4. G6PD deficiency. Glucose-6-phosphate dehydrogenase deficiency is a defect in the first step of the hexose monophosphate shunt. The main purpose of this pathway is to produce ribose SP and NADPH. NADPH is used in a variety of biosynthetic reactions and to regenerate glutathione. Glutathione detoxifies oxidants produced during infection or by oxidant drugs, such as primaquine, dapsone, and sulfonamides. Its levels are reduced in individuals with G6PD deficiency because of their diminished capacity to generate NADPH. Consequently, oxidants are free to damage cells. RBCs are especially susceptible, and hemolytic anemia may result. Alleles for G6PD deficiency are common in many populations including ones in the Mediterranean, Mrica, and India. The deficiency is believed to have been selected for because it confers an advantage against infection with plasmodium falciparum.

X-LINKED DOMINANT TRAITS A. Characteristics 1. X-linked dominant diseases are very rare, with only a few known examples. Figure IV-l-S illustrates a typical X-linked dominant inheritance pattern.

In a Nutshell A pedigree showing X-linked dominant inheritance from an affected female can look like an autosomal dominant pedigree, but transmission from an affected male is to all of his daughters but none of his sons. Figure IV-1-S. X-linked dominant inheritance pattern.

2. An affected male parent passes the trait to all of his daughters, but to none of his sons. 3. An affected female parent can pass the trait to both sons and daughters. The inheritance pattern from an affected female looks like that of an autosomal dominant. 4. Without a very large pedigree or employing molecular techniques, an X-linked dominant cannot be distinguished from an autosomal dominant.

218

meClical

---~---~--

- - - - -

Mendelian Inheritance

B. Diseases

1. Hypophosphatemic rickets is an X-linked dominant condition causing abnormal regulation of vitamin D3 metabolism and defects in renal tubular phosphate transport. Symptoms include growth retardation, osteomalacia, and rickets.

NON MENDELIAN INHERITANCE PATTERNS A. Mitochondrial inheritance

1. Mitochondrial traits exhibit a matrilineal inheritance pattern because only the mother contributes mitochondrial DNA to progeny (Figure IV-1-6).

In a Nutshell Mitochondrial inheritance exhibits a matrilineal pattern because mitochondria are inherited only from the mother.

Figure IV-1-S. Mitochondrial inheritance pattern.

2. Mitochondria, the organelles involved in the generation of ATP energy through oxidative phosphorylation, contain their own double-stranded circular DNA. Mutations in mitochondrial DNA occur at a 1O-to-20-fold higher frequency than mutations in nuclear DNA. This is thought to be a result of exposure to oxygen free radicals and the lack of an efficient repair system. The mitochondrial DNA also replicates independently of the nuclear DNA, and the organelles segregate into daughter cells independently of nuclear DNA. If a cell contains both mutant and normal mitochondria, after mitosis, daughter cells can contain different numbers of the mutant mitochondria. This phenomenon is called heteroplasmy and helps explain the variable phenotype between different tissues in mitochondrial disease. 3. Mitochondrial diseases are often expressed as neuropathies and myopathies because muscle and brain are highly dependent upon oxidative phosphorylation. a. Leber hereditary optic neuropathy was the first disease discovered that followed a mitochondrial inheritance pattern. It is a relatively common cause of acute or subacute loss of central vision secondary to optic nerve degeneration, especially in young men. b. Myoclonic epilepsy with ragged-red muscle fibers (MERRF) is a syndrome characterized by myoclonus, seizures, cerebellar ataxia, and mitochondrial myopathy.

Clinical Correlate Mitochondrial diseases affect the tissues that are the most dependent on oxidative phosphorylation (heart, skeletal muscle, brain).

B. Multifactorial or quantitative inheritance 1. In multifactorial inheritance, the phenotype is determined by the contributions of many genes as well as the interaction of the genes with the environment. Each gene contributes only a small amount toward the phenotype, and it is the net quantitative effect of all the genes that produces the phenotype. When a certain genetic threshold is exceeded and there is exposure to environmental triggers, the disease develops. Diseases or conditions impacted by many genes are said to be polygenic.

meC:lical

219

Genetics

2. Multifactorial inheritance is complex and incompletely understood. Many disorders are associated with this pattern of inheritance, including pyloric stenosis, cleft lip, and cleft palate. C. Fragile X syndrome

1. Fragile X syndrome is clearly a genetic disease, but it fails to follow the patterns described for normal X-linked recessive or dominant mutations. Completely unaffected carrier males, called nonpenetrant transmitting males (NTM), may be identified in pedigrees. The male or female offspring from such a carrier male are completely normal, but the grandchildren from the carrier's daughter may be affected. Among these grandchildren, approximately 25% of the males will be affected, and about 25% of the males will be unaffected carriers. Around 25% of the females may have mild symptoms of the disease, and about 25% of the females will be unaffected carriers. Fifty percent of the grandchildren, both males and females, will be completely normal. The expression of the phenotype seems to depend on the position of the individual in a pedigree (Figure IV-l-7).

Mild Sx

Figure IV-1-7. Fragile X syndrome.

2. The gene responsible for fragile X, called fragile X mental retardation (FMR1) is located at Xq27.3 and appears to code for an RNA-binding protein. a. There is a short series of CGG repeats that are normally transcribed as part of the 5' untranslated region of the FMRl mRNA. In normals, the repeats are present in 6 to 50 copies. b. In an individual with fragile X syndrome, the trinucleotide sequence is present in hundreds to thousands of copies. As the number of CGG repeats expands, the promoter region upstream from the expanded CGG repeat is hypermethylated, preventing transcription of the gene. In fragile X individuals, no FMRl mRNA is detected in tissues such as brain and testes, tissues in which the level of FMRl mRNA is normally substantial. c. A female carrier has an intermediate number of repeats, greater than 50 but less than 230. At this level, the 5' end of FMRl is not hypermethylated and the gene is still transcribed. The gene in this carrier female is called a premutation and is also found in NTMs. What causes the premutation to undergo an expansion of the CGG repeats is not known.

220

meClical

Mendelian Inheritance

3. Fragile X affects approximately 111,250 males and 112,000 females in all ethnic groups. It is the most common form of inherited mental retardation. In addition to mental retardation, characteristic features in males include narrow faces, prominent ears and forehead, and large testes. Physical symptoms in affected females are milder. 4. Recently, it was demonstrated that affected males have the expanded eGG repeats in somatic cells, but not in germ cells. This observation led to the hypothesis that the zygote (46,XY) possesses only the premutation. Expansion to the full mutation occurs later in embryonic development (postzygotically), after the germ cell lineage has been set aside. No expansion occurs in the germ cell lineage, and the exact magnitude of the expansion in somatic cells is not the same in each cell.

In a Nutshell Fragile Xsyndrome is the most common inherited form of mental retardation. It is caused by expansion of a eGG repeat on the mutant X chromosome in the zygote, which turns off the transcription of an essential gene called FMR 1. Other diseases caused by trinucleotide expansions include Huntington disease, myotonic dystrophy, and spinocerebellar ataxia types 1 and 3.

KAPLAIf,,_

me lea

I 221

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Chromosomal Abnormalities

Chromosomal abnormalities are not generally due to mutations in single genes, but are the result of structural alterations that can be seen under the light microscope. These alterations include the rearrangement of genetic material in a chromosome, or numerical abnormalities due to the gain or loss of a chromosome.

STANDARD CYTOGENETIC NOMENCLATURE A. The chromosomal constitution of an individual is described by a karyotype. The karyotype includes the total number of chromosomes, the sex chromosome constitution, and any abnormalities in number or morphology. A normal human karyotype is 46,XX for human females and 46,XY for human males. Karyotypes are performed when cells are entering mitosis because the DNA is condensed and can be easily observed when stained with dyes such as quinicine or Giemsa. B. Chromosomes can be classified based on the position of the centromere. A metacentric

chromosome has its centromere located at its center. A submetacentric chromosome has its centromere off-center, creating unequal arms. And an acrocentric chromosome has its centromere very near the end, creating a minute p arm and a long q arm. C. Each chromosome can be divided into three regions, the p, or short arm, the q, or long arm,

and the centromere (the primary constriction found in each chromosome). During meiosis or mitosis, the centromere is the point at which sister chromatids are held together. Each arm is further subdivided into regions by landmarks. Landmarks are consistent features that are useful to identify a given chromosome, such as the ends of the arms, the major bands seen in a specific stained chromosomal preparation, and the centromere. The area between landmarks is called a region. Regions are numbered consecutively from the centromere, and bands (both light and dark) are numbered consecutively within a region (Figure IV-2-l).

In a Nutshell The karyotype is the chromosomal constitution of the individual. It can be visualized by staining metaphase chromosomes.

meCtical

223

Genetics

3

3

p 2 1

q

2 3

Figure IV-2-1. Chromosome structure.

1. If Figure IV-2-1 represents the 17th chromosome, then 17q2S refers to the 17th chromo-

some, the long arm, the second region, band 5. Note that a period or similar division is not used between the region and the band. 2. Table IV-2-1 summarizes common symbols used in karyotype nomenclature.

Table IV-2-1. Symbols used in designating karyotypes. 1-22

X,Y (+) or (-) rep cen die inv p

q t

rob del ins dup ter

-7

mos

224

meClical

Autosome number The sex chromosomes When placed before an autosomal number, indicates that chromosome is extra or missing Reciprocal translocation Centromere Dicentric Inversion Short arm of the chromosome Long arm of the chromosome Translocation Robertsonian translocation Deletion Insertion Duplication Terminal or end (pter = end of the short arm; qter = end of the long arm) Break (no reunion, as in a terminal deletion) Break and join Separates chromosomes when the rearrangement involves more than one chromosome From-to Mosaic

Chromosomal Abnormalities

STRUCTURAL ABERRATIONS

In a Nutshell

Structural aberrations arise from the incorrect repair of breaks in chromosomes. Such breaks can be caused by viruses, chemicals, radiation, or other events. Although all cells experience chromosome breakage, in terms of human genetics, only aberrations arising within germ cells are important. Enzymes exist that can repair the breaks, but when multiple breaks occur simultaneously, mistakes can occur. The structural aberrations that can arise are grouped into three categories: deletions, inversions, or translocations.

There are three categories of structural aberrations caused by breakage of chromosomal DNA: deletions, inversions, and translocations.

A. Deletions 1. A deletion occurs when a chromosome loses a segment because of breakage. This results in partial monosomy for the affected region. If the deletion covers a large area, the affected individual will not be viable. Deletions are divided into two major categories: terminal deletions, in which the deletion removes one of the ends of a chromosome, or interstitial deletions, in which a central portion of the chromosome is lost (Figure IV-2-2).

A

A

, tc t ,B t B

Breakage

E

D

E

Partially monosomic for C and D

+

C D Degraded

Figure IV-2-2. An interstitial deletion.

2. A deletion is indicated by del with the chromosome number in parentheses followed by what is left of the chromosome (not what is deleted); e.g., 46, XY, del(l5) (pter ~ q22:) would be a male with 46 chromosomes, of which one of the 15 chromosomes has a deletion from within q22 to the qter. 3. Diseases a. Cri-du-chat (5p-) syndrome is caused by a deletion of the short arm of the fifth chromosome. It is characterized by severe mental retardation, a cat-like cry, microcephaly, hypertelorism, epicanthal folds, low-set ears, and micrognathia. Most cases are sporadic. b. DiGeorge syndrome is a hereditary absence of the thymus and parathyroid glands associated with abnormal development of the third and fourth pharyngeal pouches. It is caused by a partial monosomy of 22q 11 and is characterized by highly variable phenotypic expression in affected individuals. Severely affected patients are immunodeficient with T-cell levels between 10 and 20% of normal. Cardiac outflow tract abnormalities, abnormal facies, and hypoparathyroidism are associated features. c. Wilms tumor is usually due to a deletion of Ilp13. It is the most common malignant urinary tract neoplasm in childhood. Two-thirds of the cases are diagnosed by age 4. Primary treatment is surgical. d. Angelman and Prader-Willi syndromes are due to a deletion of 15q ll-q 13. Although the deletions causing both syndromes are identical, the phenotypes are very different. Individuals affected with Angelman syndrome (the so-called "happy puppet" syndrome) display a peculiar affect in that they are always smiling, but lack speech and are hyperactive, hypotonic, and severely mentally retarded. The face is dysmorphic, the

In a Nutshell Angelman and Prader-Willi syndromes are caused by the same deletion. In Prader-Willi syndrome, the deletion is inherited from the father, and in Angelman syndrome it is inherited from the mother. This is called imprinting, i.e., the differential expression of genetic material depending upon the parent from which it is inherited.

meCtical

225

Genetics

children suffer trom seizures, and they often walk with an ataxic, puppet-like gait. In contrast, individuals with Prader-Willi syndrome are obese, have small feet and hands, and are short in stature with dysmorphic features. They exhibit hyperphagia and other behavioral abnormalities in addition to mental retardation. The differences between the two presentations are due the parental origin of the deleted chromosome. This differential expression of genetic material, depending upon whether the DNA was originally of maternal or paternal origin, is called imprinting. Individuals with PraderWilli syndrome inherited the deletion from their fathers. In Angelman syndrome the deletion was inherited from the patients' mothers. The differences in expression are thought to be associated with differences in DNA methylation patterns that are responsible for regulation of genes at the level of transcription.

In a Nutshell Important conditions caused by deletions include cri-duchat syndrome, DiGeorge syndrome, Wilms tumor, Angelman syndrome, and Prader-Willi syndrome.

B. Inversions are rearrangements of the gene order within a single chromosome due to the incorrect repair of two breaks. The amount of chromosomal material remains the same, but recombination within the inverted region leads to unbalanced gametes. Inversions are classified with relation to whether or not the centromere is involved. An inversion in which the centromere is located outside of the inverted region is called a paracentric inversion. An inversion in which the centromere is located within the inverted region is called a pericentric inversion. 1. Paracentric inversions a. In a paracentric inversion, both breaks are on the same side of the centromere, so the overall shape of the chromosome is unchanged. If an individual inherits one normal chromosome and one inverted homologous chromosome, he or she will be phenotypically normal. These individuals are said to be heterozygous for the inversion. Problems only arise when the individual produces germ cells. b. During meiosis, recombination or crossing over always occurs between homologues. In an individual heterozygous for a paracentric inversion, if the crossovers occur outside the loop, there will not be any problems. However, a single crossover occurring within the loop (Figure IV-2-3) leads to the formation of an acentric fragment and an anaphase bridge formed by a dicentric chromatid. Formation of this anaphase bridge leads to the failure of meiosis. The cell does not progress any further in the production of germ cells, leading to reduced fertility. The severity of the effect is a function of the size of the inversion. The larger the inversion, the greater the risk that a recombination event will occur within the loop.

In a Nutshell If a single crossover occurs within a paracentric inversion, a dicentric bridge and an acentric fragment result. This terminates meiosis.

Metaphase Meiosis I

A

Anaphase Meiosis I

A

B

B

C

E

D

D

E

C

F

F Crossover is between C and D Figure IV-2-3. Paracentric inversion.

226

meclical

Chromosomal Abnormalities

2. Pericentric inversions a. Pericentric inversions have breakpoints on either side of the centromere. This changes the shape of the chromosome and the position of the centromere. Although heterozygotes for this inversion will usually be phenotypically normal, problems arise during germ cell production. b. If a crossover event occurs within the inverted segment, the chromatids undergoing the crossover will suffer from the duplication of material from one terminus and the deletion of material from the other. As with paracentric inversions, the larger the inversion, the higher the probability of the crossover occurring within the affected region (Figure IV-2-4).

Metaphase Meiosis I A

Anaphase Meiosis I

C

In a Nutshell If a single crossover occurs within a pericentric inversion, each chromatid will have one terminus duplicated and one deleted.

B B

C

A

D

D

E

E

DE Crossover is between Band C

Figure IV-2-4. Peri centric inversion.

c. Note that in the preceding examples, the crossover event occurs between two strands. It is possible, especially if the inversion is very large, that multiple crossover events involving three or even four strands will occur.

d. An inversion is indicated by the chromosome number in parentheses followed by the two breakpoints in parentheses. 46,XX, inv(5) (p12q23) would be a female with a pericentric inversion of chromosome 5 with breakpoints at bands 5p12 and 5q23. The more detailed representation of the karyotype would be 46,XX, inv( 5) (pter ~ p 12 :: q23 ~ p12 :: q23 ~ qter). C. Translocations involve the exchange of chromosomal material between two nonhomo-

logous chromosomes. Two types of translocations exist, reciprocal translocations and Robertsonian translocations. 1. Reciprocal translocations a. Reciprocal translocations result when two nonhomologous chromosomes exchange pieces. The breakpoint of a translocation is the region at which the two different chromosomes are joined. Phenotypically, a person who is heterozygous for a reciprocal translocation will appear normal because all the information is present; it is just in a novel order. Problems arise when gametic cells undergo meiosis, because now four chromosomes share homology. Using the light microscope, a configuration called a quadrivalent can be seen (Figure IV-2-5).

meCtical

227

Genetics

2

3

4

Pairing of a Reciprocal Translocation in Meiosis I

Figure IV-2-S. A quadrivalent.

b. Unlike inversions, crossing over is not as much of an issue with translocations, because recombination either produces nonviable gametes or merely switches alleles between homologous regions. c. During meiosis I, homologous chromosomes separate. However, the centromere is the structure that the cell uses to define a chromosome, so in actuality, homologous centromeres are separating. Under normal circumstances, if homologous centromeres separate, homologous chromosomes separate. In the case of a reciprocal translocation, nonhomologous material attached to a centromere confuses the issue. The major problem is how these chromosomes will segregate from each other during meiosis I. There are three possible patterns for segregation: alternate segregation, adjacent I segregation, and adjacent II segregation. (1) In alternate segregation, homologous centromeres separate such that one cell

contains two normal nonhomologous chromosomes, and the other contains both chromosomes involved in the reciprocal translocation. (2) In adjacent I segregation, homologous centromeres separate such that each cell contains a normal chromosome and a chromosome involved in the translocation. (3) In adjacent II segregation, the breakpoint of the translocation is so close to the centromere that the cell cannot identify the centromere. To our eyes, it seems that two homologous centromeres are placed in the same cell. Figure IV-2-6 summarizes these segregation patterns.

228

iiiE;&ical

Chromosomal Abnormalities

Products of Meiosis I A Iternate S egregation

L

2

Adjacent I S egregation

~

"~"

In a Nutshell

',' heterozygote

4

Different centro meres but duplications and deletions

Adjacent \I S egregation

~

~BalanCed 3 •• translocation

4

Normal

w

~ Homologous 2 centromeres . and duplications

~ and deletions

~L :

","-

-

-,

~

-

-,

If a reciprocal translocation undergoes adjacent I or adjacent II segregation, unbalanced gametes result.

-"~

Different centromeres but duplications and deletions

L

'. .1.... . " .~. .' ..

Homologous .. centro meres an d duplications an d deletions

4

Figure IV-2-6. Segregation patterns in reciprocal translocations.

d. The amount of alternate segregation is always equal to the amount of adjacent I segregation, since to the cell, they both involve separating homologous centromeres. The amount of adjacent II segregation depends upon how far the breakpoint is located from the centromere. If the breakpoint is very far from the centromere, the cell does not become confused, and only alternate and adjacent I segregation will be observed. If the breakpoint of a translocation is located very near the centromere, the cell becomes confused as to which centromere it is sending to which pole, and a significant amount of adjacent II segregation will occur. If the breakpoint involves the centromere, all forms of segregation are equally likely. e. In chronic myelogenous leukemia (CML), which causes overproduction of cells of the granulocytic series, a characteristic chromosomal abnormality called the Philadelphia (PhI) chromosome is present in the bone marrow cells in 95% of the cases. The Philadelphia chromosome usually is due to a reciprocal translocation between the long arms of chromosome 22 and chromosome 9. The breakpoint on chromosome 9 is located at q34 and occurs near a cellular oncogene called c-abZ. The translocation causes c-ab/ to move near an area on chromosome 22 called the breakage duster region. The juxtaposition of these two sequences leads to the production of a novel protein called P2IO, a tyrosine kinase thought to playa role in triggering the proliferation of CML cells.

Bridge to Heme/Lymph For more detailed information on chronic myelogenous leukemia, refer to the HematologicjLymphoreticular Pathology chapter of Organ Systems Book 1 (Volume III).

meClical

229

Genetics

2. Robertsonian translocations a. Robertsonian translocations involve any two acrocentric chromosomes that break near the centromeres and rejoin in a way that results in fusion of the q arms at the centromeres and loss of the p arms (Figure IV-2-7). There are three ways that the chromosome involved can segregate during meiosis I, and because both breaks occur at the centromere, all three types of segregation are equally frequent. Individuals who carry the Robertsonian translocations appear phenotypically normal, but experience a large number of spontaneous abortions due to the production of monosomic and trisomic embryos, which are usually nonviable.

In a Nutshell Robertsonian translocations are caused by fusion of the long arms of two acrocentric chromosomes. These translocations are most common between chromosome 14 and chromosome 21, or chromosome 21 and chromosome 22.

21 21 14

14

14/21

Figure IV-2-7. Formation of the Robertsonian translocation.

b. Down syndrome, or trisomy 21, is usually the result of a nondisjunction event during meiosis. However, approximately 4% of Down individuals actually are affected because of a translocation between chromosomes 14 and 21 or between chromosomes 22 and 21. The theoretical risk of a translocation heterozygote producing a Down offspring is 1/3. However, because 80% of trisomy 21 fetuses are spontaneously aborted, for every Down child born, 5 normal and 5 balanced translocation heterozygotes are seen. Figure IV-2-8 shows the consequences of the segregation of a Robertsonian translocation.

230

meClical

Chromosomal Abnormalities

Pairing of the Chromosomes in Meiosis I Metaphase

Possible Products at the End of Meiosis I

mW . 00 ;00 ~u~ 21 "21 'i'.

14

14/21

·I., '·"., ji:

Possible Products at the End of Meiosis II

Zygotes

i~ i~ 14

14

j:

i; ':

14

14

21

14

21

14/21

21

~

21

~

21

il 14/21

Figure IV-2-S. Consequences of a Robertsonian translocation.

3. Nomenclature for translocations

a. When the karyotype of a reciprocal translocation is written, the two chromosomes involved are listed in parentheses after t or rcp, followed by the breakpoints also in parentheses. For example, 46,XY, t(7; 9) (q31; q22) would be a male with a balanced translocation involving exchange of the segments distal to 7q31 and 9q22. The more detailed nomenclature would be 46,XY, t(7; 9) (7pter ~ 7q31 :: 9q22 ~ 9qter; 9pter ~ 9q22 :: 7q31 ~ 7qter). Alternatively, rcp can be used in place of t to be more specific. b. A Robertsonian translocation involves the loss of the short arms of two chromosomes and the centromere of one. This results in a reduction in the total number of

meClical

231

Genetics

chromosomes. 45,XX, t(l4q; 21q) would be a female with a Robertsonian translocation, with the derivative chromosome having the long arms of 14 and 21. The origin of the centromere is not indicated. A more detailed symbolism would be 45,XX, t( 14; 21) (l4qter ~ cen ~ 21qter). Alternatively, rob can be used in place of t to be more specific.

A BRIEF REVIEW OF OOGENESIS AND SPERMATOGENESIS In males and females, the products of each meiotic division have specific names. Figures IV-2-9 and IV-2-10 summarize the key stages of oogenesis (females) and spermatogenesis (males).

Bridge to General Principl~s For a more detailed discussion of spermatogenesis and oogenesis, refer to the first Embryology chapter (Gametogenesis) in General Principles Book 2 (Volume II).

A. In females, only one true egg, which contains most of the cytosol, is produced. The unequal division of cytoplasmic material is important because the zygote receives all of the initial cellular organelles, including the mitochondria, from the mother. Because the first polar body does not enter the second meiotic division, only three products of meiosis are shown for the female.

B. In males, the sperm fertilizes the ovum during the second meiotic division. While meiosis in the female is being completed, the sperm head rounds up and forms the male pronucleus. A female pronucleus and a polar body are produced at the end of the second meiotic division. When the male and female pronuclei reach each other, they fuse and form a zygote with the diploid number of chromosomes. DNA synthesis soon commences and the cell undergoes the mitotic divisions of embryogenesis.

Oogenesis

First Prophase

First Metaphase

Second Prophase

Second Metaphase

(jj~®-~~ Primary Oocyte

Second Telophase

,;§!:-(]J5

Secondary oocyte and polar body

Ovum and two polar bodies

Ovum and two polar bodies

Figure IV-2-9. Oogenesis.

First meiotic Second meiotic division division . ~ Primary ---. Secondary ---. S t·d ~ S Spermat ogonrum spermatocyte spermatocyte perma I perm

Spermatogenesis

Figure IV-2-10. Spermatogenesis.

232

meClical

Chromosomal Abnormalities

NONDISJUNCTION A. Definitions 1. The normal gametic constitution contains the haploid or n number of chromosomes. This refers to the fact that every normal gamete contains one copy of each chromosome, and hence one copy of every gene in the genome. 2. The normal somatic constitution of the cell is referred to as the diploid or 2n number. This refers to the fact that every somatic cell contains two copies of each chromosome and hence two copies of every gene in the human genome. 3. Nondisjunction is the failure of chromosomes to disjoin from each other during cell division. It can happen during either the division steps of meiosis or during mitosis. B. Timing of nondisjunction 1. The most common time for nondisjunction to occur is during meiosis 1. The mechanisms

of failure are not well understood; however, there is a strong correlation between increasing the incidence of nondisjunction and increasing maternal age. 2. If nondisjunction occurs during mitosis in the developing embryo, some of the cells will have the normal number of chromosomes, some will lack a chromosome, and some will have an extra chromosome. The cells that lost a copy of the chromosome usually die because monosomy is not well tolerated. The cells that gained a chromosome may live, producing a situation in which individual cells in the person's body will have two different karyotypes. This condition is called mosaicism, and the person is said to be a mosaic for that chromosome. 3. Nondisjunction during meiosis I is characterized by the failure of homologous chromosomes to separate. Figure IV-2-ll illustrates a failure during meiosis 1. 4. Nondisjunction during meiosis II is characterized by the failure of sister chromatids to separate. Figure IV-2-12 illustrates failures during meiosis II. 5. If certain conditions are met, it can be reliably determined in which meiotic division and in which parent the failure occurred. For an unambiguous determination, there must be a way to determine which chromosomes came from which parent and to distinguish between a parent's homologues. This can be accomplished if a restriction fragment length polymorphism (RFLP) or a staining polymorphism exists on the chromosome of interest very near the centromere. Centro meres exercise crossover suppression, so the RFLP or staining polymorphism will be a reliable marker for that chromosome. Assume that each parent has two different forms of the polymorphism and that they do not share forms with each other. If an AB mother and a CD father produce a ABC child, then nondisjunction occurred in meiosis I of the mother. She has two different forms of the polymorphism, and since the child has both of her forms, her homologues failed to disjoin. If the child born from the cross was BCC, the failure must have occurred in meiosis II of the male. The child has two forms from the male parent and they are both of the same form, so it can be concluded that sister chromatids failed to disjoin during meiosis II.

In a Nutshell If nondisjunction occurs in meiosis I, homologues fail to separate.

In a Nutshell If nondisjunction occurs in meiosis II, sister chromatids fail to disjoin.

Note A restriction fragment length polymorphism (RFLP) is a stable, inherited variation in a DNA sequence recognized by a restriction endonuclease. Each individual has a nearly unique pattern of RFLPs within his genome.

e. Results of nondisjunction 1. The term polyploidy is used to refer to the state in which there are extra sets of chromo-

somes in the cell (e.g., a triploid = 3n, a tetraploid = 4n). Euploidy refers to the condition in which the number of chromosomes is equal to some multiple of n. Hence, diploidy and triploidy are both euploid states. 2. Rarely, both triploid and tetraploid fetuses have been reported. Several triploid live births have also been recorded, but the infants do not survive very long.

meltical

233

Genetics

Metaphase of

;;Obt;;S~II~! ~ [:8 Metaphase I

..

J

2 copies of each gene on

Me~sisl / ~ . ··:~r:~~mmOT ~ro~~ase ~~c:~ t:::::::"::'~~!~~~~ =0 ' ~ ~:I;:;;: '"

t~;~~~':p~~ ~~c~~~~~~~s ~

Homologues pair with each other at the metaphase plate. During anaphase I, homologues fail to disjoin. At the end of meiosis I, one daughter cell has both homologues, and one cell lacks that chromosome.

(

)

~

(

)

J

0 copies of each gene on ~he example chromosomes 0 copies of each gene on ( the example chromosomes During metaphase II, each chromosome lines up individually at the metaphase plate. In anaphase II, sister chromatids are pulled to opposite poles.

Gametes

Figure IV-2-11. Nondisjunction during meiosis I.

2 copies of each gene on the example chromosomes

Metaphase of

Meiosis

Metaphase I I

S, G2 proPhase.

Me~sis ~

/.

:

~

~[~ J 0 copies of each gene on

~ ~ the(examPle Chromoso)mes t~;~~~':~~ ~~~~~~~~~~s ~

dJ: .: c::::=::::y11 ~

(dJ:

I

~

4 copies of each gene on the example chromosomes

2 copies of each gene on the example chromosomes

(~~ ~ ~

~~' ....--f1 I

'---V

I

Homologues pair with each other at ~ the metaphase plate. During 2 copies of each gene on 0 copies of each gene on anaphase I, homologues are pulled to the example chromosomes the example chromosomes . ( ) different poles. At the end of meiosis I, homologues will be in different cells. Dunng metaphase II, each chromosome lines up individually at the metaphase plate. In anaphase II, Gametes sister chromatids fail to disjoin and are pulled to the same pole. One gamete ends up with both chromosomes, and one does not get a chromosome.

Figure IV-2-12. Nondisjunction during meiosis II.

234

meClical

Chromosomal Abnormalities

3. Aneuploidy is the condition in which there is either an extra chromosome or a missing chromosome (e.g., trisomy or 47 chromosomes [2n + 1] chromosomes, or monosomy or 45 chromosomes). Aneuploidy is the most common type of chromosomal disorder, occurring in approximately 0.4% of live births. The incidence of autosomal aneuploidies is directly related to increasing maternal age. 4. Polyploid and aneuploid states most commonly arise from a nondisjunction event during meiosis I or meiosis II. 5. Autosomal trisomies. Although trisomy for any chromosome is possible, only three autosomal trisomies are compatible with postnatal survival, barring mosaicism. These are Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), and Patau syndrome (trisomy 13). a. Trisomy 21 (47,XX, +21 or 47,XY, +21) is the most common of the chromosomal disorders and is a major cause of mental retardation. Of the individuals affected with trisomy 21, 95% are caused by nondisjunction during gametogenesis, 4% have a Robertsonian translocation that usually involves chromosome 14 or chromosome 22, and 1% are mosaics due to mitotic nondisjunction during embryogenesis. (1) The phenotype of Down children is recognizable at birth because of their dysmorphic features, including epicanthal folds, brachycephaly, flat nasal bridge, low-set ears, and short, broad hands with a single transverse palmar crease. Mental retardation is a prominent feature, with SO% having IQs between 25 and 50. Approximately 40% have congenital heart disease, most of which is due to septal defects. Down children also have an IS-20-fold increased risk of developing acute leukemia, and in patients over 35, pathologic changes in the brain are seen that are similar to those seen in Alzheimer disease. (2) The incidence of live-born trisomy 21 is approximately 1ISOO. In SO% of cases, the extra chromosome is of maternal origin and is associated with increasing maternal age. When the maternal age is greater than 45 years, the occurrence is 1 in 25 births. b. Trisomy 18 (47,XX, +IS or 47,XY, +lS) occurs in approximately 1IS,000 births. It is estimated that the rate of conception is much higher, but that 95% of these fetuses spontaneously abort. Ninety percent of the cases are due to nondisjunction and 10% are due to mosaicism. Individuals with Edwards syndrome suffer from intrauterine growth retardation; mental retardation; severe failure to thrive; short sternum; small pelvis; cardiac, renal, and intestinal defects; and rocker bottom feet. Most die before 1 year of age. e. Trisomy 13 (47,XX, +13 or 47,XY, +13) occurs in approximately 1125,000 births. Ninety percent of the cases are due to nondisjunction and 10% are due to an unbalanced translocation. Individuals with Patau syndrome suffer from mental retardation, microcephaly, abnormal brain development (arrhinencephaly, holoprosencephaly), cleft lip and palate, ocular colobomas, postaxial polydactyly, cardiac dextroposition, and ventricular septal defect. The defects are so severe that survival past 6 months is very rare.

6. Sex chromosome aneuploidy a. In mammals, the presence of the Y chromosome determines maleness. Males are the heterogametic sex, having two different types of sex chromosomes (an X chromosome and a Y chromosome) in their normal state. b. Females are the homogametic sex because they have two copies of the X chromosome. If levels of an enzyme produced by a gene located on the X chromosome are measured

Clini(al Correlate Potential chromosomal abnormalities in a fetus can be identified by amniocentesis and chorionic villus sampling (O/S). Amniocentesis is usually performed between the 16th and 18th week of gestation. A small amount of amniotic fluid is removed and the cells are analyzed by standard cytogenetic techniques, fluorescence in situ hybridization (FISH), and biochemical and DNA analysis. Amniocentesis can also provide information concerning neural tube defects. The risk of complications due to amniocentesis is approximately 0.5%. Chorionic villus sampling has similar diagnostic accuracy to amniocentesis; however, it can be carried out at 9 to 12 weeks of gestation. This allows the results to be available much earlier; however, the risk of inducing a spontaneous abortion is approximately 3%. CVS does not provide information concerning neural tube defects.

Clinical Correlate Ruorescence in situ Hybridization (FISH) In this technique, a DNA probe that is labeled with fluorescent molecules will recognize and bind to its complementary sequence on a metaphase chromosome. The results can then be observed using a fluorescent microscope. FISH is used for a number of different applications including aneuploidy detection, microdeletion detection, and determining structural abnormalities.

meClical

235

Genetics

In a Nutshell Females are mosaics for genes on the X chromosome because in each of their cells, one X chromosome is randomly inactivated, whereas the genes on the other X chromosome are expressed.

in males and females, they are found to be the same. This phenomenon is explained by the Lyon hypothesis. It states that during early embryogenesis in the female, a decision is made in each somatic cell to turn off most of one of the X chromosomes. In fact, if the embryo contains more than two X chromosomes, the extra ones will be inactivated until only one X chromosome remains active in a given cell line. This X inactivation equalizes the expression of X-linked genes between males and females. The inactive X is visible under a light microscope as a chromatin mass called a Barr body. Since X inactivation is a random event in mammals, if a female is heterozygous for an X-linked recessive trait, she will usually have a normal phenotype because approximately one half of her cells will express the normal allele. c. Examples of sex chromosome aneuploidy (1) Turner syndrome (45,X and variants) is due to the complete or partial mono-

somy of the X chromosome. It is characterized by short stature, primary amenorrhea, infertility, webbed neck, shield chest and often, coarctation of aorta. The incidence is approximately 1/6,000 female births. (2) Klinefelter syndrome (47,XXY and variants) was the first human sex chromosome abnormality reported. It is characterized by hypogonadism, testicular atrophy, azoospermia, and a eunuchoid body with a lack of male secondary sexual characteristics. It occurs in approximately 112,000 male births. (3) XYY syndrome (47,XYY and variants) is not associated with an obviously abnormal phenotype. Individuals with this syndrome may be excessively tall and have severe acne. In newborn screening programs without ascertainment bias, XYY individuals were found to have normal intelligence, but an increased risk of behavioral problems. Fertility of these individuals is normal. XYY syndrome occurs in approximately 1/1,000 live male births.

236

meClical

Linkage

The pattern of inheritance of individual genes depends on their locations on the same or on different chromosomes. Nonallelic genes located in close physical proximity to each other on the same chromosome will tend to be inherited in association with one another, rather than assorting independently of each other.

SVNTENV AND LINKAGE A. If different genes are located on the same chromosome, they are said to be syntenic. B. When genes are located in close proximity to each other on the same chromosome such that they do not assort independently during meiosis, they are said to be linked. If genes are linked, the chance that they will be inherited together is a function of the distance between the loci. During the formation of chiasmata in prophase of meiosis I, genetic material is exchanged between homologues. If two loci are very far apart, there is a large chance that the crossover event will happen between them, and one allele will be moved to the other homologue. If the loci are very close, it is unlikely that an exchange will occur between them.

e. In Figure IV-3-1, an individual is heterozygous at both the A locus and the B locus. Although

In a Nutshell Genes located on the same chromosome are syntenic. Genes located in close proximity to each other are linked and tend to be passed together.

the A locus and the B locus are located on the same chromosome, the alleles of this heterozygote can be arranged in two different ways. The arrangement of the alleles at different loci relative to each other is called the linkage phase. If the A and B alleles are on one chromosome, and a and b alleles are on the other chromosome, the combination can be written in a shorthand form as AB//ab. When the dominant alleles, A and B, are on the same chromosome, they are said to be in coupling or in the cis configuration. If the A and B alleles are on different chromosomes, as in Ab//aB, they are said to be in repulsion or in the trans configuration.

a

o

b

a

b

A

B

A

B

a

g A

b

t

Xi B

a

b

a A

B b

A

B

0

Figure IV-3-1. Recombination frequency between homologues during prophase of meiosis I.

meClical

237

Genetics

RECOMBINATION FREQUENCY A. If a person is a heterozygote at both the A and B loci, and they are not linked, then the gametes of the heterozygote will contain AB, aB, Ab, and ab in equal frequency.

B. If A and B are linked in the cis conformation, AB//ab, four types of gametes are still made. However, they will not occur in equal frequency; it is more likely AB or ab will be passed because a recombination event between the loci is not required.

In a Nutshell Recombination frequency is the frequency of crossover events between two loci. The closer the two loci are, the less likely it is that a crossover event will happen between them.

C. In this example, the chromosomes with AB or ab are called parental chromosomes or nonrecombinant chromosomes because they are in the same configuration as in the parents. Chromosomes having Ab or aB are called recombinant chromosomes because they are the products of a recombination event and carry a different allelic conformation from that of the parents.

D. Recombination frequency is the frequency of crossover events between two loci. It can be determined experimentally in creatures like fruit flies in which a statistically significant number of progeny can be produced from a known cross. In this case, recombination frequency is equal to the number of recombinant progeny divided by the total number of progeny. E. Human families produce a small number of progeny, so an accurate determination of recombination frequency between two loci cannot be made by the simple formula, . . al test use d to d etermme "f I' recombinants . Th e I0 d score (Z)'1S t h e stattstlc 1 two oCt are tot al progeny linked or unlinked in the human genome. Data from lod scores are additive between different families, so the problem of small sample size is eliminated. 1. The lod score is defined as the 10glO of the odds that the loci are linked at a certain e or recombination frequency, divided by the odds that they are unlinked. In other words:

LOD = log

10

P(linked at a certain distance) P (unlinked)

2. Calculation of the lod score is beyond the scope of this chapter, but some useful generalizations can be made. By convention, if the lod score is equal to 3, then the odds of link_age are 1,000:1, and by convention the loci are considered to be linked. A lod score of-2 (100: 1 odds against linkage) is taken to be proof of independent assortment. To determine if two loci are linked and at what recombination frequency, the lod scores for all recombination frequencies between 0 and 0.5 are calculated. The highest lod value over 3 is considered to be the actual recombination frequency between the two loci. E Centimorgans are the standard units of genetic distance. The number of centimorgans can be calculated by multiplying the recombination frequency between the two genes by 100. G. If A and Bare syntenic but very far apart, a recombination frequency of 0.5 is seen. In this case, even though the genes are physically located on the same chromosome, they are not linked. Keep in mind that all linked loci are syntenic, but not all syntenic loci are linked.

GENETIC DISTANCE VERSUS PHYSICAL DISTANCE A. The genetic distance between two loci is a measure of the frequency of recombination between the loci. The physical distance between two loci is equal to the number of base pairs between the loci. It can be estimated from genetic distance using the following relationship: 1 centimorgan = 1 megabase pair (1 X 106 base pairs).

238

ii1eilical

Linkage

B. This relationship assumes that all regions of a chromosome have an equal probability of

recombination events. While this is often the case, some very important exceptions exist. 1. Centromeres exert crossover suppression. This effect causes recombination events in the region of the centromere to occur at a much, much lower frequency than expected. Because of crossover suppression, two loci located near a centromere will behave as if they were very closely linked even though they might be physically far away from each other.

2. The pseudoautosomal region (PAR), located near the p termini of both the X and Y chromosomes, allows for the homologous pairing of the X and Y chromosomes during meiosis I in males. In males, a chiasma always forms in the pseudoautosomal region during meiosis I. If the genetic distances between two markers at either end of the region are measured in males, PAR would be estimated to be 50 cM long. In females, chiasmata can form anywhere along the X chromosomes, since all of the material is homologous. The genetic distance between two markers at either end of the PAR in females is estimated to be less than 4 cM long. The genetic distance obtained from females correlates more closely with the actual physical distance of 2.6 Mbp found between markers at either end of PAR. 3. An illegitimate crossover event is one that occurs between nonhomologous material. During meiosis I in a male, if an illegitimate crossover occurs in addition to the crossover in the pseudo autosomal region, it can give rise to XX males and XY females. The gene Sry (sex-determining region Y) is located very close to the pseudoautosomal region. Its product is thought to be a transcription factor necessary for the activation of other genes in the testicular differentiation pathway. If illegitimate recombination occurs, Sry can be moved to the X chromosome and replaced with X material. XX males are infertile because they lack other genes necessary for fertility that are located on the Y chromosome; however, they are phenotypically normal except that they have small testes. These disorders occur at a frequency of approximately 1 in 20,000 births.

LINKAGE ANALYSIS FOR THE DIAGNOSIS OF GENETIC DISORDERS A. A restriction fragment length polymorphism (RFLP) is a stable, inherited variation in a DNA sequence within the human genome that produces a difference in the presence of one or more restriction sites. When the human genome is digested with a given enzyme, the great majority of restriction sites for that enzyme are the same between individuals because most of the DNA sequences are the same. Allelic differences between genes affect only transcribed DNA, which accounts for between 1 and 10% of the 3 X 10 9 base pair human genome. Also, the actual number of base pair differences between two alleles of a gene is very small. However, some restriction site differences exist for each restriction enzyme, just as alleles exist for almost every gene. Since 90% of human DNA is not transcribed, most of the differences will occur within this region. To be termed an RFLP, that fraction exhibiting the polymorphism must be 1% or more of a population. B. An RFLP can be detected using Southern blots. If there is variation in the presence of

restriction sites in a specific region of a chromosome, a probe with homology to the region will be able to detect several different patterns on a Southern blot of genomic DNA. These patterns are called haplotypes, and they can be thought of as the allele equivalent for RFLPs; however, they cannot truly be called alleles because an allele is defined as an alternate expression of a gene. Haplotypes of the RFLPs act as codominant alleles, since the genetic information from both chromosomes is cut and run on the gel. So if a person is heterozygous, two different haplotypes will be seen.

Clinical Correlate RFLP analysis can also be used to determine paternity or to compare a forensic sample of DNA from a crime scene with the DNA of possible suspects.

C. In many cases, a deleterious trait has been mapped to a chromosomal region, but the specific gene sequence has not been identified. In addition, there are very large genes that have been isolated, such as neurofibromatosis type I, in which many different mutations can KAPLA~.

meulCa

I

239

Genetics

occur that will lead to the same phenotype. In this case, it is too expensive and timeconsuming to sequence the entire region in the individuals in question. In both of these instances, linkage analysis between the gene of interest and a closely linked RFLP is the strategy of choice for tracing the inheritance of the deleterious allele. The following example shows the power of linkage analysis. 1. Huntington disease is an autosomal dominant disorder with delayed onset. The disease

affects approximately 1 in 10,000 individuals and is characterized by symptoms consisting of personality changes, memory loss, and chorea. A family presents to a clinic for genetic counseling. Individual II-I's father has Huntington disease and II-I is in the early stages of the disease. With this pedigree information, the best estimate that can be made as to whether or not his children, III-lor 1II-2, inherited the Huntington allele is 0.5. 2. An RFLP called GS is located 3 cM from the Huntington locus. It detects two polymorphic HindIlI sites. In Figure IV-3-2, the polymorphic sites are labeled HI and H2, and the invariant sites are labeled H. The accompanying table lists the haplotypes (A-D) at this locus and the expected bands on electrophoresis.

15.0 kb

2.5 kb 3.7 kb 1.2 kb

Haplotype H1 A + B + C

o

H

H2

+ +

Bands 15 kb, 3.7 kb, 2.5 kb 15 kb, 6.2 kb 17.5 kb, 3.7 kb 21.2 kb

Figure IV-3-2. Diagram of the G8 RFLP.

3. A Southern blot is made from the genomic DNA of the living members of the family and probed with probe GS. The following patterns are seen (Figure IV-3-3). The labels at the top of the gel indicate the haplotypes of individuals shown in the pedigree in Figure IV-3-4.

1-1

AB

11-1

BD

11-2

AC

111-1

BC

111-2

AD

markers 21.2 kb

-

17.5 kb 15.0 kb

-

6.2 kb 3.7 kb 2.5 kb

Figure IV-3-3. Southern blot analysis of a family with Huntington disease using the G8 probe.

4. Using the information from the Southern blot, it is possible to assign linkage phases to the members of this family. The B haplotype is linked to the Huntington allele (H) in individual II -1. Ninety-seven percent of the time, a parental chromosome will be passed to the progeny, and 3% of the time a recombinant chromosome will be passed.

240

meClical ----

----

Linkage

Key:

~II~

• • Huntington disease

DO

Normal

~II~ ~II~ Figure IV-3-4. Example of a family with Huntington disease.

5. Knowing that individual III-I has inherited the B haplotype from his father means that he has a 97% chance that the Huntington allele was still linked to it and a 3% chance that a crossover event occurred between the loci, giving him a normal allele (h) at the Huntington locus linked to the B haplotype. 6. By similar reasoning, individual III -2 has a 97% chance of inheriting a normal allele with the D haplotype and a 3% chance of inheriting the Huntington allele. D. RFLPs are due to single nucleotide changes that create or destroy a restriction endonuclease site. Another type of polymorphism that can be used to track a gene of interest is variable number tandem repeats (VNTRs). VNTRs are short repeat sequences that occur multiple times in a tandem array. The number of repeats in the sequence varies from chromosome to chromosome. If a restriction enzyme can be found that cuts outside the tandem array and there is a probe to that region, the different polymorphisms can be visualized on a Southern blot. The probe can be to a unique sequence, which is useful to follow a given loci, or can detect a sequence that is repeated multiple times in the genome. The pattern detected by a probe to a repeated sequence displays multiple bands on a Southern blot and is referred to as a "DNA fingerprint." DNA fingerprinting is often used in forensic DNA analysis and paternity disputes. E. VNTRs are generally analyzed using Southern blots because their tandem arrays can be several thousand base pairs long. A class of polymorphisms that can be easily analyzed using peR are called simple sequence repeats (SSRs). These repeats also occur in tandem, but the repeated sequence is only two to four base pairs in length. SSRs are now extensively used in linkage analysis.

KAPLAlf I meillea

241

Population Genetics

The previous chapters have stressed gene transmission and chromosome structure in individuals and their cells. To predict the fate of a gene, however, knowledge about how it functions in the population of organisms must be known. Important factors that influence the transmission of the alleles of a gene through time include the size of the population, the genotypes of individuals in the population, and factors that involve the interactions between the gene and the environment.

TERMINOLOGY A. A population is a community of sexually interbreeding individuals. Matings between individuals in a population can be random or assortative. Random mating or panmixis is the condition in which an individual in a population has an equal chance of mating with any other individual in that population. Assortative mating is nonrandom mating in which certain individuals of a population are more likely to mate with one subgroup of a population than they are to mate with another subgroup.

B. The gene pool of a population is the total of all the genes in the reproductive gametes of the population. Each gene in the gene pool can have different alleles. 1. The allele frequency is the frequency of a specific allele of a gene in a population. It is

sometimes confusingly called the gene frequency. 2. The genotypic frequency is the frequency of individuals with a specific genotype in the population. 3. The phenotypic frequency is the frequency of individuals with a specific phenotype in the population; this frequency can reflect contributions from several different genotypes. All of these frequencies can be calculated from population data. The following example illustrates these terms. In humans, the ABO blood loci have three alleles, A, B, and o. A and Bare codominant and are completely dominant to allele o. A specific population consists of 100 humans, each of whom carries two alleles. The total number of alleles in the population is 2(100) = 200.

a. The allele frequency refers to the frequency of that specific allele relative to the total alleles. In mathematical terms this would be expressed as: number of a specific allele all alleles at that locus

KAPLA~. I 243 meulca

Genetics

In our example, if 40 of the 200 alleles were A alleles, the frequency of A would be:

~=IO.21 200 b. The genotypic frequency refers to the frequency of individuals in the population having a specific genotype. In our example, if 12 individuals were found to be genotypically AB, then the genotypic frequency of AB individuals in the population would be 0.12. c. The phenotypic frequency refers to the frequency of individuals in the population that share a phenotype. Notice, this frequency might include more than one genotype. For example: Frequency of blood type A = frequency of genotype AA + frequency of genotype AO

HARDY-WEINBERG EQUILIBRIUM Hardy-Weinberg equilibrium states that under certain conditions, if the population is large and randomly mating, the genotypic frequencies of the population will remain stable from generation to generation. A. Hardy-Weinberg conditions. The conditions that must be met for a population to be in Hardy-Weinberg equilibrium all focus on keeping allele frequencies constant. 1. No mutations-the DNA of a given allele must not undergo any changes such as a sub-

stitution or deletion of base pairs that will convert it to a different allele. 2. No selection against one of the genotypes-all combinations of alleles must be equally viable. 3. No migration or immigration-no changes can be made to the population that will alter the existing allele frequencies.

In a Nutshell • The equation for allele frequency for a gene with two alleles is p + q = 1, where p is the frequency of the dominant allele and q is the frequency of the recessive allele. • The equation for genotypic frequencies for a gene with two alleles is p2 + 2pq + q2 = 1, where p2 is the frequency of homozygous dominant individuals, 2pq is the frequency of heterozygous individuals, and q2 is the frequency of homozygous recessive individuals.

4. No consanguineous or incestuous matings-matings between related individuals are prohibited because it causes autozygosity. Autozygosity describes the condition in which a person is homozygous at a locus because he receives two copies of the same allele from a common ancestor down both lines of descent. Overall, autozygosity leads to an increase in homozygosity at a locus, with a concomitant decrease in heterozygosity. 5. If these conditions are met, after one generation of random mating, the allele frequencies and genotypic frequencies will remain the same in each subsequent generation.

B. Equations. Hardy-Weinberg equilibrium can be expressed by the following equations. In a large, randomly mating population, there is a gene with two alleles, A and a. The A allele is completely dominant to the a allele. Let:

The frequency of A allele = p The frequency of a allele

=q

Then: The allele frequencies at that locus can be expressed as: p+q=l The genotypic frequencies at that locus can be expressed as: p2 + 2pq + q2 = 1 Where p2

= frequency of individuals with genotype AA

Where 2pq = frequency of individuals with genotype Aa Where q2 = frequency of individuals with genotype aa

244

meClical

Population Genetics

C. Examples of Hardy-Weinberg equilibrium

1. The equations for allele frequencies and for genotypic frequencies are related through the frequency of individuals with the homozygous recessive genotype, q2. The square root of q2 yields q, which can be used to solve for p. Note that individuals that have the dominant phenotype consist of two groups, those that are homozygous dominant (p2) and those that are heterozygous (2pq). Thus, if the frequency of an autosomal recessive disease is known in a population, it can be used to find the allele frequencies in the current generation, which can be used to solve for other variables. This is illustrated in the following example.

In a Northern European population, the incidence of cystic fibrosis, an autosomal recessive condition, is 4 in 10,000 births. What is the frequency of carriers of the disease in this population? We are told that the frequency of cystic fibrosis or q2 = 4110,000 = 0.0004. So, Vq2 = q = 0.02. Then 1- q = P 1 - 0.02 = P = 0.9S.

And finally the frequency of heterozygotes is expressed by 2pq and equals 2(0.98)(0.02) or 0.0392. 2. Males have only one copy of the X chromosome. If a gene is located on the X chromosome, the genotypic frequencies equation is different for males and females. In males, p + q = 1 is the equation that expresses both allele frequencies and genotypic frequencies. In females, the allele frequencies equation is p + q = 1, whereas the genotypic frequencies equation is p2 + 2pq + q2 = 1. Note that if the incidence of an X-linked recessive disease in males is given, this frequency is equal to q. For example: Red-green colorblindness, an X-linked recessive condition, occurs in S% of the male population. What is the frequency of female carriers of this trait?

In a Nutshell For an X-linked recessive trait, the frequency of the disease in males is equal to the frequency of the recessive allele.

q = O.OS P = 1- q = 0.92 Female carriers = 2pq = 2(0.92)(0.08) = 0.147.

3. If a gene has more than two alleles, variables are added to the allele frequency equation to equal the number of alleles. The genotypic frequency equation is then the expansion of the square of the allelic frequency equation. An example is: The ABO blood group has 3 alleles, A, Band o. A and Bare codominant to each other, and both A and B are completely dominant to allele O. Let: The frequency of A alleles = p The frequency of B alleles = q The frequency of 0 alleles = r Then:

The allele frequencies at that locus can be expressed as: p+q+r=1 The genotypic frequencies at that locus can be expressed as:

Where p2 = frequency of individuals with genotype AA KAPLAlf

- I

meillea

245

Genetics

Where 2pr = frequency of individuals with genotype AO Where q2 = frequency of individuals with genotype BB Where 2qr = frequency of individuals with genotype BO Where 2pq = frequency of individuals with genotype AB Where r2

= frequency of individuals with genotype 00

Note that: The phenotypic frequencies of type A blood = p2 + 2pr The phenotypic frequencies of type B blood = q2 + 2qr The phenotypic frequencies of type AB blood = 2pq The phenotypic frequencies of type 0 blood = r2 4. If two genotypes look the same phenotypically, and if additional information is known from the family history, the initial probabilities can be refined under certain conditions to include the new information. There are two conditions in which this needs to be done: if the gene has two alleles and q is very large (greater than 0.15), or if there are multiple alleles of the gene and in combination one phenotype can result from multiple genotypes. For example: In a certain population, the frequency of the A allele at the ABO blood locus is 0.4, the frequency of the B allele is 0.3, and the frequency of the 0 allele is 0.3. a. What is the probability that a person picked at random from the population will be typeAO? The answer would be 2pr or 0.24 because all we know is that the person is from the population. b. What is the probability that a type A person from this population would be AO? Now we know something about the person. Since the person is blood type A we know that he is either AA or AO. The P(AOIA) [i.e., genotype AO given phenotype A] would be:

_ P(AO) -

peA)

P(AO) P(AA) + P(AO) 2pr p2 + 2pr 0.24 0.16 + 0.24

= \0.6\

246

meClical

Population Genetics

LINKAGE DISEQUILIBRIUM

In a Nutshell

A. Linkage disequilibrium is the tendency of specific combinations of alleles at two different loci to be transmitted together. When it is found in a population between a deleterious trait and another locus that can be easily followed, linkage disequilibrium can be a useful tool in indirectly assessing the probability of a person inheriting the trait.

Linkage disequilibrium is the preferential association of a certain allele at one locus with a specific allele at a nearby locus more frequently than is possible by chance alone.

B. To understand linkage disequilibrium, consider the following scenario. In a certain population there exists an RFLP with two haplotypes, A and B. It is linked to the Huntington disease locus. Huntington disease (HD) itself is an autosomal dominant. Initially, there were only (A hd) gametes and (B hd) gametes. A mutation happened on an (A hd) chromosome that converted it to (A HD). Now three types of gametes exist within the population, (A hd), (A HD), and (B hd). Note that if no new mutations occur, (B HD) gametes will not be made until there can be crossover events between homologues that are (A HD) and (B hd). At this point in time, knowing that an individual is BB at the RFLP means that he will not develop Huntington disease. If instead the individual is AA or AB at the locus, his probability of developing Huntington disease is proportional to the frequency of the (A HD) chromosome within the population. Over generational time, the (B HD) chromosome will be made by recombination events. How long the linkage disequilibrium will last depends upon the recombination frequency between the two loci. If () is very small, the disequilibrium will persist for a long time. If () is very large, the disequilibrium will disappear rapidly (Figure IV-4-1). Linkage disequilibrium is most useful as a predictive tool if () is very small.

l00't"=============

8=0 8is small

% Disequilibrium

Note If linkage disequilibrium is known to exist between a deleterious allele (e.g., the Huntington disease allele) at one locus and a specific allele (or RFLP haplotype) at another locus, and if it is possible to determine the presence of the alleles at the second locus but not the first, then it is possible to calculate the probability that an individual carries the deleterious allele.

8 is large

o

t (generations)

Figure IV-4-1. Duration of linkage disequilibrium as a function of time.

KAPLA~. I meulca 247

----

---~------

SECTION V

Microbiology

-----------

Introduction

The human body plays host to a vast number of microorganisms. This brief introductory chapter describes the normal microbial flora that colonize different anatomical sites as well as the variety of factors that can confer virulence to selected microorganisms.

NORMAL MICROBIAL FLORA A. Properties. Normal microbial flora describes the population of microorganisms that usually reside in the body. The microbiological flora can be defined as either "resident flora;' a relatively fixed population that will repopulate if disturbed, or "transient flora" that are derived from the local environment. These microbes usually reside in the body without invasion and can even prevent infection by more pathogenic organisms, a phenomenon known as bacterial interference. The flora have commensal functions such as vitamin K synthesis. However, they may cause invasive disease in immunocompromised hosts or if displaced from their normal area.

Note Normal flora prevent pathogen colonization by occupying the receptor sites.

B. Location. Microbial flora differ in composition depending on their anatomical locations and

microenvironments. The distribution of normal microbial flora is summarized in Table V-I-I.

MICROBIAL VIRULENCE FACTORS Microbial virulence factors are gene products required for a microbial pathogen to establish itself in the host. These gene products are located on the bacterial chromosome or on mobile genetic elements, such as plasmids or transposons. Primary pathogens express virulence factors that allow them to cause disease in the normal host. Opportunistic pathogens are environmental organisms or normal flora that lack the means to overcome normal host defense mechanisms. They cause disease only when the normal host defenses are breached or deficient. Virulence factors can be divided into several categories.

KAP LA

!!._

I

meulC8

251

Microbiology

Table V-I-I. Major species of microbial flora found in different anatomic locations. Location

Ma;or Organisms of the Normal Flora

Skin

Propionibacterium acnes, Staphylococcus epidermidis, diphtheroids; transient colonization by Staphylococcus aureus Viridans Streptococci, Branhamella species, Prevotella melaninogenicus, Actinomyces species, Peptostreptococcus

Oral cavity

species, other anaerobes Nasopharynx

Oral organisms; transient colonization by S. pneumoniae,

Haemophilus species, N. meningitidis Stomach

Rapidly becomes sterile

Small intestine

Scant

Colon

Bacteroides species, Clostridium species, Fusobacterium species, E. coli, Proteus species, Pseudomonas aeruginosa, Enterococcus species, other bacteria and yeasts Childbearing years: Lactobacillus species, yeasts, Streptococcus species

Vagina

Prepuberty/Postmenopause: colonic and skin flora

A. Enzyme production can be of several types depending on the needs of the organism, its requirements for survival, and the local environment. 1. Hyaluronidase breaks down hyaluronic acid to aid in the digestion of tissue. 2. Protease digests proteins to enhance the spread of infections. 3. Coagulase allows coagulation of fibrinogen to clot plasma. 4. Collagenase breaks down collagen (connective tissues). B. Toxins 1. Exotoxins are heat -labile proteins with specific enzymatic activities produced by many

Gram-positive and Gram-negative organisms. Exotoxins are released extracellularly and are often the sole cause of disease. a. Some toxins have several domains with discrete biologic functions that confer maximal toxicity. An example is A-B exotoxin, where the B subunit binds to host tissue cell glycoproteins and the A subunit enzymatically attacks a susceptible target. b. Many toxins are ADP-ribosylating toxins, as outlined in Table V-I-2.

252

meclical

Introduction

Table V-1-2. Properties of ADP-ribosylating exotoxins. Toxin

Structure

Substrate

Effects

Diphtheria Pseudomonas exotoxin A

A-B

Elongation factor II

J- Protein synthesis

Adenylate cyclase regulatory protein

i

60S ribosome Synaptobrevin

J- Protein synthesis Nerve dysfunction

Cholera E. coli enterotoxin

Pertussis

Shiga (dysentery) Tetanus/botulinum

1 5

Adenylate cyclase

2. Endotoxin is the heat-stable lipopolysaccharide moiety found in the outer membrane of Gram-negative organisms. When released by cell lysis, the lipid A portion of lipopolysaccharide can induce septic shock characterized by fever, acidosis, hypotension, complement consumption, and disseminated intravascular coagulation (DIe).

c. Surface components may protect the organism from immune responses such as phagocytosis or aid in tissue invasion. For example, the polysaccharide capsules of Haemophilus inJluenzae type b and the acidic polysaccharide capsule of Streptococcus pneumoniae interfere with phagocytosis. Other surface proteins, such as adhesins or fIlamentous appendages (fImbriae, pili), are involved in adherence of invading microorganisms to cells of the host.

In a Nutshell Exotoxin

Endotoxin

Protein Lipopolysaccharide Gram+ and Gram- GramReleased extracellularly Heat labile

Part of outer membrane; not secreted Heat stabile

meClical

253

Bacteriology Overview

This chapter revievvs the essential features of bacterial structure with an emphasis on the differences between Gram-')ositive and Gram-negative organisms. Also discussed are important concepts relating to bacterial growth, metabolism, sporulation, and the methods by which bacteria transfer genetic information.

CLASSIFICAIION AND IDENTIFICATION OF BACTERIA A. General prOl~erties can differentiate prokaryotic (single-celled) organisms from higher eukaryotic organisms. The most distinguishable features include: 1. Prokaryotes have 70S ribosomes, whereas eukaryotes have 80S ribosomes. This difference

allows some classes of antibiotics to specifically target prokaryotic protein biosynthesis. 2. Prokaryotes have a naked, single, circular chromosome of double-stranded DNA that replicates bidirectionally. There is no true nucleus. Prokaryotes lack a nuclear membrane and their DNA does not have basic proteins (histones) associated with it. 3. Prokaryotes lack membrane-bound organelles such as mitochondria. 4. The cell wall of most bacteria is a unique, rigid, peptidoglycan layer that allows the characteristic Gram-positive and Gram-negative staining of bacteria. Gram stain (positive versus neg;ative) is based on biochemical retention of dyes by the cell's outermost layer; Gram-positives have a larger amount of peptidoglycan. Acid-fast staining is based on the ability to resist acid decolorization due to a high content of waxes in the cell wall. Mycobacteria species are acid fast and Nocardia species are partially acid fast.

Note

The mycoplasmas (including Ureaplasma) are the only bacteria that do not have cell walls. Chlamydia have cell walls that lack muramic acid. Both Mycoplasma and Chlamydia are therefore resistant to beta-Iactam antibiotics.

5. Flagella are responsible for bacterial motility. 6. Pili or fimbriae are used for attachment to host cells and are required for conjugation in Gram-negative bacteria. These structures convey adhesive properties to bacteria (adhesin). B. Classification of bacteria is defined arbitrarily by the biochemical characteristics and/or

phenotypic Jeatures that allow look-alike organisms to be differentiated from each other. The genus and species of an isolate can be determined by morphology and Gram stain, and by biochemical and nutritional traits. Some specific examples include: 1. Biochemical characteristics such as substrate specificity (e.g., B. pertussis grows specifi-

cally on Bordet-Gengou agar), the ability to ferment specific sugars (e.g., E. coli is a lactose fermentor), and the production of unique metabolic products (e.g., Mycobacterium tuberculosis produces niacin). 2. Serologic reactivity-the identification with specific antibodies in diagnostic immunoassays. 3. Bacteriophage typing is useful epidemiologically in tracing the source of epidemics. 4. Animal p:ilthogenicity 5. Antibiotk sensitivity

KAPLAlf I medlea

255

Microbiology

BACTERIAL STRUCTURE A. The cell envelope is defined as all layers that enclose the cytosol of a bacterium. It is the main structural feature that differentiates Gram-positive from Gram-negative bacteria. Grampositive bacteria have a finely patterned surface consisting of the cytoplasmic membrane, peptidoglycan layer, and sometimes, an outer capsule. Gram-negative bacteria have a complex cell envelope consisting of a cytoplasmic membrane (inner membrane), periplasmic space containing peptidoglycan, an outer membrane, and sometimes, a capsule. 1. Capsule production is usually correlated with virulence because capsules are antiphago-

cytic. Most capsules are carbohydrate in nature. 2. The bacterial cell wall is a structure unique to prokaryotes and is a major site of antibiotic attack (e.g., penicillin blocks peptidoglycan synthesis).

Table V-2-1. Bacteria by Gram-staining characteristic and shape. Gram-Negative Bacteria Cocci

Moraxella, Neisseria, Veillonella Coccobacilli or rods Bacteroides, Bartonella, Bordetella, Brucella, Burkholderia, Capnocytophaga, Citrobacter, Coxiella, Edwardsiella, Enterobacter, Escherichia, Francisella, Fusobacterium, Haemophilus, Hafnia, Klebsiella, Legionella, Mobiluncus, Moraxella, Morganella, Pan toea, Pasteurella, Porphyromonas, Prevotella, Proteus, Providencia, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Stenotrophomonas, Streptobacillus, Yersinia Spirochetes Borrelia, Leptospira, Spirillum, Treponema Vibrios Aeromonas, Campylobacter, Helicobacter, Plesiomonas, Vibrio Other

Chlamydia, Ehrlichia Gram-Positive Bacteria Cocci

Enterococcus, Gemella, Peptostreptococcus, Staphylococcus, Streptococcus Coccobacilli or rods Actinomadura, Actinomyces, Arcanobacterium, Bacillus, Bifidobacterium, Clostridium, Corynebacterium, Erysipelothrix, Eubacterium, Lactobacillus, Listeria, Mycobacterium, Nocardia, Propionibacterium, Streptomyces, Tropheryma Bacteria Resistant to Gram Stain Mycoplasma, Ureaplasma

256

meClical

Bacteriology Overview

a. Gram-positive bacteria are composed of a thick layer of peptidoglycan, lipoteichoic acids, polysaccharides, and sometimes, teichoic acid. The surface proteins bind to extracellular material. b. Gram-negative bacteria have a trilayered outer membrane anchored to the cell membrane by lipoprotein. Endotoxin (lipopolysaccharide, somatic 0 antigen, and core polysaccharide) in the outer membrane is unique to Gram-negative bacteria. Protein porin channels allow the flow of extracellular material through the cell's outer membrane. 3. The periplasm is the space between the plasma membrane and the outer membrane. It contains proteins, peptidoglycan, hydrolytic enzymes, and plasmid-controlled penicillinases. The periplasmic space is important in osmoregulation of the cell. B. Plasma (cell) membrane functions primarily as an osmotic barrier. Its composition is 60-70% protein, 30-40% lipid, and small amounts of carbohydrate. If present, the bacterial electron transport chain is located within the cytoplasmic membrane. Structures associated with the plasma membrane are membrane polyribosome-DNA aggregates and mesosomes. Mesosomes are convoluted structures of cell membrane important in cell division. C. Cytoplasmic structures 1. The nucleoid region of bacteria consists of a circular chromosome of double-stranded

DNA that lacks introns, histones, and a nuclear membrane. In contrast, eukaryotic nuclei contain all of the above. 2. Ribosomes consist of 70% RNA and 30% protein. 70S ribosomes (monomers) are attached to messenger RNA. The 70S complex can be broken down into subunits of 30S and 50S. 3. Polyamines (e.g., putrescine) are located mainly in ribosomes and serve to prevent dissociation of the 70S ribosome. 4. Cytoplasmic granules function to accumulate food reserves such as glycogen, lipids in the form of poly-~-hydroxybutyrate, and phosphate in the form of volutin granules. 5. Spores (endospores) are found in Bacillus and Clostridium species. Spores promote the survival of the organism under adverse environmental conditions since they resist heat and drying. Spores are highly dehydrated and refractile. They convert into vegetative cells via germination when conditions of the environment are more favorable. Tables V-2-1 through V-2-4 summarize the important features of bacterial structure and the key differences between Gram-positive and Gram-negative organisms.

In a Nutshell Gram-Positives

• Thick peptidoglycan cell wall overlays cell membrane • No outer membrane • Cell wall contains lipoteichoic acids and some have teichoic acid (virulence factor) Gram-Negatives

• Thin peptidoglycan cell wall overlays cell membrane • Also have an outer membrane • Space between cell membrane and outer membrane includes peptidoglycan and is called the periplasmic space • Lipoprotein in the periplasmic space connects the outer membrane to the cell membrane • Lipopolysaccharide (LPS) is a component of the outer membrane: Lipid A = toxic portion

Clinical Correlate Autoclaving is employed to ensure that spores are killed.

I KAPLAlf medlea 257

Microbiology

Table V-2-2. Features common to all prokaryotes.

• Nucleoid-single, circular, ds DNA chromosome; not called a nucleus because it lacks a membrane • No sterols in membrane (exception: Mycoplasma) • 70S ribosomes • No membrane-bound organelles • Rigid cell wall of peptidoglycan (Note: cell envelope = all layers that encircle and retain cytoplasmic contents; cell wall = peptidoglycan [PDG]) N-acetylglucosamine N-acetylmuramic acid with tetrapeptide in a BOA) linkage (lysozyme breaks this linkage) D and L alternating amino acids, tetrapeptide cross-bridges linked by transpeptidase (All of the above contribute to PDG strength) Function-shape, barrier, sieve, prevents lysis (bacteria are subjected to 2-5x the pressure that tissues face) • Amino acids found in peptidoglycan - D-glutamic acid D-alanine -

L-alanine (primarily Gram-negatives) Diaminopimelic acid

- L-Iysine (primarily Gram-positives) • Cytoplasmic membrane Structure-normal membrane - Function-osmotic barrier; active transport of nutrients via permeases; DNA attachment site for nucleoid and plasmids; iron uptake via siderophores; protein export, since Golgi and ER are lacking; site of electron transport chain, since mitochondria are lacking • Mesosome - Structure-membrane invagination-more prominant in Gram-positive (some say only in Gram-positive) • • • •

258

meClical

Function-site of DNA replication, cell division Introns absent Transcription directly coupled to translation Polygenic mRNAs Initiator tRNA is formyl methionyl-tRNA instead of methionyl-tRNA

Baderiology Overview

Table V-2-3. Features found in some prokaryotes. • Unique metabolic capacities - Obligate anaerobes -

FixN2

• Capsule - Structure-acidic polysaccharides of 2-3 sugars characteristic of the organism - Function-antiphagocytic • Flagella Structure-single proteinaceous helical filament of globular protein flagellin. Adds at the tip. 3 parts: basal body (anchored in cell membrane), filament, and hook. Counterclockwise rotation = coordinated movement toward a substance. Clockwise rotation = disorganized tumble away from a source. Function-locomotion, chemotaxis Two variations: Peritrichous-all over, e.g., Enterobacteriacae Polar-at one end, e.g., Pseudomonas Other polar nomenclature: lophotrichous-tuft of several flagella; amphitrichousboth poles have flagella • Glycocalyx structure - Structure-adhesive polymer film containing polysaccharides - Function-inhibits phagocytosis and antibody access/adhesion • Storage granules Poly ~ hydroxybutarate = lipid - Glycogen = starch - Polymetaphosphate (volutin)-phosphate • Electron transport chains are located in the cytoplasmic membrane • Spores or endospores (in some medically important Gram-positives) Cryptobiotic = dormant, no metabolic activity. Resistant to heat, dessication, freezing, etc. The inner membrane is also called the germ cell membrane. Cortex-Unique peptidoglycan in 2 layers: outer layer has few cross-links, rapid autolysis; inner layer has more cross-links and osmotic stability. Coat-Keratin-like protein layers with many cross-linking disulfide bonds Germ cell membrane Core-Dipicolinic acid, Ca2 +, chromosome, protein, and materials for resuming growth exist here

Coat has 80% of protein Coat proteins can't be solublized unless the disulfide groups are reduced-this cross-linked layer is responsible for the resistance of spores to chemicals. Ca2+ and dipicolinic acid are chelated in equal amounts in the core-helps keep the water content down by displacing water when intercalation occurs with the DNA. Seen in aerobic Bacillus and anaerobic Clostridium species KAPLAIf . ._ I me lea 259

Microbiology

Table V-2-4. Features found in Gram-positive and Gram-negative bacteria. i

260

Gram-Positive

Gram-Negative

• Cell envelope: - PDG 100-800 A - Plasma (cytoplasmic) membrane • Lipoteichoic acid: - Structure-glycerol or ribitol residues with phosphodiester links and substitutions anchored to glycolipid in membrane (so it goes all the way through the PDG) - Function-anchoring and adhesion • Proteins excreted outside cell

• Cell envelope: Outer membrane-60-180 A-sieve - PDG 20-30 A - Plasma membrane • Periplasmic space-contains PDG, enzymes, PBP, binding proteins that soak up sugars and amino acids, etc. • Membrane-derived oligosaccharide (MDO): Structure-glucan polymer secreted into periplasmic space - Function-regulates osmolality of periplasmic space - adjusts viscosity to avoid lysis • Lipopolysaccharide (endotoxin): - Structure and functions-Lipid A, which is responsible for endotoxin manifestations (hypotension and DIC), and a long oligosaccharide side chain that is group-specific and hydrophilic (this helps to exclude hydrophobic compounds) • Porins: - Structure-protein trimer bound noncovalently to PDG - Function-restricts entry of >800 MW molecules • Adhesins and/or evasins-Function: an adhesin is a gene product promoting attachment and colonization. An evasin is a gene product promoting evasion of host defenses. • Lipoprotein: - Function-covalently anchors outer membrane onto PDG; also loosely linked to LPS • Fimbriae or pili: - Structure-filament of pilin, helical arrangement, addition at base Sex pili: - Genetic exchange (plasmiddetermined) - Only see a few per cell (3-4) Common pili or fimbriae: - Adhere to host cells - Several 100 per cell - Almost all Gram-negatives have them; some Gram-positives as well Can also act as evasins or aggresins

mallical --~-----

Bacteriology Overview

BACTERIAL GROWTH How rapidly an infection can progress before host defenses respond can determine the severity of disease. In a closed system, growth is dependent upon the availability of nutrients, the external environment (e.g., temperature), and the growth rate of the specific species. Figure V-2-1 depicts a typical bacterial growth curve.

c d

b

a FigureV-2-1. Growth curve in a closed system. a =lag phase; b =exponential or log phase; c =stationary phase; d =phase of decline (death phase).

A. Bacterial growth in a dosed system 1. The lag phase is a period of no growth when the organisms are adapting to a new envi-

ronment. 2. The exponential or log phase describes the steady state of growth, typically at the organism's fastest rate. It continues until the nutrients are depleted or toxic waste products accumulate. Many antibiotics, especially those that target cell wall synthesis, are maximally effective during this phase. 3. The stationary phase occurs when nutrients are exhausted or toxins accumulate. Cell number is stable (cell loss = cell formation). Different outcomes can occur depending on the species of bacteria. Some bacteria stop growing, but remain viable for long periods of time. Some organisms cannot maintain a viable, nongrowing state. When they reach the stationary phase, they immediatedly start to die. Other organisms, if conditions do not improve, start forming spores. 4. The phase of decline is observed when the death rate increases, due to cell starvation or sensitivity to toxins.

SURVIVAL IN OXYGEN Survival in oxygen is an important parameter used to classify bacteria. All bacteria produce the superoxide ion (° 2-) in the presence of oxygen. Three enzymes are important to detoxify this ion. Superoxide dismutase converts the superoxide ion to hydrogen peroxide. Catalase or peroxidase then metabolizes the hydrogen peroxide to water and oxygen. Obligate anaerobes (strict anaerobes) lack these enzymes or have such low levels that an oxygen environment is toxic for them. Examples of obligate anaerobes are Clostridium and Bacteroides. Facultative organisms grow with or without oxygen. Whether they ferment or respire is an independent issue.

In a Nutshell The ABCs of Anaerobes

• Actinomyces • Bacteroides • Clostridium

KAPLA~.

meulCa

I

261

Microbiology

ENERGY PRODUCTION Energy production requires a source of carbon. Some bacteria are very versatile, requiring only a few essential nutrients. Many pathogenic bacteria have become so host adapted that they have lost much of their metabolic machinery. They have many additional requirements and are termed fastidious bacteria. A. Siderophores are iron (Fe 3+)-chelating compounds that are essential for growth of many pathogenic bacteria. The siderophore is a low-molecular weight material such as a catechol that is excreted from the cell. This molecule binds iron and is then bound to a cell wall or outer membrane protein and transported into the cytosol of the cell, where it is utilized in energy metabolism. B. Mechanisms of energy production 1. Fermentation is the anaerobic degradation of glucose to obtain ATP.

a. It is much less efficient than respiration for generating energy. It can be used by both obligate anaerobes and facultative organisms. End products of fermentation differ depending upon the organism and are often useful in identification. b. Most obligate anaerobes and all Streptococcus species use fermentation. Streptococcus species can only ferment because they cannot make cytochromes or catalase. However, they do have superoxide dismutase and peroxidase, so they can survive in an oxygen environment. 2. Respiration completely oxidizes organic fuels and requires an electron transport chain to drive the synthesis of ATP. a. Respiration produces almost 20 times as much ATP as fermentation. It requires a terminal electron acceptor. The usual electron acceptor is oxygen; however, alternate electron acceptors, such as nitrate and fumarate, are used by some organisms. b. Given a choice, bacteria will opt for respiration over fermentation. However, bacteria differ in their intrinsic ability to use fermentation or respiration. (1) Strict or obligate aerobes respire only and must use oxygen as a terminal electron

acceptor. Mycobacterium tuberculosis is a good example of an obligate aerobe. (2) Some bacteria ferment only. (3) The majority of bacteria use the most versatile strategy, fermentation or respiration, depending upon the conditions. Figure V-2-2 summarizes this information.

262

iileilical

Baderiology Overview

....-_ _ _ _ _---,NO Obligate Can the organism aerobes; grow without Facultative oxygen? \

Obligate aerobe -respires only (Mycobacterium tuberculosis) Ferments only (Streptococcus; no cytochromes = no catalase)

'---------'YE~ Facultative

How does the organism get ATP?

(E. coli, Strep species, Pseudomonas aeruginosa )

YES

Obligate anaerobes The enzymes for processing the superoxide ion are absent or minimal. Need superoxide dismutase, catalase, and peroxidases to detoxify the superoxide ion.

~

Ferments or respires with oxygen as terminal electron acceptor (E. coli)

Ferments only ( Clostridium species)

How does the organism get ATP? Ferments and possesses a primitive respiratory chain (uses something other than oxygen as a terminal electron acceptor, e.g., fumarate in Bacteroides fragilis)

Figure V-2-2. Bacterial survival in oxygen and methods of ATP production.

SPORULATION A. A spore is a dormant structure capable of surviving prolonged periods of unfavorable envi-

ronmental forces. Spores are capable of re-establishing the vegetative stage of growth when environmental factors become more favorable. Spores are resistant to radiation, drying, and disinfectants. Thermal resistance to denaturation is due to the high content of calcium and dipicolinic acid in the core. Spore formation is observed in Bacillus and Clostridium species.

B. Initiation of sporulation is related to the guanosine triphosphate pooL Regulation is by means of negative feedback. In addition, the availability of carbon and nitrogen sources are important to sporulation activity. C. Germination and outgrowth occurs when environmental and nutritional factors allow for

renewed cell growth. 1. Vegetative growth is triggered by the exposure to stimulants such as glucose, nucleic acids, and amino acids. 2. Activation of autolysin results in autolysis of the cortex. The synthesis of protein and structural components follows. The spore core membrane develops into the cell wall.

meClical

263

Microbiology

GENETIC TRANSFER Genetic transfer refers to three principal mechanisms that result in the movement of genetic material into a host organism. A. Transformation is the uptake and integration of naked DNA from the environment. 1. Once inside the cell, homologous recombination with the chromosome of the recipient

must occur for the transformation to be successful. 2. Transformation can be induced in the laboratory with salt and heat shock. This technique is used to make cells take up plasmids carrying genes of interest (competency). 3. There are natural transformers among both the Gram-positive and Gram-negative bacteria. The medically important natural tranformers are: Streptococcus species, Haemophilus species, Neisseria gonorrhea, and Helicobacter pylori.

B. Transduction is the phage-mediated transfer of bacterial DNA. There are two kinds of transduction: generalized transduction and specialized transduction.

Note

1. In generalized transduction, bacterial DNA is mistakenly packaged into an empty phage

The gene for diphtheria toxin is carried on a lysogenic bacteriophage.

head. This is a very low frequency event, but any gene can be transferred. Once inside the recipient cell, homologous recombination must occur for the transduction of information to be successful. 2. During specialized transduction, a lysogenic bacteriophage that is integrated into the bacterial chromsome excises itself, accidentally taking some chromosomal DNA. When the phage replicates, any bacterial gene that it has picked up is also replicated. These genes will be carried into cells that the progeny viruses infect. Specialized transduction occurs at a higher frequency that generalized transduction. C. Conjugation is the direct transfer of bacterial DNA between organisms. It requires cell-to-

In a Nutshell • Transformation ~ Uptake and integration of naked DNA fragments • Transduction ~ Phagemediated exchange of information • Conjugation ~ Direct bacteria to bacteria transfer of DNA (bacterial sex)

cell contact. It is the most important mechanism for widespread transfer of genetic information between bacteria. 1. Most conjugation is plasmid-mediated. A plasmid is an extrachromosomal piece of cir-

cular DNA that can replicate itself. It often carries genes such as those that encode resistance to antibiotics, and virulence factors such as enterotoxins or adhesins. Plasmids vary in size, copy number per cell, and host range. 2. If a plasmid can exist only within a single species, it is called a narrow-host-rangeplasmid. If it can transfer between different genera of organisms, it is called a broad-host-range plasmid. 3. All plasmids can replicate themselves within the appropriate host, but not all plasmids can transfer themselves. a. A conjugative plasmid codes for the genes involved in transfer between cells. b. A nonconjugative, or mobilizable, plasmid requires the help of a conjugative plasmid to transmit itself to another bacteria. D. Insertion sequences are small (1,000 bp) pieces of DNA that code for the enzyme transpo sase, which allows them to jump into and out of DNA. 1. A transposon consists of two insertion sequences flanking another gene or set of genes.

2. Transposons and insertion sequences can insert into target DNA without significant homology at the site of insertion.

264

m8Ctical

--------

- - -

Bacteriology Overview

3. This ability to move between chromosomes, plasmids, or bacteriophages is an efficient mechanism for moving genes through a bacterial population. In fact, transposons are frequently associated with the formation of multiple-drug resistance plasmids.

DIFFUSION ACROSS BACTERIAL CELL MEMBRANES Clearly, bacteria must transport a number of macromolecules across the cell wall for survival. Larger ions and macromolecules diffuse slowly unless transported across the membrane by one of three mechanisms: facilitated diffusion, active transport, or group translocation. A. Facilitated diffusion Substrates that cross the membrane without the use of energy. Substrates bind to carrier proteins in the membrane whose affinity is equal both outside and inside the cell, allowing transport in both directions. Glycerol is transported into E. coli by this method and ATP may be brought into Rickettsia by this method. B. Active transport

Similar to facilitated diffusion except that energy is required. This allows substrates to be transported against a concentration gradient. Sugars, amino acids, and ions are transported by this mechanism. This mechanism utilizes ATP-binding cassette transport proteins. C. Group translocation

This mechanism does not expend energy to transport, but instead modifies the substrate to be transported (i.e., via phosphorylation of the substrate). Mannitol, mannose, and glucose are examples of substrates transported by this mechanism.

KAPLAlf I medlea 265

Bacteriology: Gram-Positive Cocci There are two medically important genera of Gram-positive cocci-staphylococci and streptococci. Both are nonmotile and do not form spores. Staphylococci are catalase positive, whereas streptococci are catalase negative.

STAPHYLOCOCCUS A. Genus characteristics and classification 1. Staphylococci are Gram-positive cocci that divide perpendicular to the last plane of divi-

sion, forming clumps or clusters. Depending on the age of the culture, they can be observed singly, in pairs, in short chains, or in grape-like clusters. 2. They are hardy organisms because they are relatively resistant to heat and drying. 3. Metabolically, the staphylococci are facultative aerobes and possess both superoxide dismutase and catalase. 4. Clinically, the most important distinction is between S. aureus and all other species that are nonpathogenic members of the normal flora. The coagulase test is a simple way to differentiate S. au reus from the coagulase-negative staphylococci. 5. Although there are six species of coagulase-negative staphylococci, the most numerous species on the skin is Staphylococcus epidermidis. The other coagulase-negative species of clinical relevance is S. saprophyticus. B. Staphylococcus aureus is a common infectious agent of humans and tends to cause localized or toxin-mediated disease. transiently colonizes the nasopharynx, skin, and vagina of up to 30% of the population.

1. Staphylococcus aureus

2. Table V-3-1 summarizes the conditions commonly caused by S. aureus.

I KAPLAlf medlea

267

Microbiology

Table V-3-1. Common conditions caused by Staphylococcus aureus. Direct infection Skin: Folliculitis, furuncles, carbuncles, abscesses, cellulitis, wound infection Deep infection: Osteomyelitis (often post-trauma and/or surgery) Systemic infections secondary to above Osteomyelitis, endocarditis, lung abscesses, pneumonia Toxin-mediated disease Food poisoning, scalded skin syndrome, bullous impetigo, toxic shock syndrome

3. Staphylococcus aureus does not produce a single factor that is necessary for virulence. The best host defense against infections are PMNs. No protective immune response is raised, so one can get infections again and again. Table V-3-2Iists the multiple virulence factors of s. aureus. 4. Treatment. The drug of choice is a penicillinase-resistant penicillin (e.g., methicillin, nafcillin, or oxacillin) or a first generation cephalosporin. Methicillin-resistant s. aureus (MRSA) requires therapy with vancomycin.

Note Unfortunately, there are now vancomycin-resistant MRSA.

C. Staphylococcus epidermidis is most commonly a nosocomial pathogen. In contrast to aureus, it is coagulase negative.

s.

1. The major virulence factor it produces is a viscous exopolysaccharide biofilm (slime).

2. When foreign bodies like IVs, catheters, and prosthetic valves are inserted into the host, Staphylococcus epidermidis can grow on their surface, embedded in the biofilm. This biofilm makes it difficult for the immune system to destroy the organism. 3. Clinical manifestations are typically related to instrumentation and procedures and include endocarditis and urinary tract infections. 4. Treatment is with vancomycin. D. Staphylococcus saprophyticus

1. Coagulase negative

2. Differentiated from S. epidermidis by its resistance to novobiocin. 3. Causes urinary tract infections in newly sexually active women. 4. Treatment is with penicillin. Table V-3-2. Major virulence factors of Staphylococcus aureus. I. Binding proteins and capsules 1. Protein A: • Binds IgG by the Fc portion-IgG can't react with the Fc receptor on phagocytes 2. Fibronectin, fibrinogen, vitronectin, and collagen type II binding proteins: • Localization and adherence through binding to body proteins 3. Capsule: • Carbohydrate • Prevents phagocytosis but most adults have opsonizing antibodies

268

meclical

(Continued)

Bacteriology: Gram-Positive Cocci

Table V-3-2. Major virulence factors of Staphylococcus aureus (cont'd). II. Enzymes 1. Coagulase: Exists in a bound form and a free form • Stimulates the conversion of fibrinogen to fibrin by binding coagulase-reacting factor (CRF), a derivative of prothrombin. This complex becomes active and initiates fibrin polymerization. • Coated with fibrin, staph are resistant to phagocytosis; the fibrin deposition in an area helps localize the infection 2. DNAse: • Production highly correlated with coagulase 3. Staphylokinase: • Clot dissolution, activates conversion of plasminogen to fibrinolytic plasmin 4. Hyaluronidase: • Hydrolyzes hyaluronic acids that are present in the extracellular matrix of connective tissue 5. Lipase: • Production associated with boils II. Exotoxins 1. Hemolysins-(alpha through delta): Alpha hemolysin or Alpha toxin • Most potent of hemolysins • Dermonecrotic and lethal when injected into rabbits • Causes membrane damage that lyses red blood cells and kills white blood cells by forming pores in eukaryotic membranes ~-hemolysin:

• A sphingomyelinase made by ",,20% of isolates 2. Leukocidin: • Lethal to PMNs, disrupts their membranes through pore formation 3. Epidermolytic toxin or exfoliatin: • Cleaves stratum granulosum layer per splitting desmosomes-no inflammatory response • Scalded skin syndrome in newborns, bullous impetigo in older kids and adults • Toxin is antigenic-circulating antibody confers immunity 4. Toxic shock syndrome toxin (TSST-1): • Symptoms = fever, desquamative skin rash, vomiting, diarrhea, hypotension, multiple system involvement • Induces production of interleukin 1 and tumor necrosis factor by macrophages • Antibody associated with resistance to shock 5. Enterotoxin: • Food poisoning • Types A through E • Heat stable • 1-6 hours post-ingestion; symptoms = increased intestinal peristalis, vomiting, diarrhea, no fever, rapid recovery • Toxin appears to act on neural receptors in UGI tract ~ stimulation of vomiting center • Pyrogenic exotoxins include TSST and the enterotoxins; they are super antigens (like streptococcal pyrogenic exotoxin A or erythrogenic toxin) and cause T-cell proliferation and release of toxic cytokines such as IL-1 and TNF (Continued)

meClical

269

Microbiology

Table V-3-2. Major virulence factors of Staphylococcus aureus (cont'd). IV. Resistance to penicillin and other ~-lactam antibiotics 1. (3-Lactamase production • Most often plasmid-encoded (few strains are chromosomal) 2. Intrinsic resistance-(aka methicillin resistance) • Chromosomal • Caused by a mutation in a penicillin-binding protein

Table V-3-3. Identification of Staphylococcus aureus versus Staphylococcus epidermidis. Staphylococcus aureus Coagulase Thermostable nuclease Mannitol fermentation Ribitol teichoic acid Glycerol teichoic acid

Staphylococcus epidermidis

+ + + +

+

STREPTOCOCCUS A. Genus characteristics 1. Streptococci are Gram-positive cocci that form chains.

2. Metabolically, the streptococci are facultative anaerobes because they derive energy from fermentation only (lack cytochromes). a. The principal end product of fermentation is lactic acid, and perhaps because of this they are more acid-tolerant than most bacteria. b. Although streptococci can live in conditions where oxygen is present, they lack catalase. Catalase is a cytochrome-containing enzyme that degrades hydrogen peroxide to oxygen and water. This feature is useful for differentiating the streptococci from other Gram-positive cocci such as the staphylococci. c. Almost all medically important streptococci are auxotrophs, meaning they require

one or more vitamins, amino acids, or nucleic acids for growth and therefore are not free-living in the environment.

B. Classification. The most common classification scheme for the streptococci is based upon their reaction in blood agar. 1. a-Hemolysis-the red blood cells surrounding the colonies are intact, but there is partial breakdown of the heme, resulting in a green (viridans) pigment.

2.

~-Hemolysis-the red blood cells surrounding the colonies are completely lysed. Betahemolytic streptococci are also classified serologically into Lancefield groups (A-O) based on their cell wall carbohydrate. Clinically, the most important group is Group A.

3. y-Hemolysis-no hemolysis or color change of the red blood cells is detected.

270

meClical

- -

-------------------------

------------------

Bacteriology: Gram-Positive Cocci

TableV-3-4 Species

Classification

Common Location

Associated Diseases

Streptococcus agalactiae*

Group B

Gastrointestinal tract, vagina, some animal sources, such as raw milk

Early-onset and delayed -onset neonatal disease (meningitis, respiratory distress, sepsis), puerperal sepsis, pharyngitis

Streptococcus bovis

Group D; a-hemolytic or nonhemolytic

Gastrointestinal tract

Endocarditis in patients with gastrointestinal tumors

Streptococcus dysgalactia and Streptococcus equi

Groups C and G; j3-hemolytic

Skin, mucosae

Pharyngitis, erysipelas, impetigo, neonatal meningitis, endocarditis, septic arthritis, wound infections

Streptococcus intermedius group

Groups A, C, F, and G; variable hemolysis

Mouth, gastrointestinal tract, vagina

Sinusitis, endocarditis, brain and liver abscesses

Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguis

Wide variety of Lancefield antigens; viridans streptococci; a-hemolytic

Mouth

Endocarditis

Streptococcus mutans

No Lancefield antigens; viridans streptococci; a-hemolytic

Mouth

Dental caries, endocarditis

Streptococcus pneumoniae*

No Lancefield antigens; a-hemolytic

Upper respiratory tract of 5-70% of general population

Pneumonia, meningitis, otitis media, sinusitis, conjunctivitis

Streptococcus pyogenes*

Group A;

Upper respiratory tract of 5-20% of general population

Pharyngitis, scarlet fever, pyoderma, erysipelas, necrotizing fasciitis, toxic streptococcal syndrome, rheumatic fever, heart disease, glomerulonephritis

Enterococcus faecalis*

Group D; variable hemolysis

Gastrointestinal tract, oral cavity, gallbladder, urethra, vagina

Endocarditis, bacteremia, urinary tract infections, abdominal infections, neonatal sepsis, soft tissue infections

Enterococcus faecium*

Group D; a-hemolytic or nonhemolytic

Gastrointestinal tract

Neonatal meningitis

*Boards' favorites.

~-hemolytic

~-hemolytic

KAPLA,..leaI me

271

Microbiology

c. (J.-Hemolytic streptococci can be distinguished from each other by their inhibition or growth in the presence of optochin or bile. This group includes Streptococcus pneumoniae and the viridans streptococci. 1. Streptococcus pneumoniae, also known as pneumococci, grows in pairs or short chains. a. It is lysed by optochin or bile and is the most autolytic of bacterial pathogens. At stationary phase, an autolytic enzyme, muramidase, is activated and destroys the bacteria.

In a Nutshell

b. It is a natural transformer since it can take up pieces of DNA from the surroundings and if there is sufficient homology, incorporate it into its genome.

S. pneumoniae • Alpha hemolytic; inhibited by optochin and bile

c. Transmission. Streptococcus pneumoniae is spread person-to-person through aerosol droplets. Twenty to 40% of normals are transiently colonized in their nasopharynx.

• Virulence conferred by polysaccharide capsule

d. Clinical manifestations. It is the most common cause of bacterial pneumonia and also causes otitis media, sinusitis, bronchitis, and bacteremia. It is the most common cause of meningitis in the elderly.

• Clinical correlations: - Pneumonia (#1 cause, especially middle-aged and older adults)

e. Risk factors for infection due to Streptococcus pneumoniae include poverty, a debilitated state of health, the absence of a spleen, and certain diseases such as sickle cell anemia, Hodgkin disease, multiple myeloma, and AIDS.

- Otitis media (#1 cause)

f. The most important virulence factor of Streptococcus pneumoniae is its carbohydrate capsule. Although there are more than 85 capsule types, 23 types cause the most virulence (85%). The capsule is thought to interfere with the opsonizing activity of the alternative complement pathway. Antibody against the capsule is necessary so C3b can attach without being buried.

- Sinusitis - Meningitis (#1 cause in elderly)

g. Prevention. A vaccine of 23 of the polysaccharide antigens exists. It should be given to the elderly, those undergoing splenectomy, and those with a condition predisposing them to Streptococcus pneumoniae disease. A vaccine of 7 of the polysaccharide antigens is administered to infants at 2, 4, 6, and 12-18 months as recommended by the CDC.

- Bacteremia • Certain populations are especially susceptible: elderly, smokers, alcoholics, children, asplenics

h. Treatment. In the past, all Streptococcus pneumoniae organisms were sensitive to penicillin. Penicillin resistance due to transformation with DNA from nonpathogenic streptococci in the oropharynx is becoming a problem. These cases can be treated with vancomycin or erythromycin.

• Vaccine available • Treatment with penicillin but resistance on the rise

2. Viridans streptococci are normal oral flora. a. In contrast to S. pneumoniae, they are not inhibited by optochin or lysed by bile.

Bridge to Cardiovascular and Renal Systems • Rheumatic fever is discussed in the Cardiovascular Pathology chapter of Organ Systems Book 1 (Volume III). • Poststreptococcal glomerulonephritis is discussed in the Renal Pathology chapter of Organs Systems Book 1 (Volume III).

272

mellical

b. They produce dextran, a substance that allows them to adhere to many surfaces. c. They are the major cause of subacute endocarditis (e.g., S. sanguis) in those with

abnormal heart valves. Streptococcus mutans causes dental caries. d. Treatment of choice is penicillin. D.

~-Hemolytic streptococci are further subdivided into groups A through D and F and G based on antibodies to a heat-stable, acid-stable carbohydrate in their cell walls. This antigen is called C carbohydrate or the Lancefield antigen, in honor of Rebecca Lancefield, the scientist who devised this classification system.

1. Group A streptococci (GAS) contains only one species, Streptococcus pyogenes, but it is

the most important streptococcal pathogen. a. It can be distinguished from the other by the antibiotic bacitracin.

~-hemolytic

streptococci because it is inhibited

b. Clinical manifestations of S. pyogenes are characterized as suppurative and nonsuppurative.

- - - - - - - - -

Bacteriology: Gram-Positive Cocci

Table V-3-S. Virulence factors of Streptococcus pyogenes. I. Cell constituents

1. M protein

• Required for virulence-80+ types • Externa-extends from cell envelope as fimbriae; carboxyterminal is attached to PDG and shows little variability; aminoterminal is outside-variable • Prevents phagocytosis by preventing complement opsonization. It has a site that binds factor H, leading to the hydrolysis of C3b to inactive form. • In conjunction with LTA, binds fibrinogen, hiding the C3-binding site • Antibody to a specific M-terminus provides immunity to that M type 2. Lipoteichoic acid or fibronectin-binding molecule • Attached to M protein; binds fibrinogen 3. F protein • Allows binding to fibronectin 4. Hyaluronic acid capsule • Inhibits phagocytosis 5. Cell-bound protease • Cleaves C5a component of complement and inhibits neutrophil chemotaxis II. Extracellular products

1. Hyaluronidase (spreading factor) • Destroys hyaluronic acid-could be important in the spread through tissue during cellulitis 2. Streptolysin 0

• Oxygen labile (inhibited by oxygen)-a hemolysin that is active only in its reduced form • Works by inserting directly into the host cell membrane, forming transmembrane pores • Immunogenic-can use ASO test for evidence of recent strep infection 3. Streptolysin S

• Oxygen stable; made in the presence of serum • Hemolysin causing l3-hemolysis-produced by most group A strains • Non-immunogenic 4. Streptococcal pyrogenic exotoxins (SPE) (erythrogenic toxins) • Rash of scarlet fever • Encoded by bacteriophage • Pyrogenic exotoxins are non specific activators of the immune system (superantigens)

KAPLA~_ I meulca 273

Microbiology

In a Nutshell S. pyogenes (Group A) • Beta hemolytic; sensitive to bacitracin • Many virulence factors, including M protein, capsule, F protein, hyaluronidase, Streptolysins 0 and S, and erythrogenic toxins • Clinical correlations - Pharyngitis (strep throat) - Scarlet fever - Skin: erysipelas, cellulitis, impetigo, necrotizing fasciitis, pyoderma - Secondary diseases: rheumatic fever and acute glomerulonephritis • Treatment with penicillin

(1) Suppurative complications of pharyngitis include otitis media, peritonsillar

cellulitis, peritonsillar and retropharyngeal abscesses, and bacteremic metastatic spread. Other suppurative infections include erysipelas (skin infection), pyoderma (impetigo), scarlet fever, cellulitis, lymphangitis, perianal cellulitis, puerperal sepsis, meningitis, pneumonia, and empyema. (2) Nonsuppurative sequelae occur weeks after initial infection. Inflammation occurs in organs not originally infected. These sequelae include acute glomerulonephritis, in which edema, hypertension, and hematuria occur after pharyngeal or skin infection. Also classified within this group is rheumatic fever, occurring 7-28 days after pharyngitis and results in fever, carditis, and polyarthritis. c. Transmission and epidemiology. Group A strep is an obligate human parasite spread person-to-person by respiratory secretion via droplets, direct contact with the skin, or fomites. Pharyngitis is most common in winter and spring, with the highest incidence among preadolescents. Contaminated milk or eggs have also been the cause of foodborne epidemics of pharyngitis. Impetigo-like skin infections are most prevalent in summer and are often due to the infection of insect bites. Group A strep produces a toxic enzyme called streptolysin 0 (SLO). Patients can be diagnosed in the lab based on the presence of antibodies to SLO. d. Virulence factors. Table V-3-5 summarizes the virulence factors of S. pyogenes. The most important virulence factor to remember is M protein. 2. Group B Streptococcus (Streptococcus agalactiae) is part of normal vaginal and intestinal flora in 25% of a given population. a. It is classified into serotypes based on polysaccharide and surface-protein antigens.

In a Nutshell

b. In contrast to S. pyogenes, S. agalactiae are resistant to bacitracin.

S. agaladiae (Group B)

c. The major virulence factor is an antiphagocytic polysaccharide capsule.

• ~-Hemolytic; resistant to bacitracin

d. Infants are more susceptible to disease from the organism than adults. They aspirate the organism during passage through the birth canal, and if they lack passive resistance from maternal IgG antibody, disease may ensue.

• Antiphagocytic capsule

e. Clinical manifestations include pneumonia, sepsis, and meningitis. Group B streptococci and E. coli are the major causes of these diseases in the neonatal population (under 1 month of age).

• Causes meningitis, sepsis, and pneumonia in neonates (acquired during passage through vaginal canal where organisms are part of normal flora)

f. Risk factors for acquiring pneumonia include: recent colonization of the mother, premature birth, low birth weight, and premature rupture of the membranes. Diabetics are presumably more susceptible to infection because high glucose concentration promotes capsule synthesis.

• *1 cause of neonatal

g. Therapy for S. agalactiae is a penicillinase-resistant synthetic penicillin.

meningitis Enterococci • Variable hemolysis • Normal fecal flora • Cause urinary tract infections in hospitalized patients; rare cause of subacute endocarditis

274

meClical

3. Enterococcus, formerly group D Streptococcus, include the important pathogens Enterococcus faecalis and Enterococcus faecium. a. Both organisms are part of the normal fecal flora. These organisms were classified as group D, ~-hemolytic streptococci; however, hemolysis is not a consistent feature since it is encoded on a plasmid. b. Enterococci can grow in 40% bile and 6.5% sodium chloride, as would be expected of a fecal organism. c. These organisms can cause infection when they spread to the urinary tract. When the intestine is disrupted, they are one of the organisms found in an abscess. Enterococcus species also cause approximately 10% of cases of subacute endocarditis.

- - - - - - - - -- -

--

---

----- - - - - - - - - - -

Baderiology: Gram-Positive Cocci

d. Unlike the streptococci, enterococci exhibit penicillin tolerance since they are inhibited, but not killed, by the antibiotic. Recently, vancomycin-resistant enterococci have appeared. During peptidoglycan synthesis, vancomycin binds to the D-ala-D-ala residues and prevents the construction of the cell wall. Vancomycin-resistant enterococci use D-lactic acid instead of D-ala to form the "peptide" bridges.

Positive Positive Gram-positive cocci

Catalase Test

Staphylococcus species

Coagulase Test

Resistant

Negative

Negative

Staphylococcus aureus

- - - - - - - 1 Novobiocin

Streptococcus species

Resistant

Sheep Blood Agar

a<

AlphaHemolysis

K

Sensitive

Staphylococcus epidermidis

Viridans Streptococci

Optochin

Sensitive

BetaHemolysis

I

Staphyloco?cuS saphrophyt/cus

Streptococcus pneumoniae (+ Quellung)

Bacitracin

Other /Hemolytic streptococci

Streptococcus pyogenes

Figure V-3-1. Gram-positive cocci flow chart.

UP~~.

I 275

mvulC8

Bacteriology: Gram-Positive Bacilli The four most important genera of Gram-positive bacilli are Listeria, Corynebaderium, Bacillus, and Clostridium. Listeria and Corynebaderium do not form spores, whereas Bacillus and Clostridium do.

LISTERIA MONOCYTOGENES A. Characteristics 1. L. monocytogenes is a small Gram-positive coccobacillus that does not form spores. It is

catalase positive, a feature that distinguishes it from other Listeria and from Streptococcus. 2. Microscopically, the organism resembles nonpathogenic members Corynebacterium genus ("diphtheroids"), which are part of normal skin flora.

of

the

3. Unlike the diphtheroids, Listeria monocytogenes is motile at room temperature and produces ~-hemolysis on blood agar. B. Transmission. Listeria is a facultative intracellular pathogen; it infects phagocytic cells. It also

produces listeriolysin 0, which is a beta-hemolysin similar to streptolysin 0. Listeria is most often acquired through ingestion of contaminated meat, unpasteurized dairy products, or food contaminated with animal fecal material. C. Risk factors. Groups at risk of serious disease from Listeria monocytogenes include neonates,

pregnant women, immunosuppressed patients, and alcoholics. D. Clinical manifestations

1. Neonatal infections can arise by genital tract colonization in pregnant women with transmission to the offspring across the placenta or during delivery. a. Granulomatosis infantisepticum is an in utero infection presenting within two days of birth. It causes widespread granulomas and has a very high mortality rate. b. Meningitis in neonates will occur as a result of infection during delivery. It accounts for 10% of neonatal meningitis. 2. Adult infections

a. Meningitis in immunocompromised hosts (steroids, chemotherapy, transplants, alcoholics, etc.). b. Bacteremia in pregnant women E. Therapy is ampicillin with sulfamethoxazole/trimethoprim as an alternative.

KAPLA!._

I 277

meulC8

Microbiology

CORYNEBACTERIUM DIPHTHERIAE A. Characteristics. C. diphtheriae is a nonmotile, club-shaped, non-spore-forming, Grampositive rod. E. Diphtheria toxin is responsible for the clinical symptoms.

1. A lysogenic phage encodes diphtheria toxin.

2. Iron restriction activates toxin production. 3. It is synthesized as a single amino acid chain, but must be cleaved for activity. Fragment A has ADP-ribosyltransferase activity, and fragment B allows binding and insertion into the host cell membrane. 4. Diphtheria toxin catalyzes the ADP-ribosylation of elongation factor 2, thereby inhibiting host protein synthesis.

e. Clinical manifestations

of diphtheria include upper respiratory infection resulting in a tonsillar grayish pseudomembrane that may spread to the pharynx and larynx, and can compromise the airway. Although the organism is not invasive, the toxin is absorbed systemically and acts on other tissues. Diphtheria toxin is especially toxic to the heart and can cause cardiac failure.

D. Treatment consists of antitoxin and erythromycin and should be administered as soon as possible. E. Prevention. A vaccine containing diphtheria toxoid is administered during the first year of life. Boosters are required every ten years.

BACILLUS Bacillus species are a group of large, nonmotile, Gram-positive rods that produce spores. A. Bacillus anthracis is the major pathogenic species. 1. Spores produced by the organism persist in soil or in products from infected herbivores for many years.

Note The anti phagocytic capsule of B. anthracis is unique in that it is composed of D-glutamate, not polysaccharide.

2. Encoded on a plasmid are an antiphagocytic capsule and three virulence factors that act in concert. What is commonly called anthrax toxin is actually a combination of these three toxins. a. Protective antigen (PA) binds to target cells to promote uptake of lethal factor (LF) and edema factor (EF). b. Lethal factor is toxic to macrophages. c. Edema factor is an adenylate cyclase that increases cyclic-AMP levels in target cells following its insertion in the membrane. In a phagocyte, this decreases phagocytic activity. 3. Transmission. Infection commonly occurs through skin cuts or abrasions, although the organism may also be inhaled. 4. Clinical manifestations of anthrax may be cutaneous or systemic. a. Cutaneous anthrax accounts for 95% of all infections. The characteristic presentation is papules that develop into ulcers with necrotic centers. Regional lymphadenopathy can occur. Edema is a major complication and this infection may be fatal in 20% of untreated cases.

278

meclical

Bacteriology: Gram-Positive Bacilli

b. Systemic anthrax is acquired through the respiratory (inhalation anthrax-woolsorter disease) or gastrointestinal route, and results in lymphadenopathy and septicemia. It is almost always fatal. 5. Therapy and prevention a. The drug of choice for Bacillus anthracis infection is penicillin or ciprotloxacin. b. Anthrax is controlled through prevention and clean up of contaminated areas. e. In the United States, a killed vaccine is available for individuals with a high risk of exposure and for livestock. B. Bacillus cereus produces two enterotoxins and usually grows in foods, especially in cereal

grains such as rice. 1. The principal clinical manifestation is food poisoning of two types: a. A short incubation food poisoning (1-6 hours; emetic type) causes severe nausea and vomiting. Epidemics of this type have been known to be associated with fried rice. It is often confused with staphylococcal food poisoning due to its short incubation. b. Food poisoning of long incubation (10-24 hours; diarrheal type) is observed as abdominal cramps and diarrhea. This type may be confused with clostridial food poisoning.

Clinical Correlate Because of the threat of biologic warfare, the Department of Defense has decided to vaccinate members of the armed forces against anthrax.

2. Therapy and prevention include support for food poisoning, such as as administration of fluids. If an antibiotic is indicated, vancomycin can be used.

CLOSTRIDIA Clostridia are characterized as large, obligate anaerobic, spore-forming rods. They are Grampositive. A. Characteristics 1. Clostridia are usually found in the soil or human gastrointestinal tract and grow on blood agar or chopped meat glucose media. 2. The four major pathogenic species, C. perfringens, C. difficile, C. tetani, and C. botulinum, produce many toxins and destructive enzymes, including collagenase, protease, hyaluronidase, and lecithinase. 3. Most infections are mixed, that is, an aerobic organism grows first and reduces the environment, thus allowing the anaerobic Clostridia to grow. B. Clostridium perfringens is a fast growing, nonmotile organism found in the soil and the

intestine. 1. Characteristics a. When cultured on blood agar, a double zone of hemolysis is seen. It will not produce spores on artificial media. b. There are five types, A through E, based on the production of four toxins, alpha, beta, epsilon, and iota. All strains produce alpha toxin, a calcium-dependent phospholipase C that is also known as lecithinase. It causes the lysis of erythrocytes and other cells. 2. Transmission of C. perfringens occurs through infection of disrupted skin, bowel, or other epithelial tissues. Infection may be due to traumatic injury, surgery, or septic abortion. The organism is part of the normal intestinal flora. Spores are frequently found in soil. KAPLAlf _ I meillea 279

Microbiology

3. Clinical manifestations take on several forms depending on the site and severity of infection. A diffusely spreading organism may cause cellulitis and fasciitis and/or bacteremia. a. Gas gangrene or myonecrosis is a life-threatening illness characterized by muscle and connective tissue necrosis. Gas, an end product of fermentation, forms in the muscle tissue and causes crepitation. Approximately 80% of the cases of gas gangrene are caused by C. perfringens. b. Food poisoning can be caused by C. perfringens. It is thought to be the third most common cause of bacterial food-borne epidemics, after Staphylococcus aureus and Salmonella. The enterotoxin causes a self-limited gastroenteritis by binding to a brush border membrane receptor and interrupting ion exchange. This results in the loss of low-molecular weight metabolites and ions. Onset of clinical symptoms is 8-16 hours after ingestion of the organism. The infection is characterized by abdominal pain and diarrhea for 12-24 hours; systemic effects are uncommon. c. Skin and soft tissue infections are typically localized infections. Tissue infection is characterized by crepitant cellulitis, stump infection in amputees, perirectal abscesses, diabetic foot ulcers, and decubitus ulcers.

d. Suppurative infection is usually polymicrobial in origin. Intra-abdominal infection may cause bowel perforation and emphysematous cholecystitis. Pelvic infection may be seen as tubo-ovarian and pelvic abscesses and as septic abortion. 4. Therapy for gas gangrene consists of surgical debridement plus pencillin. Food poisoning due to the enterotoxin of type A is usually self-limiting and hence is not treated with antibiotics. C. Clostridium difficile

1. Clostridium difficile is a component of the normal bowel flora in a small percentage of adults. 2. The organism can produce two heat-labile toxins, enterotoxin (exotoxin A) and cytotoxin (exotoxin B).

3. Clinical manifestations. C. difficile causes over 25% of antibiotic-associated diarrhea and 95% of the cases of pseudomembranous colitis. a. Two antibiotics that commonly precipitate pseudomembranous colitis are clindamycin and ampicillin. b. Pseudomembranous colitis presents with a history of nausea, vomiting, abdominal pain, and voluminous green diarrhea. c. Proctoscopy demonstrates pseudo membranes with an erythematous mucosa.

d. Diagnosis is confirmed by demonstrating toxin in the stool. 4. Treatment. Oral vancomycin or metronidazole are the current treatments. D. Clostridium tetani 1. Characteristics

a. Spores are abundant in the soil. When they are inoculated into a site of injury, a drop in the redox potential leads to germination. In 50% of cases, there is no history of a wound. b. The organism produces tetanospasmin, a plasmid-encoded neurotoxin that blocks the normal inhibition of spinal motor neurons. It prevents the release of the inhibitory neurotransmitters glycine and gamma-amino butyric acid, leading to spastic paralysis. Death can occur from respiratory failure.

280

meClical

Bacteriology: Gram-Positive Bacilli

2. Clinical manifestations. Tetanus has four clinical presentations: a. Local infection can cause persistent local muscle contraction. It has low mortality and is noninvasive. b. Cephalic infection is rare in presentation. It can follow chronic otitis media and can progress to the generalized form. c. Generalized tetanus infection is the most recognized form. It is painful and presents with "lockjaw" progressing to opisthotonos. Mortality is approximately 60%. d. Neonatal tetanus infects the umbilical stump. It is the major cause of infant mortality in developing countries. 3. Therapy for tetanus includes surgical debridement of the wound, the use of human tetanus antitoxin, respiratory support (if required), and muscle relaxants (curare-like drugs). The antibiotic treatment of choice is metronidazole.

In a Nutshell . Virulence Factors of Gram+ Bacilli

Corynebacterium diphtheriae

Phage-encoded toxin that inactivates EF-2 by ADP ribosylation. Protein synthesis inhibited in host.

Bacillus anthracis

Antiphagocytic capsule

4. Prevention. Because the same toxin is found in all strains, tetanus can be prevented by immunization with tetanus toxoid. Booster shots are required every ten years. In contrast to diphtheria, a patient who survives tetanus is not immune to the disease, since the minute amount of toxin necessary to cause disease is not sufficient to elicit an immune response. E. Clostridium botulinum is found ubiquitously in soil.

1. Characteristics

+ [protective

a. It produces a powerful, heat-labile neurotoxin that is usually ingested pre-formed with improperly canned food. The toxin blocks the release of acetylcholine from motor neurons in the peripheral nervous system, producing flaccid paralysis.

Anthrax toxin

b. There are seven immunologic types of toxins, some of which are phage-encoded. 2. Clinical manifestations a. There are a constellation of clinical signs associated with C. botulinum intoxication. (1) Dilated unreactive pupils (bulbar paralysis)

Bacillus cereus

2 enterotoxins

Clostridium perfringens

4 toxins; key one is alpha toxin (lecithinase)

Clostridium difficile

Enterotoxin (exotoxin A) Cytotoxin (exotoxin B)

Clostridium tetoni

Plasmaencoded neurotoxin (tetanospasmin) blocks release of inhibitory neurotransmitters-spastic paralysis

Clostridium botulinum

Neurotoxin blocks release of acetylcholineflaccid paralysis

(2) Descending weakness or paralysis usually starting with the cranial nerves (3) Progressive respiratory weakness (4) Absence offever (5) Dry mucous membranes (6) Unexplained postural hypotension b. Infant botulism is also called floppy baby syndrome. When an infant is colonized by the organism, extremely low levels of toxin can be produced, leading to failure to thrive and eventually to progressive muscular weakness and poor motor development. Older children and adults do not seem to be afflicted. Infant botulism is the most common form in the u.s. and is the reason why infants should not be fed honey. c. In contrast to tetanus, the autonomic dysfunction that results from botulism is not

life-threatening if the patient is intubated and appropriate ventilator management is used.

antigen Lethal factor Edema factor (adenylate cyclase)

3. Therapy for botulism toxin poisoning includes respiratory support and human trivalent antitoxin therapy that targets three toxins-A, B, and E.

KAPlA~.

meulC8

I

281

~-----------

---

--------------

Bacteriology: Gram-Negative Organisms The Gram-negative bacteria are a very heterogeneous group with a large number of medically relevant species. This chapter reviews the important Gram-negative cocci (Neisseria) as well as the various groups of Gram-negative bacilli (enteric organisms, zoonotic organisms, and respiratory organisms).

GRAM-NEGATIVE COCCI A. Neisseria 1. Genus characteristics a. Neisseria are nonmotile, nonspore-forming, Gram-negative cocci. They are characteristically arranged in pairs (diplococci) with flattened adjacent sides facing each other. On Gram stain, they resemble coffee beans. b. They are oxidase positive. The oxidase test detects the c component of the cytochrome oxidase complex. It is positive in Neisseria, as well as in the Vibrios and some pseudomonads. c. Eight species have been identified. Two of these, N. meningitidis and N. gonorrhoeae,

are pathogenic for humans. 2. Isolation of the pathogenic Neisseria. The pathogenic Neisseria are fastidious organisms. They are very susceptible to heat, cold, and drying, so specimens require special handling. a. The pathogenic Neisseria require a rich media for growth. They grow nicely on a heated blood preparation called chocolate agar with 2-8% CO 2 (a candle jar). If cultures are obtained from usually sterile sites, like the cerebral spinal fluid, chocolate agar alone can be used for culture media. If the culture is obtained from a site that is usually occupied by many species of organisms, such as a cervical smear, antimicrobial agents must be added to the chocolate agar. Thayer-Martin media consists of chocolate agar plus vancomycin, colistin, and nystatin to inhibit normal vaginal flora. b. N. meningitidis and N. gonorrhoeae are differentiated based upon sugar utilization. N. gonorrhoeae can ferment glucose, but not maltose. N. meningitiditis can ferment glucose and maltose. 3. Virulence factors of the pathogenic Neisseria are detailed in Table V-S-1.

Mnemonic Think MGM (as in the studio): Meningitidis ~ glucose + maltose (gonorrheae = glucose only)

meCtical

283

Microbiology

Table V-5-1. Summary of the major virulence factors of Neisseria.

Neisseria meningitidis (Meningococcus)

Neisseria gonorrhoeae (Gonococcus)

Sugar utilization

Glucose and maltose

Glucose

Capsule

Acidic polysaccharideantiphagocytic and anticomplementary 9 serotypes; vaccine = 4 serotypes

Present, but role in virulence is uncertain

Pili

Adhesion Small antiphagocytic role Necessary for DNA uptake

Adhesion necessary for DNA uptake; contributes to resistance to neutrophil killing See antigenic phase variation (a switch between alternative states like pili or nonpili)

Porins

Present

Present

Endotoxin

Lipopolysaccharide (LPS)

Lipooligosaccharide (LOS)

IgAl protease

Present

Present

Penicillinase R plasmid

No

Yes, in some stains

B. N. meningitidis (meningococcus)

1. Features a. Ferment glucose and maltose b. The key virulence factor for N. meningitis is its antiphagocytic capsule. The capsule is the basis of serotyping and serves as the antigen for vaccines. Thirteen different capsule types have been identified. c. Endotoxin and IgA protease are also important virulence factors. 2. Transmission of meningococcus is via respiratory droplets. The carriage rate in adult nasopharyngeal tissue is approximately 10-30%. Most carriers are asymptomatic. It enters the upper respiratory tract and may eventually disseminate. The risk of disseminated disease is greatest in individuals with late complement (C6, C7, and C8) deficiencies. 3. Clinical manifestations a. Meningitis, usually of sudden and fulminant onset b. Meningococcemia, a vasculitic purpura causing disseminated intravascular coagulation (DIC) c. Meningococcus is responsible for Waterhouse-Friderichsen syndrome, characterized

284

iiie&ical ~--------

~-~-.~---

....

-------~

Bacteriology: Gram-Negative Organisms

by coagulopathy, hypotension, adrenal cortical necrosis, and sepsis, which is usually fatal. In addition, metastatic lesions may appear in lung, pericardium, joints, eyes, etc. 4. Diagnosis is made by identifying Gram-negative cocci in the spinal fluid and confirmed by culture of the CSF or blood and demonstration of oxidase production and maltose fermentation. 5. Therapy and prevention a. Penicillin G or ceftriaxone are the antibiotics of choice. b. Carriers and close contacts can be treated prophylactically with rifampin for two days. c. A quadrivalent vaccine exists to capsule types A, C, Y, and W135. The vaccine does not protect against type B, which has a capsule that is not immunogenic. C. N. gonorrhoeae (gonococcus)

1. Features a. Ferment glucose but not maltose b. Many strains produce 13-lactamase 2. Transmission of the gonococcus is via sexual contact, but may also involve fomites. It has an increased incidence of infection in sexually active young adults (15-30 years old), nonwhites, low socioeconomic groups, and urban settings. 3. Clinical manifestations are primarily associated with the urogenital tract, although disseminated disease can occur. a. Acute urethritis and gonorrhea in men is observed as a yellow purulent discharge with dysuria. Ninety percent of men are symptomatic. Common complications of acute urethritis include urethral stricture, epididymitis, and prostatitis. Proctitis is especially common in male homosexuals. b. Gonorrhea in females is asymptomatic in 20-S0% of cases, most likely due to the inability to observe a discharge. Complications include pelvic inflammatory disease (15-20% of cases), generalized peritonitis, and infertility. c. Disseminated disease can be observed as meningitis, subacute bacterial endocarditis, and arthritis. (1) Arthritis is manifested as a dermatitis syndrome called tenosynovitis. Cultures of joint and skin pustules are positive in <30% of cases. (2) Host factors that increase susceptibility to disseminated disease include deficiencies of terminal complement components C6, C7, and CS. d. Ophthalmia neonatorum results from maternal transmission to the infant during birth. Ophthalmic tetracycline, erythromycin, or silver nitrate is given to the neonate for prevention. Neonatal infection may cause gonococcal arthritis. e. Pharyngitis. Diagnosis is established by identifying Gram-negative cocci in PMNs from the infected site and is confirmed by culture. This may occur in neonates, but is much more common in individuals who engage in oral sex. 4. Diagnosis of gonococcal urethritis in males is made by the observation of Gram-negative diplococci and neutrophils in the urethral exudate. In females, culture and biochemical testing is required due to the presence of other Gram-negative coccal forms as part of normal vaginal flora. The specimen is cultured on a medium such as Thayer-Martin agar, which contains antibiotics to control the growth of the normal flora organisms. The observation of Gram-negative diplococci that are oxidase positive and able to ferment both glucose and maltose is considered definitive for the meningococcus.

meClical

285

Microbiology

5. Treatment and prevention

In a Nutshell Gonococcus Meningo· coccus Ferment

Glucose

Glucose and maltose

Spread

Sexual; passage through birth canal

Aerosol

No

Yes

Vaccine

a. Ceftriaxone is the antibiotic of choice for treating infection. N. gonorrhoeae is no longer susceptible to penicillin because of two mechanisms: a plasmid-mediated ~-lactamase, and chromosomally mediated decreased affinity of penicillin-binding proteins. b. Because combined infections are so common, treatment regime should include an antichlamydial agent (e.g., doxycycline). c. Safe sexual practices can decrease the incidence of gonorrhea. d. All cases of gonorrhea must be reported to the Public Health Department.

Treatment Ceftriaxone Penicillin

GRAM-NEGATIVE BACILLI: ENTEROBACTERIACEAE In a Nutshell Medically Important Enterobacteriaceae include: • Enterobader

• Escherichia • Klebsiella • Proteus • Salmonella • Serratia • Shigella • Yersinia

The family Enterobacteriaceae includes many Gram-negative, nonspore-forming, facultative bacilli with simple growth requirements. The majority of Enterobacteriaceae are normal gastrointestinal flora. In the gastrointestinal tract they exist in a symbiotic relationship with the host. They synthesize vitamin K and deconjugate bile salts and sex hormones for recirculation to the liver. They also prevent the colonization of intestinal mucosa by primary pathogens. Colonization is inhibited by colicin/bacteriocin synthesis and receptor competition. The normal flora of Enterobacteriaceae, which include Escherichia, Citrobacter, Klebsiella, Enterobacter, Serratia, and Proteus, generally lack the virulence factors of the pathogenic species. They act as opportunistic pathogens when they breach the normal anatomic barriers or when the host is severely immunocompromised. Opportunistic Enterobacteriaceae are the most common cause of intra-abdominal sepsis and urinary tract infections. A subset of Enterobacteriaceae are considered primary pathogens because they contain virulence factors capable of overcoming normal host defenses. They are not part of the normal flora. This subset includes Shigella species, Salmonella species, Yersinia species, and some strains of Escherichia coli. A. General characteristics. There is no universally accepted taxonomic classification for this group. The CDC scheme is based on DNA homology techniques used to determine the degree of relatedness between species. Antigenic and biochemical differences are utilized in classification. B. Physiology 1. The Enterobacteriaceae are facultative organisms; they can ferment or respire, depending

upon the conditions. 2. All members of Enterobacteriaceae ferment glucose and reduce nitrates to nitrites. 3. All are oxidase negative, meaning they lack cytochrome c, although they have an electron transport chain. 4. If motile, they have peritrichous flagella. Klebsiella and Shigella are nonmotile. 5. Enterobacteriaceae can be divided into lactose fermenters and nonlactose fermenters by their colony appearance on selective/differential media. The most commonly used media are eosin methylene blue agar (EMB) and MacConkeyagar. If the lactose is fermented, the colony changes color. a. Important lactose fermenters are Escherichia coli, Citrobacter, Klebsiella, and Serratia. b. Important nonlactose fermenters include Shigella, Salmonella, Proteus, and Yersinia. 6. Enterobacteriaceae are easily destroyed by heat and by common disinfectants or germicides. They are sensitive to drying or desiccation. In contrast, they survive best in a high-

286

meCtical

Bacteriology: Gram-Negative Organisms

moisture environment. Respiratory care or anesthesia equipment are common sources of nosocomial infections. Contaminated ice machines or water supplies may harbor these organisms, causing epidemics. C. Antigenic structure 1. K antigens (Vi antigen) are acidic polysaccharide capsules. They are antiphagocytic by

blocking the access of complement or antibodies to the organism. The K1 antigen of nephritogenic E. coli strains is associated with adherence to lectins of the GU epithelium. K1 is also associated with neonatal meningitis because it is antiphagocytic, anticomplementary, and resembles sialic acid. The capsule antigen in Salmonella typhi is called Vi antigen. 2. H antigens are the flagellar proteins and are present only in motile organisms. H antigen may exist in one of two phases in Salmonella. Shigella are not motile and do not express H antigens. 3. 0 antigen, or somatic antigen, causes a "smooth" appearance in culture and is the name for repeating oligosaccharide units found in the endotoxin in the outer membrane of Enterobacteriaceae. Certain 0 antigens promote adherence to gastrointestinal or genitourinary epithelium. D. Pathogenicity is due primarily to endotoxin (also called LPS; the lipopolysaccharide por-

tion of the cell wall) present in all Enterobacteriaceae. 1. Toxicity lies in the lipid A portion. LPS may cause endotoxic shock when the enteric

bacilli enter the bloodstream (septic shock). Endotoxic shock is characterized by blood pools forming in the microcirculation, resulting in hypotension. Vital organs lack adequate blood supply, leading to decreased tissue perfusion, acidosis, ischemia, and cellular hypoxia. DIC may also occur to further compromise the patient. 2. Enterotoxins are produced by some members of the family (some E. coli, Shigella) that exert their toxic effects on the small intestine. Enterotoxins cause secretion of fluid into the lumen, resulting in secretory diarrhea. 3. Pili, or fimbriae, promote adherence to tissues.

E. Shigella 1. Characteristics a. Shigella is an obligate human pathogen. There are four species of Shigella: S. dysenteriae, S. flexneri, S. sonnei, and-S. boydii. b. It is a slender, nonmotile organism that does not ferment lactose or produce H 2 S. 2. Transmission. The primary mode of transmission is fecal-oral. These organisms are not killed by stomach acid, therefore, only a small number of organisms «100) are needed to cause disease. 3. Pathogenesis. The site-of disea~e is in the colon, where Shigella invades and superficially destroys the intestinal mucosa:'"Virulence factors of Shigella include adhesins, invasins, and toxins. Most are encoded on plasmids.

In a Nutshell

All Enterobacteriaceae • Gram-negative bacilli • Facultative anaerobes • Ferment glucose • Reduce nitrates • Oxidase negative • Have endotoxin • Found in GI tract (exception: Y pestis)

Some Enterobacteriaceae • Motile (all except Klebsiella and Shigella) • Have polysaccharide capsule (Klebsiella, Salmonella)-K antigen • Ferment lactose (E coli, Citrobader, Klebsiella, Serratia)

Note Shigella and Salmonella typhi are the only Enterobacteriaceae that are human-only pathogens,

a. Plasmid-encoded outer membrane proteins attach by binding to integrins. Invasive plasmid antigens (ipa) mediate entry of the organism through M cells in Peyer patches, and the eventual phagocytosis by mucosal cells. b. Once inside a cell, the phagosome ruptures, and Shigella multiples within the cytoplasm. Since the organisms are nonmotile, they are pushed through the cytoplasm and into the next cell by rearrangement of the cytoskeleton. KAPLAN

'.

med lea

I 287

Microbiology

c. Shiga (S. dysenteriae) and Shiga-like toxins are the major toxins of Shigella species. They are chromosomally encoded cytotoxins. During bacterial cell lysis, they are released and bind to the surface of other cells by a glycolipid. Once absorbed byendocytosis, they exert neurotoxic, cytotoxic, and enterotoxic effects.

d. Hemolytic-uremic syndrome due to Shigella may be explained by the effects of these toxins on the microvasculature.

Clinical Correlate An examination of a fecal sample for PMNs is a key component of the workup of infectious diarrhea. If they are present, you are dealing with an invasive organism such as Shigella, Sa/manella, or Campy/abader (an inflammatory diarrhea). If there are no PMNs, you are more likely to be dealing with a toxinproducer such as ETEC or V cha/erae (a noninflammatory diarrhea).

e. Finally, like all Gram-negative bacteria, Shigella have endotoxin. This endotoxin increases the local inflammatory response, but seldom causes septic shock since the organisms do not invade beyond the submucosa. 4. Clinical manifestations (shigellosis) a. Bacillary dysentery characterized by abdominal cramps and diarrhea. The feces contain blood, polymorphonuclear leukocytes, and mucus. b. One to four weeks after the disease, a carrier state may be set up if the organism is not cleared. Carriers experience long-term, recurrent bouts of disease. 5. Therapy and prevention includes hydration and electrolyte replacement. Fluoroquinolones are now considered antibiotics of choice. a. Alternatively, ampicillin, tetracycline, or trimethoprim-sulfamethoxazole decreases the duration of symptoms and development of the carrier state. b. Prevention is facilitated through personal hygiene, proper garbage disposal, and water purification. F. E. coli are motile, ferment lactose (which helps differentiate E. coli from Shigella and Salmonella), and will form colored colonies on EMB or MacConkey agar. The gastrointestinal tract is a natural reservoir for E. coli. 1. Pathogenicity in strains causing neonatal meningitis or uropathogenic strains is related

to the presence of the Kl capsular antigen, which inhibits phagocytosis. Nephropathogenicity is associated with plasmid-mediated hemolysin production.

Note An enterotoxin is a protein exotoxin that is released locally in the intestine.

2. Enterotoxigenic Escherichia coli (ETEC) is a major cause of infant death in developing countries and is the most common cause of "traveler's diarrhea:' a. More than 100 serotypes of E. coli cause this noninflammatory, secretory diarrhea that is similar to cholera, but less severe. b. The organism is acquired through the ingestion of fecally contaminated food or water. c. Disease is noninvasive and occurs in the small intestine.

d. Major virulence factors are plasmid-encoded, including enterotoxins and pili-termed colonization factor antigens (CFA) that act as adhesins.

Note

(1) Heat-labile toxin (LT) ADP ribosylates G stimulatory (Gs) protein, destroying

LT is a classic A-B toxin. The B portion binds to the epithelium in the small intestine. The A portion is enzymatically active and ADP ribosylates the adenylate cyclase regulatory protein, thus increasing cAMP levels in the cell.

288

meClical

its GTPase activity. Without it, the alpha subunit of Gs constantly stimulates adenylate cyclase to produce cyclic AMP, leading to a loss of electrolytes and water. LT toxin shares DNA homology with cholera toxin. (2) Heat-stable (ST) toxin is a small peptide toxin that mimics the peptide hormone guanylin. It binds to the guanylate cyclase receptor and increases the cellular cGMP level, causing mucosal cells to release fluids. 3. Enteroaggregative Escherichia coli (EAggEC) causes persistent watery diarrhea in children and traveler's diarrhea.

Bacteriology: Gram-Negative Organisms

a. The organism is acquired by ingestion. b. Virulence factors include pili, an enterotoxin, and a cytotoxin. The pili promote adherence. The enterotoxin is similar to ST toxin. 4. Enteropathogenic Escherichia coli causes non-inflammatory diarrhea. a. Infants, especially in the developing world, are very susceptible. b. Enteropathogenic Escherichia coli infection causes effacement of microvilli without gross ulceration. The stools are nonbloody. It is more invasive than ETEC or EAggEC, but less invasive than the pathogens causing dysentery. e. This bacteria has no known toxins, but several kinds of adhesins.

5. Enterohemorrhagic Escherichia coli (EHEC) causes bloody diarrhea, similar to dysentery. a. Its reservoir is livestock, especially beef cattle. b. The organism is acquired by ingestion of poorly cooked ground beef. Epidemics associated with fast-food chains or processed food have occurred, although the organism has also been transmitted in milk, cider, and water. e. One serotype, 0157:H7, is the predominant cause of disease.

d. It is not significantly invasive; however, the verotoxin (Shiga-like toxin) can result in hemorrhagic colitis. e. Virulence factors include adhesins that efface the microvili and a bacteriophageencoded Shiga-like toxin. f. Enterohemorrhagic Escherichia coli can precipitate hemolytic-uremic syndrome, which is most common in kids <5 years old. 6. Enteroinvasive Escherichia coli (EIEC) causes dysentery that is clinically indistinguishable from dysentery due to Shigella. a. It lacks Shiga toxin, but contains a virulence plasmid that is essentially identical to those of Shigella. b. The organism is acquired through the ingestion of contaminated food. e. It produces a severe, but self-limited dysentery in most adults, but is often fatal in

young children. 7. Other clinical manifestations of E. coli infection a. E. coli is the most common causative organism in urinary tract infections (UTIs). b. Neonatal pneumonia is usally the result of nosocomial infection due to aspiration during the birth process. Sepsis may also occur. e. Neonatal meningitis is also caused by exposure during birth. It is a serious infection resulting in 40-80% mortality.

In a Nutshell E. coli • Most common cause of UTls • Most common cause of traveler's diarrhea • Causes neonatal pneumonia, sepsis, and meningitis

8. Therapy and prevention. Antibiotic therapy is based on the site, severity, and sensitivity of the infectious organism. a. UTI is usually treated with Bactrim (sulfa + TMP) or a fluoroquinolone. b. Pneumonia, meningitis, and sepsis are commonly treated with a third-generation cephalosporin such as cefotaxime and/or an aminoglycoside.

KAPLAlf I meillea

289

Microbiology

c. Diarrheal syndrome is usually treated with fluids and electrolytes only. Bismuth subsalicylate inactivates and binds enterotoxins. G. Salmonella. Salmonella are motile (in contrast to Shigella) and do not ferment lactose. 1. Nontyphoidal Salmonella infections cause inflammatory diarrhea with fever and vari-

able septicemia. a. Infection is acquired through the ingestion of eggs, chicken, and other contaminated food or water. Contact with reptiles can also cause Salmonella. Nausea, vomiting, and diarrhea arise 8-24 hours after ingestion of contaminated food. b. In contrast to Shigella, a large inoculum (> 1 million cells) is needed to survive gastric acid and cause disease. c. Disease is more severe in children under the age of 10.

d. Septicemia is more likely to occur in infants or the immunocompromised. Organisms especially likely to cause septicemia include S. choleraesuis and S. enteritidis.

Note Unlike the other salmonellae,

5. typhi inhabit only the human colon.

e. Known virulence factors include antiphagocytic (Vi antigen) capsules, outer membrane protein adhesins, and LPS. LPS and outer membrane proteins prevent complementmediated killing during bacteremia. Also, the organism can grow within macrophages. 2. Typhoidal Salmonella infections are caused by Salmonella typhi and Salmonella paratyphi A, B, and CI. Typhoid or enteric fever is a progressive, subacute, febrile-wasting illness. It is common in developing countries, and the most severe manifestations occur in young children. a. Transmission is by ingestion of a large inoculum of bacteria in food or water contaminated by feces from a human. b. The intestine shows local inflammation with mucosal ulcerations and perforations. The organisms spread to the bloodstream and the reticuloendothelial system. c. LPS and other outer membrane proteins inhibit complement-mediated killing. Vi

Clinical Correlate Salmonella is the most common cause of osteomyelitis in sickle cell patients.

antigen, the polysaccharide capsule, inhibits phagocytosis and may contribute to the resistance of S. typhi to complement-mediated killing. d. Clinically, there are three phases of typhoid fever. (1) The first week is expressed as fever, lethargy, constipation, and pain. (2) The second week is when bacteremia occurs. Biliary and organ involvement arise along with a sustained fever with temperature/pulse dissociation (i.e., high fever, low pulse rate), abdominal pain, rose spots, and diarrhea. This period of time is when the gastrointestinal tract is reinfected. (3) With the third week, exhaustion and fever improve unless other complications arise. Complications of typhoid fever include relapse (20%), severe bleeding, thrombophlebitis, abscess formation, pneumonia, cholecystitis (in acute and chronic colonization), and death (2-10%). e. Treatment. Third-generation cephalosporins and fluoroquinolones are the drugs of choice in developed countries. Chloramphenicol is much cheaper and is still used in developing countries. Attenuated oral vaccines and killed parenteral typhoid vaccines are available for treatment. H. Yersinia pestis is discussed along with the other Gram-negative zoonotic organisms. Yersinia enterocolitica and Y. pseudotuberculosis both cause a gastrointestinal disease clinically indistinguishable from that caused by Salmonella or Shigella species. Symptoms include severe

abdominal pain (often mimics appendicitis), diarrhea, fever, and blood in the stool.

290

meClical

Bacteriology: Gram-Negative Organisms

Complications such as septicemia or hepatic abscesses are rare and occur in patients with underlying diseases such as diabetes, pre-existing liver disease, or neutropenia secondary to steroid therapy. Wild and domestic animals are the common reservoir for humans; hence, the disease has a sporadic occurrence although local outbreaks in families or school groups have been reported. Drug sensitivities are variable, so sensitivity testing should be done; most strains are susceptible to aminoglycosides and trimethoprim/sulfamethoxazole. I. Common opportunistic Enterobacteriaceae l. Genus Klebsiella are nonmotile, lactose-fermenting rods. The colonies appear large and

mucoid due to the presence of the large capsule. The major pathogen, Klebsiella pneumoniae, causes severe lobar pneumonia in individuals with underlying conditions such as alcoholism, diabetes, and chronic obstructive pulmonary disease. 2. Genus Proteus are highly motile organisms that cause urinary tract infections. On agar plates, motility can be visualized by swarming from the initial colony. Proteus species produce urease, which raises the urinary pH to levels that promote the production of struvite. These stones obstruct urinary flow and serve as a hiding place for the organism. Up to 10% of urinary tract infections are due to this organism. The two clinically important species are P. vulgaris and P. mirabilis. 3. Other genera of Enterobacteriaceae that are normal flora, but that can cause opportunistic infections, include Citrobacter (pyelonephritis), Enterobacter (pneumonia), and Serratia (pneumonia and UTIs).

Mnemonic The As of Klebsiella • Alcoholics • Aspiration pneumonia • Abscesses in the lungs

Klebsiella pneumoniae is sometimes characterized by thick, bloody sputum classically described as "currant jelly" sputum.

GRAM-NEGATIVE BACILLI: ADDITIONAL ENTERIC ORGANISMS A. Vibrios have a highly characteristic comma-shape morphology. They are oxidase positive, a feature that differentiates Vibrio from Enterobacteriaceae. They are naturally found in both fresh and salt water and in several cold-blooded animals. l. Vibrio cholerae is the most clinically important member of this group.

a. Pathogenicity is related to the presence of a pilus that mediates adherence to the small intestine epithelium. It is a noninvasive infection with clinical effects mediated by the enterotoxin (choleragen). The principal enterotoxin effect is an increase in intracellular cyclic AMP, resulting in electrolyte/fluid secretion into the small intestine lumen. There are two major enterotoxin subunits:

Note A related organism, Vibrio vulnificus, is associated with diarrheal disease, as well as wound infections acquired by fishermen, ocean swimmers, etc.

(1) A subunit, which promotes activation of adenylate cyclase. (2) B subunit, which binds toxin to small intestinal ganglioside receptor. b. Transmission is via fecal-oral spread from contaminated water and food. c. Clinical manifestations of cholera are severe, watery ("rice water") diarrhea (20 liters/day) with the loss of sodium, chloride, potassium, and bicarbonate. Other important clinical manifestations include nausea, vomiting and abdominal cramps, metabolic acidosis, and hypovolemic shock. The mortality rate without treatment is 25-50%.

d. Therapy and prevention includes rapid rehydration and electrolyte replacement. The antibiotic of choice is either tetracycline or doxycycline. It reduces the number of organisms, decreases diarrhea, and helps eliminate the chronic carrier state of an individual. 2. Vibrio parahemolyticus resembles V. cholerae structurally as a comma-shaped organism. It is halophilic, requiring at least 2% NaCl for growth. The 0 and K surface antigens are useful for serologic typing. KAPLAlf I meillea

291

Microbiology

a. Clinical manifestations are primarily diarrhea, attributed to ingestion of raw or improperly handled seafood (especially shellfish). The incubation period is 12-24 hours. Symptoms include explosive watery diarrhea (may be bloody), headache, abdominal cramps, fever, and vomiting. Septicemia may occur, particularly with underlying liver disease.

Note

Aeromonas and Plesiomonas are two other genera belonging to the Vibrionaceae family. Aeromonas hydrophilia lives in water and can cause gastroenteritis and wound infections. Plesiomonas shigel/oides is also associated with water sources and causes gastroenteritis.

b. Therapy and prevention. Mild disease is usually self-limiting, subsiding within 2-4 days, with no therapy required. For severe cases, fluid and electrolyte replacement and antibiotics are required. Organisms are usually sensitive to chloramphenicol, tetracycline, and cephalosporins. Adequate refrigeration of raw and cooked seafood aids in prevention.

B. Campylobacter and Helicobacter. Campylobacter are small, curved Gram-negative rods. Campylobacter jejuni is the most common cause of bacterial gastroenteritis in the United States. 1. C. jejuni is spread person-to-person via the fecal-oral route, but the primary route of infection is via consumption of undercoked poultry.

a. It is a primary pathogen that causes enterocolitis, an invasive enteritis with bloody diarrhea, crampy abdominal pains, malaise, and fever. b. Inflammatory proctitis is a clinical manifestation in homosexuals. c. Reactive arthritis may follow in individuals who have HLA-B27. d. It is gut flora of many domestic animals, including chickens, pigs, sheep, goats, and cattle. e. The antibiotic treatment of choice is either erythromycin or ciprofloxacin. 2. C. fetus is an opportunistic pathogen that causes bacteremia and metastatic infections in immunocompromised patients. a. In contrast to C. jejuni, it does not grow at 42°C and is resistant to nalidixic acid but sensitive to cephalothin. b. Once in the bloodstream, C. fetus can attach to the endothelium on blood vessel walls. c. The antibiotic treatment of choice is now imipenem.

In a Nutshell H. pylori and Proteus both produce urease and create an alkaline environment.

3. Helicobacter pylori is a spiral-shaped, motile rod that produces urease and surface adhesins that promote its pathogenicity.

a. The major reservoir is humans; however, Helicobacter can be found in the intestinal tracts of other mammals. b. H. pylori exhibits age-dependent colonization rates and familial clustering. For example, it is present in the gastric mucosa of fewer than 20% of people less than 30 years old but increases to greater than 50% of people over 60 years old. c. It lives in the gastric mucus in close proximity to the gastric epithelial cells. d. Colonization may be asymptomatic; however, there is a high association with idiopathic chronic and acute antral gastritis as well as duodenal ulcer (90%). Enhanced occurrence of gastric adenocarcinoma is also observed. e. Diagnosis can be made either noninvasively by serology or breath test, or invasively via endoscopy and biopsy. f. Therapy consists of bismuth salts, metronidazole and tetracycline, or amoxicillin for 2-4 weeks.

292

meClical

Bacteriology: Gram-Negative Organisms

C. Pseudomonas is a genus of Gram-negative rods that are widespread in soil and water. They

are also a minor component of the bowel flora. 1. Characteristics and physiology. The following characteristics are relatively simple ways

to distinguish pseudomonads from enterobacteria. a. All members of the genus Pseudomonas are oxidase positive. b. Pseudomonads are nonfermentors; they do not ferment sugars. c. Pseudo monads are motile due to polar flagella.

d. They produce large quantities of extracellular proteins, including toxins and enzymes. e. Pyocyanin pigment is an important product useful in the laboratory identification of the organism and gives the organisms a blue-green appearance. f. Humans are very resistant to infection with Pseudonomas species. More than one host defense must be breached for infection to occur. The major host defenses are intact body surfaces, normal bacterial flora, complement lysis, and killing by polymorphonuclear leukocytes. 2. Pseudomonas aeruginosa is the most important member of the genus. It is an important nosocomial infection in immunocompromised and chronically ill patients. a. It possesses many virulence factors, and no single factor is decisive for virulence. Virulence factors include: (1) Pili-adhesin (2) Slime layer capsule-antiphagocytic alginate capsule; seen mainly in cystic fibro-

sis patients (3) Endotoxin (4) Exotoxins A and S inhibit protein synthesis. Exotoxin A works by ADP ribosylating (inactivating) EF2 and thus shutting down host protein synthesis. This is the same mechanism used by diphtheria toxin, but the two toxins are unrelated. (5) Many enzymes (e.g., gelatinase, collagenase, phospholipase C, elastase); elastase is an important virulence factor because it allows the organism to invade blood vessels. Elastase allows P. aeruginosa to cause ecthyma gangrenosum. (6) R factors b. Risk groups incude immunocompromised patients and hospitalized patients with underlying disease. Groups at particular risk are radiation therapy patients, burn patients, patients with metastatic or metabolic disease, patients on prolonged immunosuppressive or antimicrobial therapy, patients with prior instrumentation or manipulation, and patients with cystic fibrosis. c. Clinical manifestations

(1) Wound and burn infections (2) Ecthyma gangrenosum-skin lesion with vascular invasion leading to hemor-

rhage and necrosis (excreted elastase is important in the development of this lesion). Usually seen in neutropenic patients. (3) Ear infections-otitis extern a (swimmer's ear, mild); in diabetics-malignant otitis externa (4) Pulmonary infections-especially in cystic fibrosis patients and the immunocompromised

meClical

293

Microbiology

(5) Corneal infections in contact wearers (6) Urinary tract infections d. Treatment will include antipseudomonal penicillin (e.g., ticarcillin, piperacillin) and aminoglycoside (e.g., tobramycin). 3. Pseudomonas pseudomallei are small, nonmotile, aerobic rods. The organism is a common soil inhabitant in Southeast Asia and is transmitted to humans through local abrasions or inhalation. It causes a disease called melioidosis that resembles glanders in equine animals. Recommended therapy for P. pseudomallei is ceftazidime.

ZOONOTIC ORGANISMS There are four genera of Gram-negative bacilli that are harbored by animal reservoirs: Yersinia, Francisella, Pasteurella, and Brucella. Borrelia, a zoonotic spirochete, is discussed in Chapter 9, Bacteriology: Spirochetes. A. Yersinia pestis is a member of family Enterobacteriaceae. It is a bipolar-staining, Gramnegative bacillus that can multiply intracellularly. "", 1. Pathogenesis. A virulence plasmid is responsible for intracellular survival and growth.

2. Transmission a. Animal reservoirs include rats, squirrels, prairie dogs, mice, cats, and rabbits. b. The organism is transmitted by infected fleas. c. In the United States, all plague cases have occurred in the Southwest. 3. Clinical manifestations. Disease can present with several clinical pictures. a. Bubonic plague is characterized by fever and a painful bubo caused by lymphadenopathy. b. Septicemic plague is characterized by fever and hypotension without bubo. c. Pneumonic plague presents with cough and hemoptysis. This form can be transmitted by respiratory droplets from human to human and is highly contagious with a high mortality rate (>90% if untreated). 4. Treatment and prevention a. Treatment consists of streptomycin or gentamicin with chloramphenicol or tetracycline as alternatives. b. A killed vaccine is available for travelers to endemic areas or those who work with the organism. B. Francisella

1. Characteristics. Francisella are small, nonmotile, Gram-negative coccobacilli with bipolar staining. They are aerobic organisms that require cysteine or cystine for growth. The clinically-important species is Francisella tularensis, the cause of tularemia.

2. Transmission. Francisella tularensis can be transmitted by the bite of an infected arthropod, usually a tick, or by direct contact with tissues and body fluids of infected animals. 3. Clinical manifestations. Tularemia is plague-like with lymphadenopathy and sepsis. Skin lesions often occur at the site of entry. They begin as a small papule, and progress to an ulcer with central necrosis.

294

meclical

Baderiology: Gram-Negative Organisms

4. Treatment and prevention. Antibiotic of choice is streptomycin. A live, attenuated vaccine exists for people at high risk.

e. Pasteurella 1. Characteristics. Pasteurella are nonmotile, Gram-negative coccobacillary and rod-shaped

organisms that exhibit bipolar staining. They are facultative anaerobes that live as commensals in the oropharynx mariyaflimals and birds.

of

2. Transmission. The human pathogen, Pasteurella multocida, is acquired from animal

bites, especially cat bites. 3. Clinical manifestations. Infections in humans are usually localized soft tissue infection

(cellulitis), the majority of which arise from bites. 4. Treatment consists of penicillin G with doxycycline as an alternative.

D. Brucella 1. Characteristics. Brucella are facultative intracellular parasites that can replicate inside

macrophages. Other animals are the primary hosts. The clinically important species and their primary hosts are B. melitensis (goats), B. abortus (cattle), B. suis (swine), B. ovis (sheep), and B. canis (dogs). 2. Transmission. Humans usually acquire the organism through handling infected animals, or through the ingestion of unpasteurized dairy products. It is typically an occupational

disease of farmers, veterinarians, butchers, and slaughterhouse workers. 3. Clinical manifestation. Symptoms of brucellosis are nonspecific, including fever,

headache, and arthralgia. If untreated, the fever begins to follow an undulating pattern, from which the disease takes its name, undulant fever. 4. Treatment and prevention. Treatment consists of ~o~~s:I~E~ phlS~~~!~~~~~" or§
RESPIRATORY PATHOGENS A. Haemophilus 1. Characteristics. Haemophilus are small, p~~~:p'~~.c."c~c<:~bacilli.

a. All species require factors X, V, or both. (1) X is defined as hematin or precursors from the cytochrome biosynthetic pathway. It is heat stable.

(2) V is nicotinamide adenine dinucleotide (NAD). It is heat labile.

(3) Haemophilus species are cultured on chocolate agar in which blood has been heated to release factor X. NAD is supplied by supplementation. (4) Alternatively, X and V can be added to the media, or blood agar plated with Haemophilus can be cross-streaked with Staphylococcus. Haemophilus species can grow near the Staphylococcus because the latter releases enough X and V for the needs of the Haemophilus. This is called satellitism. b. Many species of Haemophilus are normal flora of the human pharynx. c. The major pathogen of the group is Haemophilus injluenzae.

KAPLA~. I meulCa 295

Microbiology

d. Serotyping in Haemophilus inJluenzae is based on the polysaccharide capsules. There are six serotypes, designated a through f. Disease due to Haemophilus inJluenzae is caused chiefly by serotype b, which is composed of polyribosyl ribitol phosphate (PRP). This capsule type prevents phagocytosis of the organism. 2. Clinical manifestations. Diseases caused by Haemophilus inJluenzae type b include meningitis, otitis media, and epiglottitis. Primarily children under the age of 5 are affected. a. Meningitis. Until the development of a vaccine, Haemophilus inJluenzae type b was the most common cause of bacterial meningitis in the 3-month to 6-year age group. b. Acute epiglottitis occurs with rapid onset and can compromise the airway. c. Nontypable strains cause pneumonia and otitis media. Haemophilus ducreyi is the cause of the veneral disease chancroid. It is characterized by painful, nonindurated, ragged ulcers confined to the genitalia and perianal areas. 3. Treatment. Recommended treatment for Haemophilus inJluenzae includes cefotaxime or ceftriaxone for meningitis or epiglottitis, and amoxicillin plus clavulanate for non-lifethreatening illnesses. Resistance due to ~-lactamases is encountered in greater than 30% of isolates. 4. Prevention. A vaccine now exists for Haemophilus inJluenzae type b. PRP is conjugated to a carrier, such as tetanus toxoid or an outer membrane protein of N. meningitidis. This vaccine is given to children in four doses (2 months, 4 months, 6 months, and 15 months).

In a Nutshell

Pertussis toxin, cholera toxin, and the heat-labile toxin of E coli (LT) all work by ADPribosylation of G protein and the consequent increase in cAMP. There is a difference, however: cholera toxin and E coli heat-labile toxin act by activating the stimulatory unit of G protein (Gs); pertussis toxin acts by inactivating the inhibitory unit of Gprotein (G).

B. Bordetella 1. Characteristics. Bordetella are very small, Gram-negative fastidious coccibacilli. They are

strict aerobes. a. B. pertussis causes whooping cough, and B. parapertussis causes a relatively mild disease, parapertussis. b. B. pertussis contains many virulence factors. (1) Attachment to the host is mediated by pili, also called fimbriae or filamentous

hemagglutinins. (2) Toxins include pertussis toxin, adenylate cyclase toxin, tracheal cytotoxin, and lipopolysaccharide. Pertussis toxin blocks the inhibition of host cell adenylate cyclase by ADP-ribosylation of G inhibitory portion of G protein (GJ The resulting inactivation of Gi in effect stimulates adenylate cyclase, leading to an increased cAMP level in the cell. B. pertussis-encoded adenylate cyclase also enters mammalian cell membranes, raising the internal cAMP levels. It is responsible for the systemic manifestations of the disease, such as increased insulin sensitivity. Tracheal cytotoxin causes ciliostasis. c. Clinical manifestations. Whooping cough is the clinical manifestation of B. pertussis infection. This disease is highly communicable by the respiratory route, and humans are the only known reservoir. Whooping cough develops in three stages following an incubation period of 7-10 days. (1) Catarrhal or prodromal state occurs as a mild upper respiratory infection. (2) Paroxysmal cough, followed by the characteristic whoop on inspiration, then develops. Children may be anoxic, and vomiting may occur. (3) Convalescence is characterized by a slow decline in the "whoop." This stage lasts for months.

296 m~ical

---------~-

~----------------------

Bacteriology: Gram-Negative Organisms

d. The diagnostic gold standard is isolation of the organism on Bordet-Gengou agar. Direct fluorescein-labeled antibody detection has been developed as a rapid diagnostic alternative. e. Treatment and prevention. Erythromycin is the drug of choice for Bordetella pertussis infection. However, antibiotic treatment has no effect on the course of disease if therapy is begun after the catarrhal stage. The best protection is immunization. There are two vaccines for pertussis, a killed-cell vaccine and an acellular vaccine. The acellular vaccine was developed to avoid the other serious side effects of the killed-cell vaccine.

e. Legionella is a genus of Gram-negative bacteria that has over 40 species. The most important is Legionella pneumophila, a facultative intracellular parasite that uses the complement receptor to infect macrophages and then multiplies within the phagosome. It causes a wide spectrum of disease in humans. 1. Characteristics

Clinical Correlate We have now reviewed all three elements of the DPT vaCCIne: • Diphtheria • Pertussis

a. Legionella pneumophila is widely distributed in aquatic environments.

• Tetanus

b. It is an aerobic organism that requires iron and cysteine for growth. It will not grow on routine agar, but must have a buffered (pH 6.9) charcoal yeast extract agar instead.

DPT is administered in 5 doses:

c. Legionella pneumophila is flagellated, and positive for catalase, oxidase, gelatinase, and ~-lactamase. It also contains a high proportion of branched-chain fatty acids.

2. Transmission is by aerosols.

2 months, 4 months, 6 months, 15 months, and upon entry to school (4-6 years). DT boosters are recommended at lO-year intervals thereafter.

a. The source for humans is often air conditioning equipment, respiratory therapy devices, humidifiers, shower heads, or whirlpools. b. It is not transmitted person-to-person. c. Risk factors include immunocompromised status, alcohol abuse, and cigarette smok-

ing. 3. Clinical manifestations. Disease caused by Legionella pneumophila varies greatly in its intensity. Infection can be asymptomatic. a. Pontiac fever is a mild, febrile illness without pneumonia caused by Legionella pneumophila. b. It causes a mild, atypical pneumonia that may progress to Legionnaires disease. c. Legionnaires disease is a severe, often fatal, pneumonia.

4. Diagnosis. Direct fluorescent antibody staining is most commonly employed. Serology showing a four-fold rise in antibody titer is more useful in epidemiologic studies than clinical decision making. 5. Treatment. The drug of choice for Legionella pneumophila is erythromycin.

meCtical

297

Bacteriology: Anaerobes

Obligate anaerobes are bacteria that require a reducing environment. They cannot survive in an environment that contains oxygen because they are unable to detoxify the superoxide anion.

GENERAL CHARACTERISTICS A. Morphology and physiology 1. This group includes Gram-positive and Gram-negative cocci, bacilli, and coil-shaped

spirochetes that are often pleomorphic. 2. What unites these diverse organisms is the inability to detoxify the superoxide ion. The most essential enzyme in this process is superoxide dismutase. It catalyzes the conversion of the superoxide ion to hydrogen peroxide. The hydrogen peroxide can be further processed to water and oxygen through the action of catalase or peroxidase. Almost all obligate anaerobes lack these enzymes and hence require a reducing environment. 3. The greatest natural defense against anaerobic infection is healthy tissue that has a high oxidation-reduction potential and hence will not support the growth of the anaerobes. 4. Obligate anaerobes are normal inhabitants of anaerobic niches in the oral cavity, the vagina, and the gut. They can cause opportunistic infections in tissues adjacent to their normal habitat when they are displaced due to tissue injury or vascular compromise.

In A Nutshell Oxygen is toxic to obligate anaerobes because they lack superoxide dismutase and catalase/peroxidase.

B. Pathology 1. The primary pathology is frequently purulent malodorous abscess formation.

2. Culture of the abscess most often reveals a polymicrobial infection with multiple facultative and anaerobic species. C. Treatment. Therapy includes surgical drainage plus antibiotics. Penicillin G, clindamycin, metronidazole, and chloramphenicol are usually the most effective antimicrobials. Cefoxitin is also effective against most anaerobes.

ANAEROBIC GRAM-NEGATIVE BACILLI A. Bacteroides are the primary organisms of the colon, accounting for 30% of fecal isolates. Bacteroides are the most frequent cause of anaerobic infections and Bacteroides fragilis is the most common clinical infectious isolate.

meCtical

299

Microbiology

Clinical Correlate

1. Bacteroides fragilis is a Gram-negative, non-spore-forming, nonmotile bacilli.

Bacteroides are part of the

a. It inhabits the intestinal and genital tracts.

normal gastrointestinal flora. They can cause disease when there is a break in the surface of the mucosa.

b. It has four major virulence factors. (1) Unlike other group members, B. fragilis contains a polysaccharide capsule that is antiphagocytic and chemotactic. Capsular antigen alone can produce an abscess. (2) Because it is a Gram-negative organism, Bacteroides fragilis contains endotoxin (although it is much less toxic than the endotoxin produced by E. coli). Bacteroides fragilis also has the ability to deconjugate bile salts, making it resistant to bile. (3) It can produce a small amount of superoxide dismutase, allowing it to survive oxygen exposures for long periods of time.

(4) Bacteroides fragilis contains

~-lactamases

that confer resistance to penicillin.

c. Clinical manifestations include intra-abdominal infections, including abscesses and peritonitis. It is a primary cause of Gram-negative bacteremia. d. Therapy consists of metronidazole with clindamycin or chloramphenicol alternatives. Surgical debridement and drainage may be necessary to eradicate the organisms.

2. Prevotella melaninogenicus, formerly Bacteroides melaninogenicus, is a small coccobacilli that can be found primarily in the oral pharynx. a. On blood agar, it forms black pigmented colonies. b. The virulence factors of Prevotella melaninogenicus include an antiphagocytic capsule and collagenase. c. Prevotella melaninogenicus is an important agent in oral and pulmonary infections.

B. Fusobacteria are pleomorphic, Gram-negative rods with tapered ends. They normally inhabit the mouth, gastrointestinal tract, and female genital tract. 1. Fusobacteria do not have a capsule, but do possess an extremely potent endotoxin.

2. Fusobacterium nucleatum is the most common isolate. It is an important agent in oral infections, lung abscess, and other pleuropulmonary infections. 3. Fusobacterium necrophorum is often found in liver abscesses. It contains both a leukocidin and a hemolysin. 4. Most isolates of Fusobacterium are susceptible to penicillins, cephalosporins, and clindamycin.

ANAEROBIC GRAM-POSITIVE BACILLI A. Spore-forming. Clostridium species were discussed previously in Chapter 4, Gram-Positive Bacilli, in this section. B. Non-spore-forming 1. Propionibacteria normally inhabit the skin. These organisms may infect shunts and prosthetic devices, and are a cause of acne.

2. Actinomyces-see next chapter.

300

mectical

-------------------------.-------------------------------

Bacteriology: Anaerobes

ANAEROBIC COCCI A. Anaerobic Gram-positive cocci 1. Peptostreptococcus is the only genus of anaerobic cocci important in clinical infections.

a. They are almost always isolated from a mixed infection. b. As a group, they are frequently recovered from cutaneous, oral, respiratory, and female genital tract infections. c. Pep to streptococci are sensitive to penicillin, cephalosporins, clindamycin, and

metronidazole. B. Anaerobic Gram-negative cocci. Veillonella species are small bacteria that resemble the Neisseria species. Part of normal mouth, nasopharynx, and vaginal flora, they are rarely a cause of infection. They may be confused with Neisseria on Gram stain.

meclical

301

Bacteriology: Mycobacteria and Actinomycetes The mycobacteria are acid-fast bacilli notable for the high-lipid content of their cell wall. They are responsible for tuberculosis (M. tuberculosis), disseminated disease in AIDS patients (M. aviumintracellulare), and leprosy (M. leprae). Actinomycetes are Gram-positive organisms with a characteristic branching filament growth pattern that resembles fungi. These organisms are abundant in soil. The actinomycetes include two genera of clinical relevance: Actinomyces and Nocardia.

MYCOBACTERIA A. Mycobacterium tuberculosis 1. Characteristics

a. Obligate aerobes and Gram-positive acid-fast bacilli (AFB). Once stained, M. tuberculosis resists decolorization with an acidic alcohol rinse (hence the name acid-fast, based on ability to retain the stain). This property is dependent on the waxy lipid cell wall, which includes mycolic acids. The organism is facultatively intracellular (can grow inside of host cells). Mycobacteria often grow within monocytes and/or macrophages within the immune system. b. The cell wall contains lipoproteins or glycolipoproteins essential for tuberculin activity and confers the ability to induce type IV hypersensitivity reactions (DTH, delayed-type hypersensitivity). c. The organism expresses cord factor (trehalose-6,6-dimycolate), which correlates with

virulence. Cord factor inhibits leukocyte migration and stimulates immune responses (important in reactivation of disease).

~~id~e, . to.I.I1II11~~olC).1Y Type IV hypersensitivity reactions are discussed in detail in the Basic Immunology chapter of the Immunology section of this book.

d. M. tuberculosis is slow-growing, requiring 20-60 days before growth can be visualized; its doubling time is 18 hours. e. These organisms produce niacin, a characteristic that distinguishes them from other mycobacteria. 2. Antigenicity a. Purified-protein derivative (PPD) of the cell wall is the preferred antigenic material for skin testing. b. Amphipathic polar lipids in the plasma membrane (termed phosphatidyl inositol mannosides) are also immunogenic and serologically active.

c. Wax D, or muramyl dipeptide, is a heterogeneous group of peptidoglycolipids from the cell wall that elicit strong immune responses. KAPLA~. I meulca

303

Microbiology

3. Pathogenicity a. Cord factor is associated with virulence and characteristic serpentine grouping pattern that is seen in virulent strains. Cord factor inhibits polymorphonuclear leukocytic migration, elicits granuloma formation, and attacks mitochondrial membranes. b. Sulfatides are responsible for neutral red reactivity and act synergistically with cord factor. Sulfatides inhibit phagolysosomal membrane fusion in macrophages, thus protecting the microorganism from attack by the hydrolytic enzymes of the lysosome. 4. Epidemiology. M. tuberculosis is found only in humans. It is more common in lower socioeconomic groups. There has been a rapid, recent rise in cases in the U.S., partially related to the AIDS epidemic and to immigration patterns. 5. Transmission a. Occurs primarily through droplet nuclei inhalation. b. The most infectious person is one with untreated cavitary pulmonary tuberculosis who is actively expelling bacilli. c. The risk of infection is much greater than the risk of disease (i.e., more people are infected than have clinical disease) since disease may be related to weakened immune responses. 6. Pathogenesis is dependent on immunologic responses. First, delayed-type hypersensitivity (a T-cell immune response) occurs within 3--4 weeks after infection and correlates with positive tuberculin reaction. Acquired cellular immunity is associated with "resistance" or protection from reinfection.

Clinical Correlate The primary lung infection is usually found subadjacent to the pleura in the lower part of an upper lobe or in the upper part of a lower lobe of one lung. This localization reflects the areas receiving the greatest flow of air.

(1) Exudative type occurs when the organism is inhaled and spreads via alveolar macrophages to the hilar lymph nodes. Hematogenous dissemination may occur at this time; however, signs of infection are minimal and immune-competent hosts will successfully limit the organism to the pulmonary location. (2) Productive type is characterized by the tubercle forms (with or without caseation) and depends on the host's immune response. (3) The primary site of infection forms calcified lesions (referred to as Ghon complex). Delayed-type hypersensitivity develops and infection becomes quiescent in pulmonary as well as metastatic sites (the PPD test is now positive). Immunocompromised or debilitated patients may have progressive primary disease from local sites or more distant sites without the disease becoming quiescent. b. Secondary infection (reactivation) is usually localized, particularly in lung apices, due to the higher oxygen tension (P0 2 ). Tubercle formation occurs histologically with caseation, necrosis, and fibrosis. Secondary infection results from either a breakdown of quiescent foci or from new infection, despite the acquisition of cellular (T-cell) immunity.

Note Eighty percent of pulmonary TB cases in adults are due to reactivation of an infection acquired years or even decades earlier.

304

a. Primary infection

meClical

7. Clinical features of tuberculosis. The clinical presentation may include nonspecific constitutional symptoms such as fatigue, weight loss, anorexia, weakness, fever, and night sweats. a. Pulmonary TB. Eighty-five percent of cases are pulmonary, although infection may involve any organ of the body. Pulmonary disease may present with cough, hemoptysis, and pneumonitis.

Bacteriology: Mycobacteria and Actinomycetes

b. Miliary or disseminated masses develop anywhere, but some sites are favored, signifying an advanced stage of the disease. These favored sites include bone and joints (osteomyelitis), meninges (meningitis), kidneys, peritoneum, and lymph nodes. 8. Diagnosis a. Abnormal chest x-ray b. Acid-fast bacteria (AFB) in sputum; culture of M. tuberculosis on Lowenstein-Jensen medium c. Skin testing (1) PPD-S, the designated standard, containing 50,000 tuberculin units per mg of

protein (purified-protein derivative) is injected under the skin. Tuberculin tests should be read from 48-72 hours after intradermal injection. (2) The interpretation is based on diameter of induration and recorded in millimeters: ~5 mm in HIV-positive patients or in anyone with recent TB exposure; ~1O mm in high-risk populations (intravenous drug users, immunocompromised patients, those at the poverty level); ~15 mm in low-risk populations. (3) False negative reactions are usually the result of tuberculin injections too deep into skin. Other causes include the improper storage or handling of PPD, concurrent infectious diseases, or immunosuppression or anergy due to overwhelming infection, steroids, malignancy, etc. (4) False positive reactions are usually caused by hypersensitivity to mycobacteria other than M. tuberculosis. False positives can also result from previous immunization with BCG (Bacille Calmette-Guerin) vaccine (not administered in the United States, but widely used outside U.S.). (5) Diagnostically, a positive PPD test does not prove active disease only exposure. The tubercle bacilli must be isolated and identified in culture. Microscopic examination of a specimen stained with Ziehl-Neelsen or Kinyoun stain will give presumptive evidence of active disease if acid-fast organisms are seen. Cultures are then initiated by inoculation of either egg-potato-base media (LowensteinJensen) or agar-base media followed by incubation in 5-10% CO 2 atmosphere for 3-6 weeks. Unlike other mycobacteria, M. tuberculosis produces niacin. 9. Treatment. Therapy should be for a period of 6-9 months with a combination of at least three antituberculous drugs. If the patient is HIV-positive, therapy should be continued for a longer period (9-12 months). The emergence of drug-resistant strains, particularly in large metropolitan areas, is a growing problem that can best be controlled by sensitivity testing of the isolate from the patient. When resistance is detected, additional drugs (ethionamide, streptomycin, ciprofloxicin) may be added to the regimen.

10. Prevention a. Isoniazid (INH) prophylaxis of household contacts of patients with newly diagnosed active disease or treatment of recently converted (within the past two years) PPD-positive individuals. Children under 5 years of age are treated without exception while adults greater than 55 years old are generally followed clinically. Treatment of adults 35-55 years of age is based on the clinical risk of infection outweighing the risk of INH -induced hepatitis. b. BCG immunization. This treatment is given only to PPD-negative individuals in countries where incidence of tuberculosis is high. It is used to establish cell-mediated immunity to TB, although it must be remembered that individuals will become skin test-positive for PPD (thus eliminating its utility as a diagnostic tool). BCG is a live attenuated strain of Mycobacterium bovis.

Clinical Correlate Emergence of multiple-drugresistant strains has prompted the Centers for Disease Control to recommend four drug regimens. Currently, the drugs considered "first line" include: • Isoniazid • Rifampin • Ethambutol • Pyrazinamide KAPLA~.

meulCa

I

:505

Microbiology

B. Mycobacterium bovis 1. M. bovis is the etiologic agent of tuberculosis in cattle. It can also cause human TB, usu-

ally through the ingestion of unpasteurized, contaminated milk. 2. The most common clinical manifestations are lesions in the cervical and mesenteric lymph nodes with possible dissemination to the bones and joints. Pulmonary TB is also possible through the inhalation of infected droplets (for example, by dairy farmers). 3. In contrast to M. tuberculosis, M. bovis does not produce niacin. It will, however, result in a positive PPD skin test. 4. The BCG vaccine is derived from a live, attenuated M. bovis strain. C. Nontuberculous mycobacteria ("atypical"). The nontuberculous mycobacteria encompass

approximately 20 species, including M. kansasii, M. marinum, M. scrofulaceum, M. fortuitumchelonei and most importantly, M. avium-intracellulare. They cause mycobacteriosis. The agents of mycobacteriosis are not spread from human to human; they are environmental agents and found predominantly in immunocompromised individuals. 1. General characteristics of non-TB mycobacteria. There is no known primary animal

host. The organisms usually occur as natural inhabitants of the soil. 2. Non-TB mycobacteria are very diverse. a. Most are slow growers like the TB bacillus, but M. fortuitum has a growth rate equal to most other bacteria. b. M. avium and M. intracellulare are nonchromogenic, as is M. tuberculosis, but other species produce yellow pigments under particular growth conditions (e.g., M. kansasii develops pigmented colonies when grown in the presence of light). 3. Clinical manifestations a. Pulmonary disease is usually found in older white men with chronic bronchitis and emphysema. Pathogens include M. kansasii, M. avium-intracellulare, and M. fortuitum-cheloni complex. b. Lymphadenitis is caused by M. scrofulaceum and most commonly occurs in children. c. Cutaneous lesions are caused by M. marinum when this organism contaminates an open wound; the disease is called "swimming pool" granuloma. d. Disseminated disease can arise from M. kansasii or from M. avium-intracellulare, particularly in patients with acquired immunodeficiency syndrome (AIDS). 4. Treatment of nontuberculous mycobacterium. Many of the mycobacteria are resistant to the usual antituberculosis drugs. Antibiotic regimens may require as many as six drugs, including rifampin (which is quite effective against M. kansasii) and clarithromycin (good against M. avium-intracellulare complex). Surgical resection is also recommended on occaSIOn. D. Mycobacterium leprae

1. Species characteristics a. Cannot grow in vitro on any culture medium. b. Organisms are acid fast and induce a delayed-type hypersensitivity in patients. 2. Leprosy (Hansen disease) a. Leprosy is endemic in Mrica, South and Southeast Asia, and South America. Small endemic areas are found in the U.S. (Hawaii, Texas, California, Louisiana, and Florida).

306

meClical

Baderiology: Mycobaderia and Adinomycetes

b. Transmission is through contact with organisms from nasal secretions or ulcer exudates from infected individuals. Lesions classically involve the cooler regions of the body, including the skin of nasopharynx, cartilage, eyes, testicles, and larynx. Incubation period for the infection to progress to clinical disease is 5-7 years. c. Disease forms (1) Tuberculoid leprosy is indolent and nonprogressing. Clinical findings show mature granuloma in the dermis, which also contains epithelioid cells, giant cells, and lymphocytes (predominantly TDTh cells). Acid-fast bacilli are usually not found in lesions of the skin. Schwann cells are the targets of bacterial colonization. Nonspecific nerve damage occurs with asymmetric involvement. Tuberculoid leprosy reveals a positive lepromin skin test (cell-mediated immunity intact). (2) Lepromatous leprosy is a progressive and invasive disease. Pathologic examination shows foamy histiocytes with an absence of epithelioid and giant cells. Cellmediated immunity is suppressed (negative lepromin skin test) and T8lymphocytes infiltrate the skin lesions. Numerous acid-fast organisms are present in the lesions. Schwann cells are infected, but less nerve damage occurs in comparison with tuberculoid leprosy. Skin lesions are of an invasive and nodular nature. d. Immunity to M. leprae is mediated primarily by CD4+ T helper 1 cells. The disease is marked by a low infectivity rate and occurs more frequently in individuals with defective cell-mediated immunity. (1) Tuberculoid leprosy exhibits strong delayed-type hypersensitivity immune

responses where CD4+ T helper 1 cells infiltrate the skin lesions. (2) Lepromatous leprosy arises primarily in patients with a loss of cell-mediated immunity. Patients will be anergic (unresponsive) to skin testing with the antigen (i.e., no delayed-type hypersensitivity). There is a strong serologic (antibody) response marked by polyclonal hypergammaglobulinemia (T helper 2 response). e. Lepromin test determines the immunologic response or status of the patient. The test is not for diagnostic use and there is no serological test for diagnosis due to delayedtype hypersensitivity in the patient. f. Diagnosis is made by acid-fast staining analysis of scrapings from the affected skin or nasal mucosa. A biopsy of infected nerve or cartilage can be histologically diagnostic. g. Therapy requires long-term (3-5 years) antibiotic treatment to eradicate the organism. Tuberculoid leprosy responds to dapsone plus rifampin. Lepromatous leprosy is treated with dapsone plus rifampin and clofazimine. Close contacts should also be treated.

ACTINOMYCETES Actinomycetes (Actinomyces, Nocardia, Streptomyces) are characterized by their filamentous form. A. Actinomyces. Several species from this genus can cause actinomycosis. The most important

species are A. israelii and A. naeslundii. 1. Species characteristics a. A. israelii organisms are anaerobic, nonacid-fast, Gram-positive bacilli.

IlAPLAN"dme leaI

307

Microbiology

b. They are part of the normal oral flora (not found in soil) and are usually pathogenic only after oral trauma. 2. Clinical manifestations (termed actinomycosis) a. Cervicofacial actinomycosis causing lower jaw involvement following the development of dental caries (about 50% of actinomycotic infections) or after dental work. Pyogenic abscesses may develop with swelling, tenderness, and erythema (lumpy jaw). Granules consist of Gram-positive mycelial filaments surrounded by eosinophils and leukocytes. Osteomyelitis (bone infection) is a frequent occurrence. Chronic, poorly healed oral cavity abscess will spread by direct extension. Fortunately, the incidence of oral actinomycosis is declining due to improved oral hygiene practices. b. Thoracic actinomycosis is caused by the extension from cervicofacial infection in approximately 20% of cases. c. Abdominal actinomycosis is due to traumatic perforation of the intestinal mucosa (approximately 20% of cases), such as a ruptured appendix or perforated ulcer.

d. Pelvic actinomycosis arises in women with intrauterine devices. The organism may simply colonize the tissue or produce infection. 3. Diagnosis is made by examination of secretions or tissues for granules. Crushed "sulfur granules" contain Gram-positive, nonacid-fast rods. Actinomycetes are grown anaerobically in thioglycolate broth. 4. Therapy consists of penicillin or ampicillin for several weeks. Infection may require surgical drainage because antibiotics achieve poor penetration through abscesses. B. Nocardia. N. asteroides is the most common isolate found in soil and aquatic environments. 1. Characteristics. Nocardia species are aerobic, Gram-positive, partially acid-fast, filamen-

tous organisms. 2. Clinical manifestations (nocardiosis) a. Half of patients have underlying disease. It is an opportunistic infection in patients with hematological malignancies or immunodeficiencies. Seventy-five percent of cases occur in males.

In a Nutshell

• Actinomyces is an anaerobe; Nocardia is not. e

Nocardia is partially acidfast; Actinomyces is not.

b. Infection begins as a chronic lobar pneumonia following inhalation of the organism and may be subclinical. c. The central nervous system (brain) is the most common site of metastatic infection, occurring hematologically. The kidneys and skin may also become involved (in metastatic infection). Abscess formation is the most common pathologic finding.

3. Diagnosis is determined from the evaluation of histological appearance of specimens, culture, and modified acid-fast stain. 4. Treatment is usually with sulfonamides and may require surgical drainage of abscesses.

308

meClical

Bacteriology: Rickettsiae and Chlamydiae Rickettsia and Chlamydia are obligate intracellular parasites. Rickettsial diseases include Rocky Mountain spotted fever, typhus, cat scratch disease, and Qfever. Chlamydia species are responsible for ocular trachoma and are the number one bacterial cause of sexually transmitted diseases in the United States.

RICKETTSIACEAE A. General characteristics and physiology

1. Rickettsiaceae are a family of small, pleomorphic Gram-negative coccobacilli. They Gram stain poorly but can be stained using the Giemsa or Macchiavello methods.

2. The Rickettsiaceae family comprises four main genera: Rickettsia, Bartonella, Coxiella, and EhrZichia. The members of the family share several unique characteristics. a. All members are transmitted by arthropods, except Bartonella (where direct contact is the mode of transmission) and Coxiella burnetii (acquired by humans primarily through aerosol inhalation). b. Most species are obligate intracellular parasites of endothelial cells (Rickettsia) or leukocytes (other genera). c. Most cause zoonotic disease with humans as an accidental host. Chlamydia trachomatis is the exception; it is a human pathogen with no reservoirs. d. All are susceptible to tetracyclines (e.g., doxycycline). 3. Each genus has a set of common pathogenic features associated with the diseases caused by its members. a. The genus Rickettsia is disseminated through the bloodstream, causing vasculitis, thrombosis, and capillary leaks leading to the characteristic rash. Release of the endotoxin leads to fever, disseminated intravascular coagulation, and shock. b. Coxiella burnetii acute disease presents as pneumonia. Rare chronic infections include hepatitis and endocarditis. c. EhrZichia invasion of PMNs and monocytes causes leukopenia. Thrombocytopenia is

also seen. The acute symptoms resemble Rocky Mountain spotted fever, but a rash is far less common; often seen as coinfection with Lyme disease. d. The Bartonella organism invades the blood and lymphatic vessel walls and causes local lymphadenopathy and angiomas.

IlAP~~.

I

m.ulC8

309

Microbiology

Note

B. Rickettsial diseases

Tick-borne infections can include bacteria from other groups, including Borrelia burgdorferi, human granulocytic ehrlichiosis (HGE), and Babesia. Also, ticks may carry one or more of these organisms at the same time

Clinical Correlate Lesions in the palms of the hands and the soles of the feet are valuable clinical clues. Causes include RMSF, meningococcemia, and secondary syphilis.

1. Rocky Mountain spotted fever (RMSF) is caused by R. rickettsii and accounts for 95% of

rickettsial diseases in the United States. a. Epidemiology. RMSF is found throughout the United States, particularly in the south central and eastern portions of the country. b. Transmission. The arthropod vectors of R. rickettsii are various species of ticks. The bite of the infected tick transmits the organism. The reservoirs for the agent include rodents, dogs, and ticks. c. Clinical features (1) After a 3-12 day incubation period, the disease begins with malaise, frontal headache, and fever.

(2) Skin manifestations appear within 2-4 days after onset of symptoms. Initially, a maculopapular rash appears on the hands and feet (including palms and soles) and spreads centripetally to involve the trunk. The rash will initially appear petechial, but may coalesce to become hemorrhagic. (3) Other manifestations may include splenomegaly, hepatomegaly, thrombocytopenia, and disseminated intravascular coagulation. 2. Epidemic typhus (louse-borne typhus) is caused by Rickettsia prowazekii. a. Transmission. The agent is transmitted by the human body louse. Rickettsia prowazekii are found within the louse feces and enter individuals via skin excoriations. The louse is the vector, but not a reservoir, for Rickettsia prowazekii (humans are the primary reservoir). b. Clinical features are similar to those of RMSF but the rash is less prominent and spares the palms and soles. c. Brill-Zinsser disease is the recurrent form of R. prowazekii infection. R. prowazekii can remain dormant in lymph nodes, and the individual is asymptomatic during the intervening time period. Years after the primary attack, a milder form of the illness may occur. Symptoms include headache, myalgia, and malaise with an erratic fever. 3. Endemic or murine typhus is caused by R. typhi cycled by the rat and its ectoparasites (through feces). a. Transmission. The rat flea is the most common vector. It becomes infected when it feeds on the reservoir-the rat-during the acute part of the rat's illness. Organisms are passed in the flea feces, and can be scratched in at the site of the bite. b. Clinical manifestations are similar to but less severe than in epidemic typhus. 4. Scrub typhus is caused by Orienta (formerly Rickettsia) tsutsugamushi. It occurs endemically in Japan, China, Southeast Asia, Australia, the South Pacific Islands, and the Asian subcontinent. a. Transmission. O. tsutsugamushi is transmitted by mites (chiggers). Since there is transovarial passage of the organism, the mite is a reservoir as well as the vector. Other reservoirs include rats, field mice, and shrews. Humans are accidental hosts. b. Clinical features. Clinically, the disease resembles epidemic typhus. 5. Q fever is caused by Coxiella burnetii.

310

meClical

Baderiology: Rickettsiae and Chlamydiae

a. Transmission. Q fever does not have an arthropod vector in the human disease cycle. It is spread mainly by inhaling infected dusts, by handling infected hides or tissues, or by drinking milk contaminated with Coxiella burnetii. The organism is widely distributed in nature. Reservoirs include ticks, small wild animals, sheep, cows, and goats. b. Clinical features (1) Q fever is characterized by vague symptoms such as fever and chills, headache, malaise, and myalgia. It is unique among the Rickettsial diseases in that it does not cause a skin rash. (2) Many cases are asymptomatic or present with a self-limited febrile illness. (3) The classic presentation of the disease is as a pneumonia with fever and no pulmonary symptoms. Atypical pneumonia and a rapidly progressive pneumonia also occur.

Note

(4) Chronic Q fever can manifest as myocarditis or hepatitis. 6. Human ehrlichiosis is caused by Ehrlichia chaffeensis. It has a tick vector. The clinical course can range from asymptomatic to fatal. The classic presentation includes fever, headache, and myalgia. Important laboratory features are leukopenia, thrombocytopenia, and anemia. Fewer than half of patients develop a rash. It is commonly found as a coinfection with Lyme disease because it is spread via the same tick vector. 7. Bartonella henselae causes cat scratch disease (a benign local lymphadenopathy that follows contact with cats) and bacillary angiomatosis. The latter occurs in AIDS patients and is characterized by proliferative vascular lesions in the dermis (resembles Kaposi sarcoma) and internal organs. C. Diagnosis of rickettsial disease

1. The diagnosis is based on clinical presentation and epidemiological information. Due to the danger of infection to ancillary personnel, Rickettsiaceae are cultured in reference laboratories. 2. Diagnosis is most often aided by serology using convalescent serum to detect IgM- and IgG-specific antibodies to Rickettsiaceae. A four-fold or greater increase in antibody titers between acute and convalescent serum supports acute infection. The obvious disadvantage of this method is that serologic evidence of infection occurs no earlier than the second week of illness.

Q fever is an outlier among the Rickettsiaceae:

• It does not have an arthropod vector. • It does not cause a rash. • It does not elicit a positive Weil-Felix test.

In a Nutshell Organism R. rickettsii

Vector Tick

Disease Rocky Mountain spotted fever (think rashpalms/soles moving to trunk)

R. prowozekii Louse

Epidemic typhus (think rash-trunk spreading to extrem ities)

R. typhi

Flea

Endemic (murine) typhus

a. Patients with rickettsial disease will have antibodies that react with Proteus vulgaris antigens and will agglutinate these organisms.

O. tsutsugomushi

Mite

Scrub typhus

b. The procedure is simple and inexpensive but lacks specificity for Rickettsiaceae.

£ choffeenis Tick

Ehrlichiosis

C. burnetii

Qfever (no

3. Some hospital and public health laboratories use direct immunofluorescence to demonstrate organisms in skin biopsy specimens. 4. These Rickettsia-specific techniques have largely replaced the Well-Felix test. This test is based upon cross-reactions between anti-rickettsial antibodies and Proteus polysaccharide o antigen (Proteus OX-19 and OX-2).

c. Coxiella burnetii does not elicit a positive Weil-Felix test. D. Treatment. Treatment with antibiotics has greatly reduced the morbidity and mortality due to rickettsial infections. Whereas all rickettsial diseases were traditionally treated with tetracycline, newer antibiotic regimens include doxycycline, ciprofloxacin, or chloramphenicol for Rickettsia and Ehrlichia. Coxiella burnetii can be treated with doxycycline or ciprofloxacin.

None

rash!)

B. hense/oe

None

CJt scratch fever and bacillary angiomatosis

meltical

311

Microbiology

CHLAMYDIAE A. General characteristics and physiology 1. Chlamydiae are obligate intracellular parasites infecting birds and mammals.

2. They possess a gram-negative envelope that contains a genus-specific lipopolysaccharide but lacks muramic acid. 3. Their genome is very small, so they lack the capacity to carry out many metabolic pathways, including the production of ATP. This makes them dependent upon their host for energy. 4. Three species of Chlamydia are pathogenic for humans: Chlamydia trachomatis, Chlamydophila psittaci, and Chlamydophila pneumoniae (TWAR).

In a Nutshell Life Cycle of Chlamydia Elementary body (EB): • Extracellular • Infectious • Cannot multiply • Enter cells through endocytosis Reticulate body (RB): • Intracellular • Can undergo binary fission

5. The life cycle of Chlamydia contains morphologically distinct infectious and reproductive forms. The infectious form is known as the elementary body (EB). It is incapable of multiplication. The intracellular form capable of binary fission is called the reticulate body (RB). This form is not infectious. a. The first step is the attachment of the elementary body to the host cell and endocytosis without lysosomal fusion. b. Next, the elementary body is transformed into the reticulate body inside an endosome. c. Eventually, the reticulate bodies undergo DNA condensation and disulfide bond

bridgings of the major outer membrane protein (MOMP) to form EBs from RBs. Shortly thereafter, the elementary bodies are released by cell lysis. B. Chlamydia trachomatis. Chlamydia trachoma tis is transmitted by fomites, sexually or perinatally. It infects humans only. Infected cells develop oval vacuolar inclusions that contain glycogen and hence will stain with iodine. 1. Clinical features a. Ocular trachoma is the leading cause of blindness in developing countries. It is caused by repeated infections that result in chronic conjunctivitis, leading to scarring, eyelid deformities, and progressing to pannus formation and total blindness. It is associated with serovars A, BI and B2 , and C. b. Inclusion conjunctivitis (1) In infants, it is associated with maternal genital infection. If untreated, it may develop into pneumonia. (2) Adult conjunctivitis is often associated with contaminated swimming pools and hot tubs. (3) Associated with serovars D-K. c. Reiter syndrome is strongly associated with genitourinary or gastrointestinal infection with C. trachoma tis. Reiter syndrome is characterized by conjunctivitis, urethritis, and

reactive arthritis. d. Sexually transmitted diseases. C. trachoma tis is the number one cause of bacterial STDs in the U.S. (1) In men: nongonococcal urethritis, epididymitis, prostatitis, and proctitis. Associated with serovars D-K.

312

meClical ~-.----------

- - - - -

Baderiology: Rickettsiae and Chlamydiae

(2) In women: cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. Associated with serovars D-K. (3) Lymphogranuloma venereum (LGV) is a venereal disease. LGV is more common in males and blacks. There are three antigenic types (Ll, L2, L3) differentiated by immunofluorescence. The primary lesion is a painless, vesicular site on any part of the genitalia, anus, or rectum. The lesion becomes painful and suppurative and can spread into the inguinal and femoral lymph nodes. 2. Treatment and prevention. C. trachomatis has been traditionally treated with tetracyclines or erythromycin. Its spread can be minimized through improved hygiene and safe sexual practices. C. Chlamydophila psittaci. Chlamydophila psittaci is transmitted by inhalation of the organ-

isms from infected birds and their droppings. 1. Risk factors. Anyone coming in contact with an infected bird is at risk, but those that have close contact with birds such as veterinarians, pet store employees, poultry farmers, or bird handlers are at an increased risk. 2. Clinical features. The disease has a 5-15-day incubation period. Severity of the illness ranges from asymptomatic disease to a fatal systemic illness with severe pneumonia.

In a Nutshell C

trachomatis

• Obligate intracellular parasites; cytoplasmic inclusions stain with iodine. • Diseases - Trachoma (serovars A-C) - Neonatal conjunctivitis and pneumonitis (serovars D-K) - Ongonococcal urethr~is (D-K) - Cervicitis, salpingitis, PID (D-K) - Lymphogranuloma venereum (L 1-L3)

a. Pulmonary symptoms include a nonproductive cough and rales. The pulmonary exudate is predominantly monocellular and chest x-ray suggests atypical pneumonia or bronchopneumonia.

In a Nutshell

b. CNS manifestations are characterized by severe frontal headaches. Toxic encephalitis may occur, leading to death.

C psittaci.

D. Chlamydophila pneumoniae. Chlamydophila pneumoniae (formerly TWAR) is a humanonly pathogen that is believed to be transmitted by inhalation. This organism is a cause of pharyngitis, bronchitis, and a relatively mild atypical pneumonia, although life-threatening infections have occurred. E. Diagnosis of chlamydial diseases 1. Chlamydia trachomatis can be identified through cell culture, serology, antigen detection,

or nucleic acid hybridization. Inclusion bodies can be detected in cell culture after staining with Giemsa, Macchiavello, or Gimenez stain, or by using immunofluorescence. 2. Chlamydia psittaci is cultured in reference laboratories. F. Treatment. The drugs of choice for chlamydial infections are tetracyclines (doxycycline) or

erythromycins.

Birds + pneumonia, think

Note Epidemiologic studies have also associated atherosclerosis and coronary artery disease with antibody to

Chlamydophila pneumoniae, and this organism has been found in plaques of coronary arteries. However, its role in the pathogenesis of atherosclerosis is still unknown.

KAPlAN'iII me 1C8

313

Bacteriology: Spirochetes The spirochetes are motile, helically coiled organisms that divide by transverse fission. Spirochetes contain an axial fibril, an outer sheath, a protoplasmic cylinder (cell wall and membrane), and cytoplasm. There are three genera of spirochetes that can cause disease in humans: Treponema (syphilis, yaws, pinta, bejel), Borrelia (Lyme disease, relapsing fever), and Leptospira (leptospirosis). This chapter focuses primarily on the two most clinically relevant spirochetes: Treponema pa//idum, the etiologic agent of syphilis, and Borrelia burgdorferi, the tick-borne cause of Lyme disease.

TREPONEMA A. Treponema pallidum is the most important species. It has a capsule-like outer coat and a tapered end. Organisms are highly motile and constantly rotate about an axial filament. An important characteristic is that T. pallidum does not grow on artificial media and therefore cannot be cultured in the laboratory. T. pallidum is the etiologic agent of syphilis. 1. Transmission and epidemiology a. Syphilis is primarily a sexually transmitted disease. It can also be transmitted across the placenta and rarely, from blood transfusions. b. Risk groups include individuals with multiple sex partners and infants born to infected mothers. 2. Clinical manifestations a. Primary syphilis arises within 2-10 weeks following exposure. Organisms spread locally from the site of inoculation to the lymph nodes and bloodstream. A chancre forms at the site of inoculation. Initially, the chancre is a firm, painless reddish lesion with a raised border. The center usually erodes into an ulcer that heals within 3-6 weeks without scarring. The chancre contains numerous spirochetes. Symptoms may be overlooked in females owing to poor visualization of the genitourinary tract. Lymph nodes may be enlarged but nontender. If untreated, disease progresses to the secondary stage. b. Secondary syphilis occurs 1-3 months following primary syphilis. Symptoms represent disseminated disease and commonly include rash, fever, sore throat, headache, and generalized lymphadenopathy (especially in the epitrochlear region). White patches occur on mucous membranes (condylomas). Condyloma lata occur in moist areas, including the anus, vagina, axilla, and mouth. These lesions are highly infectious and are teaming with Treponema.

Clinical Correlate The rash of secondary syphilis appears on the palms of the hands and the soles of the feet (as in Rocky Mountain spotted fever and meningococcemia).

meClical

315

Microbiology

----~----------------------~--------------- -~-----

c. Latent syphilis develops in 30-40% of infected individuals. Mucocutaneous relapses are most common, with the lesions remaining infectious. After two years, contagious lesions rarely develop. d. Tertiary (late) syphilis arises in approximately 30% of untreated cases of primary syphilis. (1) Benign tertiary syphilis is marked by indolent granulomatous lesions (gummas) in the skin, mucocutaneous tissue, liver, or skeletal system. Spirochetes are rarely seen. (2) Cardiovascular syphilis is manifested as aortitis and occurs 10-20 years after untreated primary disease. Medial necrosis can occur with the destruction of elastic tissue. Saccular or fusiform aneurysms may develop and rarely dissect. (3) Neurosyphilis. Can occur more than 20 years after the initial infection. One or more CSF abnormalities may be apparent, including pleocytosis, elevated protein, or decreased glucose. Other neurologic symptoms are ''Argyll-Robertson pupils" and tabes dorsalis. In the former condition, the pupil constricts on accommodation but fails to react to light. Tabes dorsalis, or the demyelination of posterior columns, causes the loss of proprioception. Tabes dorsalis is observed as a widebased gait with long "slapping" motions of legs upon ambulation. e. Congenital syphilis results from the transplacental transmission of spirochetes to the developing fetus. It most commonly occurs if the mother has primary or secondary disease. There is approximately 25% mortality if left untreated, with a high incidence of spontaneous abortions and stillbirths. Adequate treatment of the mother will prevent disease in the fetus. Early manifestations in the newborn include hepatosplenomegaly, hemolytic anemia, pneumonia, skin lesions, and snuffles (obstructed nasal breathing). Late manifestations may include Hutchinson triad (abnormal teeth, interstitial keratitis, eighth-nerve deafness), saddlenose deformity, mulberry molars, and central nervous system and developmental abnormalities. 3. Serology diagnostics use two methods of analysis: antigen nonspecific and antigen spe-~ cific. Whereas darkfield microscopy can be used to visualize organisms from lesions, these serologic tests are the most common method of diagnosis. a. Nontreponemal tests use cardiolipin as the antigen and can be performed as a complement fixation (CF) test or as a flocculation test (called VDRL or RPR assays). (1) Patients' antibodies are of the IgM and IgA class and are termed reaginic antibodies. The CF test will fix (consume) complement if the IgM reaginic antibodies are present and combine with antigen. (2) The flocculation test causes lipid antigen to clump when combined with reagin. Tests are positive within 2-3 weeks after infection and revert to negative 6-18 months after appropriate treatment. (3) False positive reactions are an important problem with these serologic tests. The tests may show false positive results using serum from patients with other acute illnesses such as pneumonia, hepatitis, vaccination, viral exanthems, and collagen vascular diseases. Some drugs can also cause a false-positive VDRL. b. Fluorescent antibody tests (fluorescent treponemal antibody, FTA) are specific for treponemal antigens. The assay detects specific antibodies and is used for the confirmation of positive nontreponemal tests. Once positive, it remains positive for life and therefore cannot be used to monitor therapeutic response.

316

meClical

Bacteriology: Spirochetes

4. Treatment and prevention a. Long-acting benzathine penicillin is used for early syphilis, and penicillin G is still used for urogenital late syphilis. b. Erythromycin, tetracycline, and ceftriaxone are less-suitable alternatives. c. Long-term follow-up is required to see that reaginic antibody serology decreases. d. Spread can be minimized through use of safe sexual practices. e. Like gonorrhea, cases of syphilis must be reported to the Board of Health. f. Many patients with secondary syphilis have a Jarisch-Herxheimer reaction (fever, headache, and myalgia) 2-12 hours after penicillin G therapy. It is caused by spirochete lysis and release of endotoxin. Plasma concentrations of tumor necrosis factor IL-6 and IL-8 are increased. B. Other treponema! diseases 1. Yaws is caused by T. pertenue in the tropics and is transmitted by direct contact. It is main-

ly a disease of children and is manifested as a painless erythematous lesion usually on the arm or leg. 2. Pinta is caused by T. carateum and is acquired by person-to-person contact, rarely via sexual transmission. 3. Bejel is caused by T. pallidum subspecies endemicum. Poor hygiene plays a role and is responsible for endemic syphilis in the Middle East, Africa, and Southeast Asia. Transmission is by direct contact; skin lesions are highly infectious.

BORRELIA A. Genera! characteristics 1. Borrelia species are transmitted by arthropods (ticks) to humans.

2. Borrelia species are coarse, irregular coils that are very flexible and motile. They can be visualized with light microscopy using Wright or Giemsa stains. Silver stains like WarthinStarry are also useful.

B. Lyme disease 1. Transmission and epidemiology. Lyme disease is caused by Borrelia burgdorferi. a. The organism resides in tick vectors (Ixodes) that have fed on infected deer or mice reservoirs. b. The highest incidence of disease is found during the spring and summer when the nymph and adult stages of the tick require blood meals. c. The disease was first described in Lyme, Connecticut, and is now found throughout the U.S. and in Europe and Australia.

d. It is not uncommon for the patient to have no memory of a tick bite. 2. Clinical manifestations a. The hallmark initial finding is erythema chronicum migrans (a red macule, progressing to annular erythema with central clearing, "bull's-eye") at the site of the tick bite. The rash occurs within the first 10 days of infection and fades within 3-6 weeks. Infection, however, is still active. Constitutional symptoms include fever, headache, malaise, myalgias, adenopathy, and mild meningeal irritation. These early symptoms

meClical

317

Microbiology

typically last for approximately 4 weeks. b. Untreated infection may lead to late neurologic and cardiac disease. (1) Neurologic symptoms include severe headaches, meningitis, cranial nerve palsies (Bell palsy), and painful peripheral neuropathies. These symptoms resolve after several months. (2) Cardiac symptoms include fluctuating cardiac arrhythmias (resolving after several weeks), myocarditis, and pericarditis. c. Late-stage disease is marked by arthritis, occurring weeks to years after disease onset.

3. Diagnosis of Lyme disease is made from the clinical picture and a history of tick bite or exposure, in combination with laboratory identification by serology. a. Spirochetes have been seen in CSF, synovia, blood, and skin lesions, but culture is difficult. b. Serology is marked by a peak in IgM in the third to sixth week (followed by IgG) typically determined by ELISA assays. Because of the frequency of false positives, a Western blot is used to confirm the diagnosis. 4. Therapy consists of doxycycline for early disease; also amoxicillin, cephalosporins, and erythromycin. High-dose intravenous penicillin is used for cardiac, neurologic, and arthritic complications. Ceftriaxone is effective in refractory cases. C. Relapsing fever is named for its numerous antigenic shifts and variations. It is caused by B.

recurrentis and is transmitted by the human body louse. 1. Clinical manifestations a. The incubation period is approximately one week (3-10 days) and is marked by the acute onset of high fever, severe headache, myalgia, photophobia, cough, and meningismus. Symptoms are associated with spirochetes in the bloodstream. b. The initial fever lasts 3-6 days and can be associated with hemorrhage, rash, and neurologic manifestations. c. The initial fever is followed by an afebrile period of 4-10 days. d. Subsequent fevers are less intense and last a shorter period of time. Antigenic shifts occur during this time. The afebrile times allow a new virulent variant to emerge and cause disease. 2. Diagnosis is made by the observation of spirochetes by darkfield microscopy, or by Giemsa-stained or Wright-stained blood smears of febrile patients. 3. Therapy. Treatment with tetracycline or erythromycin rapidly leads to recovery.

LEPTOSPIRA A. General characteristics

1. Leptospira species are visible by darkfield microscopy and appear as a tightly coiled spiral with right angle hooks at both ends. 2. Two principal species exist. a. L. interrogans is pathogenic and includes many of the serovarieties that cause leptospirosis.

318

meclical

Bacteriology: Spirochetes

b. 1. biflexa are the saprophytic or water forms. B. Pathogenicity is mediated by a thermolabile hemolysin produced by virulent strains. Some

strains contain small amounts of endotoxin. C. Transmission and epidemiology

1. Domestic animals (dogs, sheep, goats, cattle, horses, etc.) and rats are major sources for human infection. Ingestion or contact with water contaminated by urine from infected reservoirs is the primary mode of infection.

2. Leptospira enters the host through broken skin or mucous membranes. 3. The organism is an occupational hazard for farmers, veterinarians, abattoir workers, and hunters and jet skiiers. D. Leptospirosis

1. Leptospirosis occurs within a lO-12-day incubation period. The clinical expression is marked by an abrupt onset of fever, chills, headache, conjunctival suffusion, gastrointestinal complaints, and myalgia. At this stage, spirochetes enter the bloodstream. It is a biphasic illness.

a. The first leptospiremic stage is approximately 1 week in duration. The Leptospira infect organs, especially the liver and kidneys, leading to jaundice, bleeding, and uremia. The termination of fever and leptospiremia corresponds with a humoral immune response to the organism. b. The second stage usually occurs about 2 days after the fever disappears and lasts 4-30 days. The second stage is associated with a rise in IgM titers. Aseptic meningitis, hepatitis, nephritis, uveitis, myositis, and rash may develop. 2. Weil syndrome (icteric leptospirosis) is hepatitis associated with elevated CPK. Renal failure occurs as a result of tubular damage; glomeruli usually remain unaffected. 3. Diagnosis is made by the isolation and culture of organisms from blood or CSF in the first stage of illness. a. Direct examination is performed by darkfield microscopy and must be done early in infection. b. Serologic responses peak 5-8 weeks after acute illness. A rapid screen can be performed by a slide agglutination test. E. Treatment and prevention. Treatment includes penicillin, tetracycline, and macrolide antibiotics. A vaccine is available for at-risk individuals.

meClical

319

Bacteriology: Mycoplasma and

Ureaplasma Mycoplasmataceae are the smallest free-living organisms. They are prokaryotic cells resembling Gramnegative bacteria, but they lack the cell wall. The family contains the genera Mycoplasma and Ureaplasma. The organisms are classed into one or the other genus based on the ability to hydrolyze urea. The species of medical importance in humans are Mycoplasma pneumoniae, Mycoplasma hominis, and Ureaplasma urealyticum. Disease in humans involves the respiratory and urogenital tracts.

MYCOPlASMATACEAE A. General characteristics and physiology 1. Morphologically, they are filamentous and pleomorphic. They lack cell walls and are

therefore penicillin resistant. 2. They are facultative organisms that are mainly fermentative.

3. They grow very slowly, and may require up to 3 weeks to visualize colonies on agar. 4. These organisms are unique among the prokaryotes because they require sterols for

growth and their cell membranes contain cholesterol. B. Mycoplasma pneumoniae 1. Epidemiology and transmission. Mycoplasma pneumoniae is found throughout the world. Transmission occurs via aerosol droplets. Clinical illness is more likely in ages 5-20. 2. Pathogenesis. Once in the upper respiratory tract, Mycoplasma pneumoniae are found

among the cilia of the epithelial cells. They attach by way of neuraminidase-like adherence protein to a sialic acid containing glycoprotein on the host cell. Oxidants produced by the organism, especially hydrogen peroxide and the superoxide ion, injure the mucosa. 3. Clinical manifestations. M. pneumoniae is the most common cause of pneumonia in

young adults (walking pneumonia). a. Frequent clinical manifestations of this atypical pneumonia include nonproductive cough, low-grade fever, and an insidious headache. b. Nonpurulent otitis media or bullous myringitis occur in about 20% of patients with mycoplasmal pneumonia. The presence of these conditions concomitantly with pneumonitis in a teenager is a strong indication of M. pneumoniae infection. c.

Rare extrapulmonary manifestations include myocarditis, pericarditis, aseptic meningitis, meningoencephalitis, Guillain-Barre syndrome, peripheral neuropathies, and polyarthralgias.

meClical

321

Microbiology

4. Diagnosis is primarily based on clinical findings and serology.

In a Nutshell M.

pneumoniae

• Lack cell walls • Transmitted via respiratory droplets • Cause atypical pneumoniacause of pneumonia in young adults

*1

• Cold agglutinins (lgM) used in presumptive diagnosis • Treatment: erythromycin, tetracycline, or fluoroquinolones

a. During acute infection, cold agglutinins are produced. These agglutinins are capable of clumping human red blood cells at 4.0°C, but this process can be reversed by heating the blood to 37.0°C. These cold agglutinins are IgM antibodies directed against the I antigen on the surface of the erythrocyte. Although cold agglutinins do not develop in every patient with M. pneumoniae, this remains a rapid test for acute disease. b. Another useful antibody is the Strep MG agglutinin, which many patients produce. The antibody agglutinates Streptococcus salivarius strain MG at normal incubation temperatures. c. Confirmation of the diagnosis can be accomplished using the complement fixation test.

5. Treatment consists of macrolides (e.g., erythromycin or azithromycin), tetracyclines, or fluoroquinolones. Because the family Mycoplasmataceae lacks a cell wall, all members are resistant to the ~-lactam antibiotics. C. Mycoplasma hominis

1. Sexually transmitted agent 2. A major source of infection in postpartum women. 3. Clinical manifestations include postabortal and postpartum fevers and bacteremia, as well as pelvic inflammatory disease. 4. Treatment with tetracyclines; in contrast to other Mycoplasma species, it is resistant to macrolides. D. Ureaplasma urealyticum

1. Sexually transmitted agent 2. Unlike other Mycoplasma species, it produces urea 3. A minor cause of nongonococcal urethritis 4. Treated with tetracycline or erythromycin

322

meClical

Virology

Viruses are the smallest agents of infection, ranging from 20-300 nm in diameter and consisting of either RNA or DNA surrounded by a protective protein shell (capsid). The protein shell may be surrounded by an envelope containing lipid and protein. Virus multiplication occurs only within host cells and is accomplished by the separate synthesis and subsequent assembly of component parts. Some viruses have the unique capacity to become latent and even to integrate their genomes into the host cells. In this situation, the integrated viral genome is replicated as part of the host genome and is transmitted to each daughter cell without production of infectious virus.

CLASSIFICATION AND IDENTIFICATION Classification and identification of viruses is based on common characteristics shared by viral families (such as the presence of single- or double-stranded viral nucleic acids, DNA or RNA). A. Morphology is based on structures and components (nucleic acids, envelope, etc.) common to the viruses and is a basis for viral classifications (see Table V-ll-l). 1. Terminology a. Virion is a term used to describe the complete infectious virus particle. b. Capsid is the protein shell that encloses and protects the nucleic acid genome (either RNA or DNA). The individual protein units are called capsomeres. These structures protect the viral genome from destruction in the extracellular environment. Capsids also control the host range and cell tropism of the naked viruses as they are the molecules that adsorb to the cell surface.

In a Nutshell • Smallest infectious agents • Only one type of nucleic acid (DNA or RNA) • No metabolic activity outside of living cells; obligate intracellular parasite

c. Nucleocapsid denotes the protein shell plus the nucleic acid.

d. Peplomers are the protein spikes found in the envelope of some viruses. 2. Nucleocapsids have characteristic symmetry that is usually helical or icosahedral (see Figure V-ll-l).

KAPLA~. I meulCa 323

Microbiology

Nucleic acid } ~~~rlltr-Protein

Nucleocapsid

Envelope Glycoprotein spikes Enveloped helical nucleocapsid

Capsid {

• • •

~

~~ ~~

~

• •

~:t.

-=

:t.

~

Nucleic acid Capsomere

~~

Icosahedral nucleocapsid

Figure V-11-1. Viral nucleocapsids.

a. A helical nucleocapsid is marked by an extended nucleic acid cavity surrounded by helically arranged proteins with an outer lipid envelope. Examples of viruses with helical structure include ortho- and paramyxoviruses and rhabdoviruses. b. Icosahedral symmetry is marked by condensed nucleic acids forming a central portion of cuboidal nucleocapsid structure. The icosahedral structure may be enveloped or naked. Examples of icosahedral symmetry include parvovirus, adenovirus, herpesvirus, and picornavirus. 3. Envelopes are lipid-containing structures surrounding some viral particles. a. Envelopes are derived from nuclear or plasma cell membranes acquired during viral maturation. The viral envelope is usually acquired when the viral nucleocapsid buds through the host's membrane. b. Envelopes lack rigidity and appear heterogeneous by electron microscopy.

Note Antibody to the gp 120 glycoprotein of HIV is used to monitor the course of HIV infection. The level of the capsomere protein p24 is used to determine the virus load in the blood.

324

meCtical

c. Viral glycoprotein peplomeres are contained in the viral outer envelope. Glycoproteins serve an important role in antigenic structure and host immune responses. These viral attachment proteins (VAP) mediate viral binding (see below) and entry into host cells. d. Lipids are the major component of the viral envelope. They are a complex mixture of phospholipids, glycolipids, and neutral lipids that are a part of the host cell membrane in which the virus multiplied. 4. Virus classification (see Table V-ll-!) is based on nucleic acid composition such as the presence of single-stranded or double-stranded DNA or RNA. Positive-sense RNA (+ RNA) serves directly as mRNA, whereas negative-sense RNA (-RNA) must use an RNA polymerase to synthesize a complementary positive strand to serve as mRNA.

Virology

Table V-II-I. RNA versus DNA viruses. RNA viruses Arenavirus Bunyavirus Calicivirus Coronavirus Filovirus Flavivirus Orthomyxovirus Paramyxovirus Picornavirus Reovirus Retrovirus Rhabdovirus Togavirus DNA viruses Adenovirus Hepadnavirus Herpesvirus Papovavirus Parvovirus Poxvirus

Enveloped helical nucleocapsid (-RNA) Enveloped helical nucleocapsids (-RNA) Naked icosahedral nucleocapsid (+RNA) Enveloped helical nucleocapsid (+RNA) Enveloped helical nucleocapsid (-RNA) Enveloped icosahedral nucleocapsid (+RNA) Enveloped helical nucleocapsid (-RNA) Enveloped helical nucleocapsid (-RNA) Naked icosahedral nucleocapsid (+ RNA) Naked icosahedral nucleocapsid (dsRNA) Enveloped icosahedral (+ RNA) Enveloped helical nucleocapsid (-RNA) Enveloped icosahedral nucleocapsid (+ RNA)

Naked icosahedral nucleocapsid (dsDNA) Enveloped icosahedral nucelocapsid (dsDNA with ss portions) Enveloped icosahedral nucleocapsid (dsDNA) Naked icosahedral nucleocapsid (dsDNA) Naked icosahedral nucleocapsid (ssDNA) Enveloped dsDNA with complex structure

5. Viral proteins are important in the initial contact with the host cell; they dictate which cells will be infected. Viral proteins also determine the antigenic structure of a virus and protect the genome against host nucleases. a. Hemagglutinins are viral proteins that agglutinate red blood cells. The hemagglutinin of influenza virus promotes attachment of the virus to host cells. It can also function in the fusion of the virion with the host cell membrane. They are excellent vaccine antigens. b. Enzymes serve several biological functions for the virus. (1) Neuraminidase acts to hydrolyze sialic acid. It functions to help release virus particles from the cells in which they were formed to further propagate infectivity. (2) RNA polymerase is required for viral replication of RNA viruses. This enzyme must be brought into the cell as a part of the virion in negative-sense RNA viruses. (3) Reverse transcriptase in retroviruses transcribes single-stranded RNA into double-stranded DNA. Transcribed DNA can then be integrated into the host genome by an integrase enzyme. B. Replication. Viruses are dependent on the host cell to provide the synthetic mechanisms and metabolic machinery for replication. 1. The replication cycle of the virus may either lyse the host cell or form a stable interaction that allows the host cell to survive. A stable interaction causes the viral genome to become

In a Nutshell • All RNA viruses have singlestranded RNA except for Reoviruses Cds) • All RNA viruses are enveloped except Reoviruses, Caliciviruses, and Picornaviruses • All DNA viruses have double-stranded DNA except Parvoviruses (ss); Hepadnavirus has ss regions in the DNA • All DNA viruses have an icosahedral nucleocapsid except Poxviruses • All viruses with helically symmetrical nucleocapsids are RNA viruses KAPLAN· . I medlea 325

Microbiology

incorporated into the host cell. The virus may be latent (e.g., herpesvirus) or it may continue to multiply without causing overt disease. The cycle of viral replication has discrete stages: a. Adsorption, or attachment, is the first step. It is highly specific, involving the attraction and physical interaction of viral surface proteins (capsomere or peplomere units) with a host cell surface receptor. Adsorption is host-specific and tissue-specific (e.g., poliovirus attaches primarily to eNS and GI tract cells). b. Penetration and uncoating follows the adsorption step and is typically mediated by receptor-specific endocytosis. The virus usually loses its coat or envelope directly following penetration. Uncoating separates the capsid (and envelope) from the nucleic acids.

In a Nutshell Positive-sense RNA viruses are mRNA and can directly encode all the proteins needed for replication. Other viruses require enzymes in the core such as RNA-dependent or DNA-dependent RNA polymerases to produce mRNA for viral replication.

c. The synthetic period begins when uncoating is complete. Its time course is variable depending upon the virus. Specific mRNA must be transcribed from viral nucleic acid by host cell mechanisms. Macromolecular synthesis begins with an eventual accumulation of viral components (including empty capsids or nonenveloped nucleocapsids). Minus-strand RNA virus, double-stranded RNA viruses, and DNA viruses initiate nucleic acid synthesis to produce mRNA, whereas positive-sense RNA viruses can immediately initiate protein synthesis. This stage is marked by an orderly sequence of synthetic events (with some proteins synthesized early and others later in the synthetic processes). d. Production of viral proteins (1) In positive-sense RNA viruses like polio, the viral RNA (mRNA) is read directly

by the host cell ribosome and enzymes for RNA synthesis are produced. Other proteins that are synthesized at this time will inhibit host biosynthetic processes, whereas others will serve as capsid (structural) proteins. (2) In other viral groups, the viral genome (-RNA, double-stranded RNA, or DNA) must first synthesize the messenger RNA molecules. They do this by an RNAdependent RNA polymerase (transcriptase) that is contained in the virion and encoded by the viral genome in the case of RNA viruses; or by transcription of the viral DNA to synthesize the mRNAs necessary for protein synthesis. Some of the proteins will be structural units (capsomeres, peplomeres), others will be enzymes necessary for progeny DNA synthesis (DNA polymerase). e. Replication of viral genome (nudeic acid) (1) Plus-stranded RNA viruses can immediately begin protein synthesis without nucleic acid replication or transcription events. RNA synthesis will occur when sufficient amounts of RNA polymerase are formed (using the host cell machinery for synthesis). A minus-strand copy is made from the parental strand RNA that serves as the template (replicative intermediate) for the transcription of plus-strand progeny_

Note • All DNA viruses replicate entirely in the nucleus except for Poxviruses. • All RNA viruses replicate entirely in the cytoplasm except for Influenza viruses and Retroviruses.

326

meClical

(2) Minus-strand and double-stranded RNA viruses must first synthesize mRNA for the eventual translation into viral proteins. The minus-strand acts as a negative template for synthesis of mRNA. These viral genomes carry RNA-dependent RNA polymerase required to synthesize mRNA from the (-) strand. (3) Double-stranded RNA viruses (Reo and Rota) synthesize a positive strand of RNA from the negative strand of the parent. This acts both as mRNA and as the replicative intermediate to make the negative-sense RNA that will be assembled with a complimentary positive strand to make the progeny genome. (4) Retroviruses use the negative strand of the DNA intermediate to make positivesense progeny RNA.

Virology

(5) Double-stranded DNA viruses replicate by the same process employed by the host cell; each strand serves as the template for synthesis of the complimentary DNA copy. Hepatitis B virus contains a viral RNA-dependent DNA polymerase (a reverse transcriptase) that uses the viral mRNA as a template to synthesize the missing portion of the viral genome, which is then duplicated by host cell DNA polymerase. (6) Single-stranded DNA viruses (Parvovirus) synthesize a double-stranded intermediate to use as the replicative template for the single-stranded DNA progeny. f. Viral assembly occurs toward the end of the synthetic period. Complete intracellular virus assembly begins. The viral genomes and capsid polypeptides assemble, forming infectious viral offspring. g. Release of the complete nucleocapsid is the final stage of the replication cycle.

In a Nutshell Viral Growth Cycle • Attachment of virus to cell • Penetration of cell • Uncoating of viral genome • Transcription of genome into mRNA

(1) Enveloped viruses are released gradually by a budding process. Nucleocapsids bud through virally altered membrane patches, thus gaining viral specific glycoproteins.

• Translation into proteins

(2) Poxviruses and naked capsid viruses burst out rapidly from the cell, causing the cell to disintegrate.

• Assembly of particles into new viruses

• Replication of viral genome

• Release of virus

DNA VIRUSES A. Adenoviruses

Mnemonic

1. Characteristics. Adenoviruses are medium-sized linear double-stranded DNA viruses

with a naked icosahedral nucleocapsid. They contain penton fibers that serve as viral attachment sites. There are 41 adenovirus serotypes, with approximately one third accounting for the bulk of human disease. After infection, adenoviruses typically travel and replicate in local lymph nodes. 2. Transmission usually occurs person-to-person through respiratory and ocular secretions (usually infecting mucous membranes or lymphoid tissue). Humans are the only known host. 3. Clinical manifestations a. Acute respiratory disease (ARD) that may induce a latent infection in tonsils, adenoids, and other lymphoid tissues. Most infections are acute and self-limited. Influenza-like illness occurs in the late fall and winter, characterized by pharyngitis, fever, cough, and malaise.

To remember which viruses are RNA versus DNA, just memorize the DNA viruses (there are fewer) and know that all the rest are RNA viruses. To help remember the DNA viruses, think about how HHAPPP you will be once you finish studying. The tough part is recalling that the PPP represents papova-parvo-pox (picorna and paramyxo are RNA viruses).

b. Acute febrile pharyngitis may be seen in infants and children and military recruits. c. Conjunctivitis ("pink eye")

d. Diarrhea and gastroenteritis e. Pneumonia and pharyngoconjunctivitis are less frequent complications except in the military, where frequent epidemics may occur. 4. Diagnosis is made by clinical presentation, culture of stool, urine, throat swabs, or conjunctival scrapings. Serology can be performed by ELISA or complement fixation techniques. 5. Treatment and prevention a. Treatment is supportive. KAPLA~. I meulCa 327

Microbiology

b. A vaccine consisting of live, nonattenuated viruses is used in the military. It is administered in three separate doses (one for each serotype, 4, 7, and 21) in enteric capsules that protect the virus from the pH of the stomach. An asymptomatic intestinal infection results that induces respiratory tract immunity. The viruses are administered separately because they interfere with each other if given simultaneously. B. Papovavirus family includes Papilloma, Polyoma, and Vacuolating viruses. 1. Characteristics. Papovaviruses are small double-stranded circular DNA viruses with a

naked icosahedral nucleocapsid. 2. Human papillomaviruses (HPV) are distributed worldwide. They cause skin warts (prevalent in children) and genital warts (condyloma acuminata) and are the most common cause of viral STD. a. Transmission occurs via contact with warts. b. In addition to warts, HPV has also been implicated in benign laryngeal papillomas. c. Serotypes 16 and 18 are associated with penile, laryngeal, and, especially, cervical cancer. d. Treatment includes removal by electrocautery, cryotherapy, or chemicals. Recurrence is common. 3. BK virus is distributed widely in the human population, with 75% serologic positivity in children aged 6 and older. Infection remains latent. Disease (virus in urine) is limited to patients undergoing immunosuppressive therapy and is thought to be a reactivation of infection acquired in childhood. 4. JC virus is a polyoma virus associated with human progressive multifocalleukoencephalopathy. 5. SV40 is one of the serotypes of the simian vacuolating viruses. Transcription products of infection include tumor (T) antigens required for infectivity and oncogenesis. Late proteins synthesized are viral capsid proteins. Transformation of the host cell involves the integration of viral DNA into cellular DNA. This virus has not been associated with disease in humans. C. Herpesviruses 1. Characteristics. Herpesviruses are large double-stranded DNA viruses with an enveloped

icosahedral nucleocapsid. Herpesviruses are characterized by latent infections with recrudescence of disease (increasingly prevalent in immunocompromised patients). 2. Herpes simplex virus, types 1 and 2 (HSV-I and HSV-2) cause oral and genital lesions by infecting epithelial cells. Upon the resolution of acute illness, latent infections are commonly found in neurons. Humans are the only known host. Direct contact with the infected lesion or secretions are necessary for transmission. a. HSV-I is typically acquired early in life. It is usually associated with oral lesions ("fever blisters") and with neurologic disease. HSV-l is the leading cause of sporadic encephalitis in the U.S. Note that HSV-l can cause genital lesions as well. b. HSV-2 is acquired after the onset of sexual activity. It is associated with genital lesions and with neurological disease. It is transmitted through sexual contact.

328

meClical

-----

- - -

----

----

Virology

c. Primary infection may be asymptomatic or characterized by vesicular lesions with

edema, leading to ulceration and crusting. The lesions heal without scarring. Infection is characterized by primary gingivostomatitis (HSV-I) or by primary herpes genitalls (HSV-2). Neonatal herpes may be acquired in utero or during birth. d. Recurrent infection occurs at the site of primary infection. It involves the activation of latent virus from neurons of cervical or sacral ganglia. Stresses that lead to reactivation include hormonal changes (menses), fever, sunlight, physical trauma, and immune suppression. e. Diagnosis is made by identification of the clinical lesions as well as with viral isolation by tissue culture. The Tzanck smear will demonstrate multinucleated giant cells on the stains of scraped lesions (differential includes varicella-zoster virus). Immunofluorescent stain of the lesions will demonstrate viral intranuclear inclusion bodies. The latter test is preferred. f. Antiviral therapy with acyclovir is instituted upon diagnosis in serious infections (see the Antimicrobial Agents chapter of this section). 3. Varicella-zoster virus (VZV) is isolated from patients with chickenpox and shingles. Shingles is reactivation of latent varicella infection. a. Chickenpox (varicella) caused by herpes zoster virus is a mild self-limited illness in children expressed as fever followed by a macular then papular eruption on skin and mucous membranes. Chickenpox usually occurs in epidemics and is highly contagious. The virus is spread by respiratory secretions with approximately a 2-week incubation period. The papules are pruritic and become vesicular on skin and mucous membranes (all stages of the lesions are found simultaneously). Disease is more severe in adults, with pneumonia common in immunocompromised patients. Encephalitis is a rare but severe complication. b. Shingles is a recurrent infection, usually in adults, that may be activated by trauma, neoplasm, drugs, or immunosuppression. Shingles occur from virus that remains latent in the sensory ganglia of spinal or cranial nerves. Severe dermatomal pain occurs with vesicular eruption, fever, and malaise. Pain and neuralgia may precede dermal eruption by 1-3 days. The pain may persist for months (postherpetic neuralgia), especially in older patients. c. Diagnosis is primarily made by serologic assay and by the history of exposure to the

virus. Scrapings oflesions stained with fluorescein-labeled antivaricella antibodies will reveal multinucleated giant cells with viral intranuclear inclusions.

Note Both herpes simplex and varicella-zoster encode a thymidine kinase. The enzyme is the starting point for the antiviral action of acyclovir. It is phosphorylated to an active triphosphate form that blocks viral DNA polymerase.

Clinical Correlate Due to the delay betvveen the onset of pain and the development of skin lesions, zoster should be considered in anyone presenting with pain in a dermatomal distributioneven if the skin appears normal.

d. Treatment and prevention (1) Varicella infections can be treated with acyclovir. Second choices include foscar-

net or vidaribine. (2) Immunosuppressed patients can be treated prophylactically with human varicellazoster immunoglobulin (VZIG). (3) An attenuated varicella vaccine was approved for use in the U.S. in 1995. Children should be vaccinated between 12 and 18 months of age because maternal antibody can interfere with attenuated viral vaccine efficacy. 4. Epstein-Barr virus is the etiologic agent of infectious mononucleosis (1M). 1M is spread by saliva and respiratory secretions and is initiated in the oropharynx. EBV replicates in epithelial cells prior to infecting the B lymphocytes. EBV polyclonally transforms B lymphocytes, leading to the secretion ofIgA, IgG, or IgM. Heterophile antibodies that agglutinate sheep erythrocytes are present in 90% of adults during the illness. EBV infection is

meClical

329

Microbiology

Note The Monospot test is a rapid serologic procedure that detects agglutinins to formalinized RBCs in patients with Epstein-Barr infection.

Clinical Correlate Due to the severe splenomegaly, patients with mononucleosis may undergo splenic rupture during physical exam.

associated with Burkitt lymphoma and nasopharyngeal carcinoma in particular populations. It is also the etiologic agent of hairy oral leukoplakia in immunocompromised hosts. In Burkitt lymphoma cell lines the c-myc oncogene translocates from chromosome 8 to one of the chromosomes involved in immunoglobulin synthesis, where it is activated by the enzymes involved in the VDJ recombination events. The gene product is thought to act as an activator of genes involved in cell proliferation. a. Clinical manifestations last 2-4 weeks and include fatigue, malaise, tender lymphadenopathy, pharyngitis, fever, headache, and splenomegaly. Hepatitis and meningitis are less frequent clinical symptoms. Atypical lymphocytes with a foamy cytoplasm (Downey cells) are noted on peripheral blood smear. (These are reactive T cells.) b. Nonspecific serologic responses to EBV infection are due to the viral infection of B lymphocytes. Heterophile antibodies that arise will agglutinate sheep and horse red blood cells (as measured by the classic Paul-Bunnell test). c. Specific antibodies also arise to the viral capsid proteins. IgM class antibodies indicates recent infection, whereas IgG antibodies persist for more than 1 year.

Epstein-Barr Virus

d. Diagnosis. Ten percent or more of patients fail to make heterophile antibodies. In these individuals diagnosis is made by ELISA assay, isolation of virus, and/or nucleic acid hybridization technology.

• Herpes virus

e. Treatment is supportive. Acyclovir can be used in severely ill patients.

In a Nutshell

• Causes infectious mononucleosis • Hairy oral leukoplakia in immunocompromised patients • Associated with Burkitt lymphoma and nasopharyngeal carcinoma • Heterophile antibodies; Monospot test • Atypicallymphocytes (reactive T cells)

5. Cytomegalovirus (CMV). CMV is species-specific (i.e., human CMV only infects humans). a. CMV infection of nonimmunocompromised (normal) humans elicits a mononucleosis illness that is clinically indistinguishable from EBV-mononucleosis. The majority of human infections are subclinical (with no overt symptoms) but may lead to lifelong latent infection. CMV has a 4-8 week incubation period. Less common clinical sequelae include pneumonia, hepatitis, myocarditis, and meningoencephalitis. b. Illness in immunosuppressed patients (cancer, transplant [especially kidney), chemotherapy-induced, and AIDS patients) is often due to the reactivation of previously acquired infection. Infection in the immunosuppressed is more severe and is marked by fever, adenopathy, leukopenia, hepatosplenomegaly, and myalgias. Interstitial pneumonia, hepatitis, and gastrointestinal ulceration may also arise in immune compromised individuals. CMV retinitis is seen in AIDS patients. c. Congenital disease arises when the fetus acquires CMV virus transplacentally, causing cytomegalic inclusion disease. Infection can be acquired during any trimester. A severe fatal form of the illness is characterized by large intranuclear inclusion bodies in salivary glands, kidneys, brain, liver, and lungs. Periventricular calcifications are seen in the CNS; the infant will be deaf and have hemorrhagic cutaneous lesions (blueberry muffin baby). Breast milk is a significant source of CMV infection from mother to newborn.

Note In contrast to EBV, CMV mononucleosis is heterophile antibody (monospot) negative.

d. Diagnosis is confirmed by tissue culture and isolation of CMV (usually from pharyngeal swabs or urine). Serology by ELISA is also simple and virus-specific. Histological examination of specimens will reveal the classic inclusions within infected cells when stained by immunofluorescence techniques. CMV infection is common. Surveys indicate that 40-100% of adults have been infected with CMV at some time in their life. e. Antiviral therapy is instituted in severe disease with ganciclovir. Mononucleosis in normal patients is usually self-limiting without antiviral treatment.

330

meClical

Virology

6. Human herpesvirus-6 (HHV-6) is a lymphotrophic human herpesvirus. It is thought to be the etiologic agent of pediatric "sixth disease;' or roseola infection (exanthem subitum). 7. Human herpesvirus-8 (HHV-8) is a cofactor in Kaposi sarcoma. D. Poxviruses 1. Characteristics. Poxviruses are the largest of all the viruses. They are linear doublestranded DNA enveloped viruses that replicate entirely in the cytoplasm of infected cells. They appear ovoid or brick-like in shape. The virion contains several enzymes for replication, including both DNA and RNA polymerases. 2. Variola virus (smallpox virus) is confined to humans and is spread by direct person-toperson contact. Smallpox is now officially considered eradicated after years of worldwide vaccination. 3. Molluscum contagiosum causes umbilicated wart-like skin lesions and satellite nodules on the periphery of the parent nodule. In children, the lesions are more commonly found on the trunk, face, or limbs. The sexually transmitted virus will be expressed as genital lesions. a. The disease is typically benign and self-limiting but may require up to 3 years to resolve (particularly genital lesions). The lesions may be numerous in immunocompromised (especially AIDS) patients. b. The diagnosis is usually clinical, although the virus can be seen on electron microscopy. 4. Cowpox is an occupational disease transmitted to humans by contact with infected cow udders. Infection is usually restricted to fingers and hands and is self-limited. The disease is usually milder in vaccinated individuals. E. Hepadnaviruses are DNA viruses that include the hepatitis B virus (HBV), which is discussed in the section on hepatitis viruses, beginning on page 339. F. Parvoviruses are small single-stranded DNA viruses. Serotype B19 is the only human pathogen in the family. It causes erythema infectiosum ("fifth disease") in children (charac-

terized by slapped cheek rash), aplastic crisis in individuals with chronic hemolytic diseases such as thalassemia and sickle cell (the virus infects premature RBCs and kills them), and fetal infections that may cause hydrops fetalis or stillbirth of fetuses that are profoundly anemic.

RNA VIRUSES A. Picornaviruses are small (+) single-stranded RNA viruses with a naked nucleocapsid. The RNA genome is positive sense (can serve as mRNA) and replication of picornaviruses occurs in the cytoplasm of the host cell. The picornaviruses can be divided into enteroviruses (poliovirus, coxsackie A and B, echovirus, and enterovirus) and rhinoviruses. Enteroviruses are acid-resistant and are able to survive in the gastrointestinal tract; rhinoviruses are acidsensitive. 1. Polioviruses bind to receptors in the gut and on neurons. Disease occurs only in pri-

mates, with the majority of infections expressed as subclinical disease. Three antigenically distinct immunologic subtypes exist. Serologic positivity is found most commonly in low socioeconomic areas.

Note Vaccinia virus, thought to be an attenuated variant of cowpox, is being extensively studied as a vaccine vector for carriage of multiple immunogens. The work involves inserting DNA that encodes toxins, adhesins, etc. into the viral genome.

Note Other erythemas are measles, rubella, scarlet fever, roseola, and exanthem subitum (HHV-6).

Note Polioviruses are polycistronic, which means that the RNA is read as large polyprotein messages (i.e., no stop codolls in the genome). The individual proteins are produced by the action of a viral protease that "chops up" the polyprotein product of translation. Similar replicative processes occur in HIV; the newest AIDS drugs being used today are protease inhibitors.

meclical

331

Microbiology

a. Transmission. Poliovirus is excreted in the feces and transmission occurs primarily by person-to-person contact and via contaminated water sources. b. Pathogenesis. Poliovirus is ingested, replicates in oropharyngeal and intestinal mucosa, and drains to the cervical and mesenteric lymph nodes. Transient viremia ensues and the virus then spreads systemically. CNS involvement may lead to the destruction of motor neurons in the spinal cord, resulting in flaccid paralysis.

c. Clinical findings are expressed as three syndromes with an incubation period of 7-14 days. (1) The first stage, termed abortive poliomyelitis, is a mild infection (most common form of disease) causing flu-like symptoms that resolve without sequelae. This stage is only diagnosed when virus or antibody is detected. (2) The second stage is aseptic meningitis, which includes the above symptoms with meningeal signs without paralysis.

Clinical Correlate Polio vaccination schedule: currently IPV is given at 2,4, and 6 months together with DPT, Hib, and Hep B. A booster is given between the ages of 4 and 6.

(3) The third stage is paralytic poliomyelitis expressed as flaccid paralysis from lower motor neuron infection (poliovirus primarily attacks the anterior horn cells but may involve posterior horn and dorsal root ganglia). d. Prevention. Live attenuated virus vaccine (oral; OPV; Sabin) or the killed virus vaccine (IPV; Salk vaccine) are used to induce immunity. Both vaccines induce serum antibodies; only the oral vaccine induces gut immunity and sIgA synthesis. 2. Echoviruses (.Enteric Cytopathic Human Qrphan) consist of 32 serotypes recognized by viral capsid antigen differences. All echoviruses infect the gastrointestinal tract although not all cause human disease. a. Transmission and epidemiology. Echoviruses are acquired by ingestion or inhalation. The initial infection occurs in the throat, followed by gastrointestinal tract infection. The incidence of clinical disease is increased in the summer months. b. Clinical manifestations. Disease is observed as aseptic meningitis, fever, rash, enteritis, common colds, and/or acute hemorrhagic conjunctivitis. Less common symptoms include paralysis, pleurodynia, encephalitis, myocarditis, and respiratory illness. 3. Coxsackieviruses are divided into A and B groups according to their pathogenesis in suckling mice. Coxsackie A causes diffuse myositis of skeletal muscle; coxsackie B causes focal necrosis of skeletal muscle and degeneration of brain and other tissues. a. Transmission and epidemiology. Epidemics occur in the summer and fall. Coxsackie is transmitted by nasopharyngeal secretions and by the fecal-oral route. Coxsackie infection is usually an asymptomatic or benign illness with the following exceptions. b. Clinical manifestations (1) Coxsackie A may cause herpangina, with headache, sore throat, dysphasia, stiff

neck, fever, anorexia, and abdominal pain. Discrete vesicles are seen in the oropharynx. It is also the etiologic agent of hand-foot-and-mouth disease. (2) Coxsackie B may cause myocarditis, pericarditis, and pleurodynia. (3) Both viral groups may cause meningitis in humans. 4. Enterovirus 72 is the etiologic agent of hepatitis A. It is discussed in hepatitis viruses, beginning on page 339. Other enteroviruses are associated with exanthema and aseptic meningitis. 5.. Rhinoviruses are associated most frequently with the common cold.

332

meclical

Virology

a. Transmission and epidemiology. Rhinoviruses infect only human hosts and are commonly isolated from the nose and throat. The incubation period lasts 2-4 days, with clinical illness lasting up to a week. Over 100 serotypes have been identified, each conferring type-specific immunity. b. Clinical manifestations include upper respiratory tract irritation, headache, nasal discharge, cough, malaise, chills, and myalgia. Fever is usually limited or nonexistent, as is cervical lymphadenopathy. c. Treatment and prevention. Treatment is supportive. The large number of serotypes makes a vaccine impractical. B. Orthomyxoviruses include influenza viruses A and B. Orthomyxoviruses are mediumsized, negative-sense, single-stranded, segmented RNA with an enveloped nucleocapsid. 1. Structure. Influenza viruses are composed of eight separate segments of RNA.

a. The M (matrix) protein is an abundant inner surface membrane protein important in viral maturation. b. Another major viral protein is the internal nucleocapsid protein (NP protein). c. Hemagglutinin (HA) and neuraminidase (NA) are spike glycoproteins (peplomeres)

along the outer surface of the virus that are the principle targets of the immune response and are excellent immunogens. 2. Classification into types A and B is based on NP protein and M protein antigens. No cross-reactivity between the individual proteins exists. Immunologic variants within each type are characterized by differences in spike glycoproteins, neuraminidase, and hemagglutinin. 3. Antigenic variation frequently occurs in serological types resulting from rapid changes in HA and NA antigenicity. Rapid antigenic variation is an important mechanism for the virus to evade immune system destruction, and is the reason for yearly change in vaccine composition. a. Antigenic drift is defined as minor antigenic changes in the HA or NA proteins (due to the mutation of genes for these proteins) and is responsible for epidemics. b. Antigenic shift is defined as major antigenic changes in the virus. New antigenic types are usually unrelated to the previous types and involve either of the spike glycoproteins, HA or NA. Influenza A has shifted 4 times since 1933. The likely mechanism is the genetic reassortment of genes between human and nonhuman influenza viruses (swine, bird) and is responsible for pandemics. 4. Transmission is by inhalation. Epidemics occur with antigenic drifts while pandemics arise from antigenic shifts. 5. Clinical manifestations. Influenza type C causes symptoms of the common cold with an incubation period of 1-4 days. The symptoms of influenza types A and B are more severe and include: a. Fever, chills, myalgia, and lassitude all occur abruptly b. Sore throat, headache, nasal congestion, and dry cough c. A potential progression to viral pneumonia or secondary bacterial infection (especially

Staphylococcus). These symptoms usually occur in the elderly and debilitated. d. Influenza B is one of the many viruses associated with Reye syndrome.

meclical

333

Microbiology

6. Treatment and prevention a. Treatment with amantadine or rimantadine is only effective for type A infections, whereas newer drugs (oseltamivir and zanamivir) target the neuraminidase and are effective for types A and B. b. Vaccines composed of inactivated virus are designed to elicit immunity against the existing serotypes in the population. Vaccines change from year to year based on the particular serologic determinants of the virus. C. Paramyxoviruses are negative-sense RNA viruses with an enveloped nucleocapsid.

Paramyxoviruses are genetically stable (no antigenic shifts or drifts). Initial infection is via the respiratory tract. These viruses are the most common cause of respiratory infections in children. 1. Structure a. The envelope proteins consist of the HN protein (neuraminidase and hemagglutinin activity together) that is responsible for attachment of the paramyxovirus to the invaded cell surface. b. M protein lines the inner layer of the virus and the F protein mediates cell fusion and hemolytic activity (essential factor in the invasive process).

Clinical Correlate Children suffering from croup often have a characteristic "barking" cough.

2. Parainfluenza viruses are ubiquitous and spread by aerosolized droplets. Parainfluenza is the etiologic agent of croup. a. Adults usually have neutralizing antibody to all four major serotypes. However, reinfection may occur despite the presence of neutralizing antibody. Reinfections are typically mild compared to the primary infection, which usually occurs during the first 6 years of life. b. Clinical manifestations are usually associated with febrile illness (viremia is uncommon). Laryngo-tracheobronchitis (croup), or an obstruction due to swelling oflarynx and trachea, is caused by serotypes 1 and 2. Bronchiolitis and pneumonia in infants is caused by serotype 3. c. Treatment. Treatment is symptomatic; steam/nebulized air reduces symptoms.

Note Koplik spots = measles = paramyxovirus

3. Measles virus (rubeola) is a highly contagious childhood infection characterized by fever and maculopapular exanthem. Rubeola is transmitted by respiratory secretions. The virus multiplies in the oropharynx then spreads to lymphoid tissue followed by further viral replication throughout the reticuloendothelial system. Infection leads to permanent immunity because only one antigenic type exists. a. Clinical manifestations. Koplik spots, bluish-white specks on a red base found on buccal mucosa, are pathognomonic for measles. Other clinical symptoms include an abrupt onset of anorexia, nausea, fever, malaise, and the three Cs: coryza, conjunctivitis, and cough. A maculopapular, erythematous rash, lasting about 5 days, originates on the face and may spread to the torso.

Note MMR vaccine is given at 15 months to avoid interference with viral replication by traces of maternal antibody.

334

meclical

b. Complications of measles infection include encephalomyelitis (1 in 1,000 cases), pneumonia (in immunodeficient patients), otitis media, and subacute sclerosing panencephalitis (SSPE). SSPE is a fatal disease developing many years after acute infection. Measles infections are also thought to precede the onset of multiple sclerosis. These complications are thought to be due to an autoimmune response against nervous tissue that is induced by the measles virus.

Virology

c. Treatment and prevention

(1) A live, attenuated vaccine is available as part of the measles-mumps-rubella vaccine that is administered at 15 months and again at entry to school. (2) Unvaccinated children exposed to measles can be treated with pooled serum globulin because most donors have measles antibodies in their serum. 4. Mumps virus causes an acute contagious, nonsuppurative parotitis (either unilateral or bilateral). Less frequent clinical sequelae include orchitis, occurring in 20-35% of postpubertal males, and aseptic meningitis. However, as many as one third of cases are asymptomatic. Mumps is prevented by immunization with live attenuated virus as part of the measles-mumps-rubella (MMR) vaccine. 5. Respiratory syncytial virus (RSV) is the primary cause of lower respiratory tract infections in infants. a. Transmission occurs via aerosolized droplets; it can also be spread by fomites. b. RSV replicates in the upper respiratory tract and persists in older children and adults. In infants and young children, the virus spreads to the lower respiratory tract, causing bronchitis and pneumonia.

In a Nutshell Paramyxoviruses • Parainfluenza • Measles • Mumps • RSV

c. The spectrum of disease ranges from common cold-like symptoms to severe lower respiratory illnesses (especially in infants). Impaired immune functions will allow recurrent and prolonged infections with RSV. d. Diagnosis is made by serology although syncytial cells form in RSV-infected tissue culture and in vivo. e. Treatment includes supportive care and aerosolized ribavirin in more severe cases. Virus-specific immunoglobulin injections are also being employed in the treatment of severe disease. D. Togaviruses elicit diseases that range from febrile illness to encephalitis or severe bleeding disorders. Togaviruses contain (+) single-stranded RNA in an enveloped nucleocapsid with glycoprotein hemagglutinins in the envelope. 1. Alphaviruses multiply in many arthropods (arthropod-borne) and vertebrates. They are

zoonotic agents spread to man by insect vectors. Alphaviruses include encephalitis viruses. a. Eastern equine encephalitis (EEE) virus causes a severe disease with 50-70% mortality. The illness is marked by the abrupt onset of headache, fever, and nuchal rigidity, with nausea, vomiting, and drowsiness. The individual may be left with neurologic deficits. b. Western equine encephalitis virus causes less severe disease and is seen more in children. 2. Rubivirus or rubella (3-day measles) is the virus causing German measles. It is the only togavirus not transmitted by an arthropod vector. a. Rubella consists of a single antigenic type that resembles measles. However, rubella is an infection of shorter duration and the disease is much less severe. The virus infects the upper respiratory tract and then spreads throughout the body via a viremia. b. A morbilliform rash occurs 2-3 weeks postinfection. c. Congenital rubella. Rubella virus may be transmitted across the placental barrier.

This has serious consequences if it occurs in the first trimester. If the fetus survives, neurologic and other congenital abnormalities are common, including mental retardation, heart abnormalities, blindness, motor abnormalities, and encephalitis. KAPLAlf I medlea 335

Microbiology

d. Live attenuated virus vaccination is effective for prevention of infection (as part of MMR [measles-mumps-rubellal). E. Flaviviruses are very similar to togaviruses and specifically resemble alphaviruses. They are

arthropod-borne viruses. 1. Yellow fever is a mosquito-borne tlavivirus infection with an incubation period of 3-6

days. a. Two viral forms exist, urban (human reservoir) and jungle (monkey reservoir) types; both may result in severe disease affecting liver and kidney (with icterus and hemorrhage). b. Yellow fever is characterized by the acute onset of fever, jaundice, proteinuria, vomiting (vomitus is characteristically black) and the hemorrhage of internal organs with necrosis. Severe symptoms may lead to hypovolemic shock and death. c. A safe and effective attenuated vaccine (strain 17D) is available. 2. Dengue fever is also a mosquito-borne illness characterized by fever, rash, arthralgia, and lymphadenopathy. Recurrent infection with Dengue involves hemorrhagic manifestations. Several antigenic serotypes are recognized and clinical complications may result in death (10% fatality rate). Dengue fever occurs primarily in the tropics. 3. St. Louis encephalitis and Japanese B encephalitis are arthropod-borne viruses that do not cause hepatitis and hemorrhagic disease seen in dengue and yellow fever. Their symptomatology is restricted to the CNS. F. Bunyaviruses are segmented (-) single-stranded RNA viruses with an enveloped nucleocapsid. Bunyaviruses are transmitted by arthropod vectors (mosquitoes) and humans are only accidentally infected hosts.

1. California encephalitis viruses cause an abrupt fever and severe bifrontal headache. Other sequelae include CNS involvement requiring prolonged convalescence but with low morbidity and mortality. 2. Hantavirus causes hemorragic fever and acute respiratory distress syndrome with a high case fatality rate. They are natural pathogens of rodents. Humans become infected by inhalation of infectious urine or feces. It is prominent in the desert Southwest United States. G. Rhabdoviruses include only one significant human pathogen, rabies virus. Rabies is a bulletshaped enveloped virus with (-) single-stranded RNA. It has a nucleocapsid with protruding glycoprotein spikes; the virus replicates in host cell cytoplasm. The rabies virus is of a single immunologic type eliciting neutralizing antibodies against the surface glycoproteins.

Note Negri bodies = rabies = rhabdovirus

1. Transmission is dependent on the persistence of the virus in a wild animal reservoir (e.g., skunks, raccoons, bats, foxes), making this virus practically impossible to eradicate. 2. Pathogenesis. The virus enters through breaks in the skin produced from the bite of a rabid animal. The virus replicates in the muscle and connective tissue with an incubation period of 2-16 weeks. The virus moves through the axoplasm of peripheral nerves to the CNS, including the basal ganglia. Rabies may also inhabit the salivary glands and other tissues along its retrograde movement through the peripheral nerve pathways. Negri bodies, the cytoplasmic viral inclusions in neurons of the hippocampus, are pathognomonic for the infection. 3. Clinical manifestations. The clinical course is described in four discrete phases: prodrome, sensory, excitement, and paralytic. Prodrome symptoms include paresthesia at wound site, irritability (including fever and a change in mood or temperament), and a tlu-

336

meclical

Virology

---------~~~- -~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

like illness. Pharyngeal spasms may arise, resulting in drooling (hydrophobia). Terminal disease involves seizures, coma, and, eventually, death. 4. Diagnosis is confirmed by the recovery of virus from saliva, by serology (direct immunofluorescence), or by immunofluorescence of Negri bodies in neural tissue (especially Ammon's horn). 5. Treatment and prevention a. Vaccines consist of inactivated virus from infected human diploid cells. Pets and highrisk individuals should be vaccinated, as should anyone with a history of being bitten by an animal that is possibly rabid. b. Human rabies immunoglobulin (HRIG) should be given immediately in cases of probable infection.

H. Coronaviruses are (+) single-stranded RNA viruses with an enveloped nucleocapsid and distinct club-shaped projections. Coronaviruses cause common cold-like illness similar to those caused by rhinoviruses. Infections are usually found in older children and adults. I. Caliciviruses are (+) single-stranded RNA viruses with naked nucleocapsids. 1. Norwalk agent is a calicivirus with a single structural protein. Norwalk agent accounts for about one third of gastroenteritis outbreaks (epidemics) throughout the year. The patient population is 2 years and older. The infection usually originates from contaminated water or food and is also associated with shellfish. The incubation period is usually 48 hours with symptoms lasting 12-24 hours. Symptoms include diarrhea, nausea, vomiting, low-grade fever, crampy abdominal pain, and headache.

2. Hepatitis E is very similar to Norwalk agent and will be discussed later in this chapter.

J. Arenaviruses are segmented (-) single-stranded RNA viruses with an enveloped nucleocapsid. Infection with arenaviruses may induce a chronic carrier state in natural hosts such as rodents. The most clinically important arenaviruses are the lymphocytic choriomeningitis virus (LCMV) and Lassa fever virus. 1. Lymphocytic choriomeningitis virus (LCMV) is transmitted to humans from the house mouse or from laboratory rodents.

a. Clinical symptoms include fever, headache, myalgia, malaise, and conjunctivitis with a relatively long incubation period of 18 to 21 days. The disease may progress to aseptic meningitis, disorientation, and/or somnolence. b. The disease is more severe in adults and often subclinical in children. c. Infection is usually self-limiting and mortality is rare.

2. Lassa fever virus is present in West Africa and causes chronic infection in its rodent reservoir. a. It is acquired by ingestion of infectious urine. b. Lassa fever is a highly virulent disease with symptoms of a fulminant, fatal infection marked by fever, shock, hemorrhage, renal failure, and myocardial involvement. c. Mortality rate is high.

d. The infection requires careful isolation procedures to prevent its spread between humans. K. Filoviruses are (-) single-stranded RNA viruses with enveloped nucleocapsids. They have a characteristic filamentous shape. These viruses include the Marburg virus and the Ebola virus. They are so dangerous that they require very sophisticated containment facilities. KAPLAtr I medlea

337

Microbiology

1. Transmission and epidemiology. These viruses are endemic to Africa. Monkeys are the

primary reservoir. 2. Clinical manifestations include a flu-like illness followed by nausea, vomiting, and diarrhea. Eventually, extensive tissue necrosis occurs, leading to widespread hemorrhage and shock. The mortality rate is nearly 100%. 1. Reoviruses are double-stranded, segmented RNA viruses with a naked nucleocapsid. Reoviruses may be isolated from healthy or symptomatic humans. Reoviruses are responsible for upper respiratory and gastrointestinal disease. 1. Rotavirus is the major cause of diarrhea in infants and children under 2 (infantile gas-

troenteritis). The disease is seasonal and classically occurs in the fall and winter months. Rotavirus infects the cells of the small intestine, destroying the transport mechanisms of villi. GI tract infection leads to cell sloughing and impaired absorption of sodium and glucose. 2. Orbiviruses are the cause of Colorado tick fever, the only tick-borne viral disease in the U.S. Symptoms include myalgia, particularly involving legs and back, with no rash. The infection is usually mild and self-limiting. M. Retroviruses are diploid (+) single-stranded RNA, enveloped viruses associated with tumors, oncogenesis, and immunodeficiency disease (AIDS). 1. Retroviruses contain two copies of single-stranded RNA, and viral-encoded reverse tran-

scriptase produces double-stranded DNA from the RNA. 2. The viral genome encodes three groups of proteins: Pol protein (reverse transcriptase and integrase), Env protein (type-specific envelope proteins), and Gag protein (type-specific viral core proteins).

Bridge to Antimicrobials Treatment options for HIV are discussed in the chapter on Antimicrobial Agents later in this section.

Bridge to Immunology HIV is discussed in detail in the Clinical Immunology chapter (in Section VI) of this book.

Note The high level of antigenic variation among the HIV proteins is the result of inaccuracies in the action of reverse transcriptase, leading to incorporation of incorrect nucleotides.

338

meltical

3. This viral family includes human T-cell leukemia viruses (HTLV I, II) and human immunodeficiency virus (HIV). HTLV viruses are categorized as oncoviruses, whereas HIV is considered a lentivirus and is not oncogenic. 4. Oncoviruses. These viruses encode oncogenes that promote cell growth. The most important oncogenic retrovirus is human T-Iymphotropic virus 1 (HTLV 1). This virus infects CD4+ T cells (helper and delayed-type hypersensitivity). Infection can progress to acute T-cell lymphocytic leukemia (ATLL) (aka adult T-cell leukemia). HTLV II causes hairy cell leukemia. 5. HIV is the etiologic agent of acquired immunodeficiency syndrome (AIDS). AIDS was initially recognized as a clinical syndrome in 1981 followed by the isolation of HIV in 1983. HIV is a retrovirus that infects millions of people worldwide. HIV infects helper T cells via attachment to cell surface proteins (termed CD4), resulting in severe immunodeficiency. The virus also infects macrophages via an interaction with CD4 and CCR5 on the macrophage membrane. Patients are prone to opportunistic infections, malignancies, and wasting syndromes. Total CD4 T-cell counts are directly correlated with the degree of disease and infection. Opportunistic infections typically occur when the CD4 count drops below 200. 6. HIV is classified as a lentivirus (nononcogenic, cytocidal, retrovirus). HIV has a cylindrical, conical capsule that contains two positive, single-stranded RNA genomes with a glycoprotein envelope. The gp120 glycoprotein spike protrudes from this envelope and is the ligand for the CD4 molecule. Reverse transcriptase enzyme, critical to its replication, is inside the virion particle. The envelope proteins demonstrate considerable variation from virion to virion. This property makes it difficult for immune responses to clear the virus and complicates the development of a vaccine. Besides HIV, the lentivirus subfami-

Virology

ly also includes SIV (simian), FlV (feline), and BIV (bovine) immunodeficiency viruses. Although this viral family shares common features (such as genes for reverse transcriptase), these viruses are infectious only in their respective species.

HEPATITIS VIRUSES Hepatitis viruses include both DNA (hepatitis B) and RNA (hepatitis A, C, D, E, and G) viruses. A summary appears in Table V-1l-2.

Bridge to Gastrointestinal Pathology Hepatitis is also reviewed in detail in the Gastrointestinal Pathology chapter of Organ Systems Book 2 (Volume IV).

A. Hepatitis A virus (HAV) is a picornavirus. 1. Transmission. Hepatitis A is transmitted fecal-orally after a 15- to 40-day incubation period. Hepatitis A is associated with epidemic and endemic spread of infection (usually occurring as familial or institutional outbreaks). Most childhood infections are asymptomatic, but adult disease can be severe.

2. There are typically no extrahepatic manifestations and there is no chronic hepatitis or carrier state. Infection is not associated with either cirrhosis or hepatic carcinoma. Fulminant hepatitis occurs only in 1--4% of clinical infections. 3. Diagnosis is made by the presence of anti-hepatitis A IgM appearing soon after the elevation of transaminases. IgG class antibodies appear toward the end of the symptomatic period. Life-long immunity usually follows infection. 4. There is a killed virus vaccine against hepatitis A that is recommended for individuals traveling to endemic areas, food handlers, and day-care workers. Pooled gamma globulin can be administered to travelers if there is insufficient time for active immunization. B. Hepatitis B virus (HBV) is an enveloped double-stranded DNA virus with single-stranded segments. It is classified as a hepadnavirus. 1. Characteristic viral antigens include: a. Surface antigen (HBsAg) is found on the surface of the virion. Its presence in serum indicates active viremia (infectivity). b. Core antigen (HBcAg) is found in the capsid. c. E antigen (HBeAg) is another epitope found in the capsomere proteins. 2. Antibodies to HBV-specific antigens are diagnostic markers of disease activity. a. Antibodies to the surface antigen (HBsAg) are considered protective antibodies and are detected after the disappearance of the virus. b. Antibodies to HBcAg are not protective. They are detected just after the appearance of HBsAg and are used to confirm infection when HBsAg and anti-HBsAg are absent (window phase). c. Antibodies to HBeAg are associated with a low risk of infectivity. 3. Transmission is parenteral or sexual. The majority of affected patients recover from the illness. 4. Clinical manifestations include: anorexia, nausea, vomiting, headache, fever, abdominal pain, dark urine, and, sometimes, jaundice. a. Liver function tests will indicate elevated transaminases, hyperbilirubinemia, and elevated alkaline phosphatase. b. Pathology is observed as the accumulation of inflammatory cells and parenchymal necrosis in liver. KAPLA~.

meulCa

I

339

Microbiology

In a Nutshell

c. Extrahepatic manifestations include arthralgia, arthritis, nephritis, and dermatitis.

Childhood Vaccines Against Viral Illnesses

d. Ten to fifteen percent continue to carry the virus and may develop chronic persistent hepatitis or chronic active hepatitis with subsequent fibrosis, cirrhosis, and hepatocellular carcinoma.

• -

Polio (lPV) = 4 doses 2 months 4 months 6-18 months 4-6 years (before school)

• Mumps-measles-rubella (MMR) = 2 doses - 12-15 months - 4-6 years (before school) • Chickenpox ryZV) = 1 dose - 12-18 months • Hepatitis B (HBV) = 3 doses - Birth-2 months - 2-4 months - 6-18 months For review of all childhood immunizations, see Table V-1l-3.

5. Prevention. Recombinant HBsAg vaccine is available and is recommended for all individuals at risk (e.g., health care workers). It is also now part of the protocol for childhood immunizations. Children should receive three doses: one at birth (up to 2 months), one at 2-4 months, and a final dose at 6-18 months. Children born of hepatitis B carriers should be injected at birth with hepatitis B-specific immunoglobulins (HBIG). C. Hepatitis C virus (HCV) is a positive-sense, single-stranded RNA virus classified as a £lavivirus. It is associated with post-transfusion hepatitis. HCV has been characterized as the

etiologic agent for the majority (>50%) of non-A-non-B (NANB) hepatitis infections. Screening of blood for HCV has significantly reduced the incidence of transfusion-related hepatitis. HCV develops a chronic carrier in nearly 80% of patients and is associated with cirrhosis and hepatocellular carcinoma. D. Hepatitis D (delta agent) is a defective RNA virus that can replicate only in cells concurrently infected with hepatitis B. This virion requires the presence of hepatitis B enzymes to

replicate. It occurs in Italy and in the Near East. E. Hepatitis E is caused by a single-stranded RNA virus that is similar to Norwalk agent (calicivirus). Clinically, it causes a disease similar to hepatitis A, but it can become fulminant in pregnant women (20% mortality). It is spread via fecal-oral route. It occurs primarily in the Far East.

Table V-11-2. Summary of hepatitis viruses.

Note Not surprisingly, yet another hepatitis virus was recently identified (1996). Hepatitis G (HGV) is an RNA virus associated with both acute and chronic hepatitis. It has a global distribution and is transmissible by transfusion. It appears to be distantly related to hepatitis C.

Virus

HepA

HepB

HepC

HepD

HepE

+RNA Flavivirus Parenteral

-RNA Delta agent Parenteral

+RNA Calicivirus Fecal-oral

Rare

DNA Hepadnavirus Parenteral Perinatal Sexual Rare

Rare

Frequent

Never No

Often Yes

Very often Yes

Very often

In pregnant women Never No

Genome

+RNA Picornavirus Transmission Fecal-oral Fulminant course Chronicity Oncogenic

Note Creutzfeldt-Jakob disease is considered a "prion" disease. Another spongiform encephalopathy, kuru, was associated with cannibalism and is now extinct.

340

meClical

PRIONS Prions do not have genomes or virion structures. They are unique infectious protein particles with a mode of replication that is not entirely known. The major human disease caused by a prion is Creutzfeldt-Jakob disease (C]D; a spongiform encephalopathy [mad cow diseaseD. Outbreaks of CJD were reported with the use of contaminated growth hormone preparations. This disease is progressive and always fatal. It is preceded by dementia and motor/movement disorders. Confirmation of diagnosis can be made at autopsy only.

Virology

Table V-11-3. Recommended childhood and adolescent immunization schedules-United States, 2007. DEPARTMENT OF HEALTH AND HUMAN SERVICES· CENTERS FOR DISEASE CONTROL AND PREVENTION

Recommended Immunization Schedule for Persons Aged 0-6 YearS-UNITED STATES • 2001 Age...

VaccineT Hepatitis B'

Birth Hep8

Rota

Rotavirus' Diphtheria, Tetanus, Pertussis'

DTaP jm ...................... !

Range of recommended ages

DTaP

•

....................... !.......... m.................... m..............

Haemophilus influenzae type b'

Hib

Pneumococcal'

PCV

Inactivated Poliovirus

IPV

Hib

Catch-up immunization

Influenza'

Certain high-risk groups

Measles, Mumps, Rubella' Varicella' Hepatitis A' Meningococcal'·

Recommended Immunization Schedule for Persons Aged 7-18 YearS-UNITED STATES • 2001 Age"

Vaccine,..

Tetanus, Diphtheria, Pertussis1 Human Papillomavirus Meningococcal

2

7-10

years

11-12 YEARS

13-14

years

15

years

16-18

years

see footnote

1 see footnote

2

3

Pneumococcal' Influenza 5 Hepatitis AS Hepatitis 81

Range of recommended ages

-

Catch-up immunization

Certain high-risk groups

Inactivated Poliovirus8 Measles, Mumps, Rubella 9 Varicella 10 This schedule indicates the recommended ages for routine administration of currently licensed childhood vaccines, as of December 1, 2006, for children aged 7-18 years, Additional information is available at http://www.cdc.gov/nip/recs/child-schedule.htm. Any dose not administered at the recommended age should be administered at any subsequent visit, when indicated and feasible. Additional vaccines may be licensed and recommended during the year. Licensed combination vaccines may be used whenever any components of the combination are indicated and other components

of the vaccine are not contraindicated and if approved by the Food and Drug Administration for that dose of the series, Providers should consult the respective Advisory Committee on Immunization Practices statement for detailed recommendations. Clinically significant adverse events that follow immunization should be reported to the Vaccine Adverse Event Reporting System (VAERS), Guidance about how to obtain and complete a VAEAS form is available at http://www.vaers.hhs.govorbytelephone, 800-822-7967.

mettica.

341

Mycology

Fungi are eukaryotic organisms that can be classified as either yeast or molds based on their morphology and their mode of reproduction. The simplest way to review medically important fungi is to divide them into groups based on their clinical presentation: cutaneous mycoses, subcutaneous mycoses, systemic mycoses, and opportunistic mycoses.

INTRODUCTION A. Morphology. Fungi are eukaryotes possessing a cell wall (composed of glucose and mannose polymers called chitin) and a cell membrane (containing ergosterol). Capsules are found only with Cryptococcus neoformans. The fungi are divided into yeasts or molds based on shape and mode of reproduction. 1. Yeasts have round or oval morphology and reproduce by budding. 2. Molds have tubular structures called hyphae. Molds grow by branching and longitudinal extension to form mycelial structures. Hyphae can be either septate (divided into nucleated cells) or nonseptate (coenocytic). 3. Dimorphic fungi grow in the host as a yeast-like form, but grow as molds at room temperature in vitro. B. Reproduction can be sexual and/or asexual. Asexual spores form through mitosis. This

form of reproduction is referred to as an "imperfect" state. Most pathogenic fungi are found only in the imperfect state. C. Immunity. The T-cell response is protective in fungal diseases; antibodies are not, although

they may have some role in serodiagnosis.

DERMATOPHYTOSIS (CUTANEOUS MYCOSES) Dermatophytosis is a mycotic infection of any keratinous structure of the skin and its appendages by Trichophyton (T.), Microsporum (M.), or Epidermophyton (E.). Candida can cause cutaneous infections but will be discussed later under Opportunistic Mycoses. A. Stratum corneum infections are characterized by location. Ringworm or tinea is another name for these cutaneous mycoses. The clinical syndrome includes scaling associated with pruritus. 1. Tinea corporis-body 2. Tinea cruris-groin (jock itch) 3. Tinea pedis-feet (athlete's foot)

meClical

343

Microbiology

4. Tinea manuum-hands 5. Tinea capitis-scalp B. Infections of the hair include tinea capitis (scalp) and tinea barbae (beard). These infections are commonly due to organisms from the genera Trichophyton or Microsporum. Ectothrix is when fungi are present outside of the hair shaft. Endothrix is the term to describe fungi within the hair shaft.

Clinical Correlate

C. Infections of the nail are primarily due to tinea unguium. Trichophyton rubrum and T. mentagrophytes are the two most common etiologic agents.

The major dermatophyte that spreads from person to person is M. audouinii. M. audouinii and M. canis are the two species that fluoresce in ultraviolet light.

D. Diagnosis is made by microscopic examination of skin scrapings using KOH, by fungal cultures, or by UV (Woods) lamp examination (to detect fluorescent tinea capitis caused by M. canis and M. audouinii). E. Treatment consists of topical imidazoles such as miconazole or dotrimazole (only for stratum corneum infections) or oral griseofulvin. Tolnaftate may also be used for skin infections.

SUBCUTANEOUS MYCOSES Subcutaneous mycoses typically result from implantation of the organism by means of some sort of trauma.

Note If a question mentions roses, there is a very good chance the answer is Sporothrix

schenckii.

A. Sporotrichosis is caused by the dimorphic saprophytic fungus, Sporothrix schenckii. Sporotrichosis has worldwide distribution with all ages and both sexes affected. S. schenckii is isolated from soil, living plants, or plant debris. It is classically associated with rose thorns and is often called "rose gardener'S disease:' 1. Clinical disease is generally limited to the skin and regional lymphatics (lymphocuta-

neous sporotrichosis). S. schenckii enters through skin breaks in the fingers or hands, causing a chancre, papule, or subcutaneous nodule with erythema and fluctuance. Nodular, ulcerating lesions appear along lymphatic channels, but the lymph nodes are not commonly infected. Culture of drainage or aspirated material for the presence of S. schenckii is usually diagnostic. 2. Treatment consists of potassium iodides for subcutaneous disease and amphotericin B for relapsing cutaneous disease as well as pulmonary and disseminated forms. B. Mycetoma (Madura foot, maduromycosis) is a local chronic progressive destruction of skin, subcutaneous tissue, fascia, muscle, and bone. 1. Transmission is by soil contamination of a wound. Lesions usually occur on the feet or

hands. 2. Infection results in a suppurative granuloma with multiple sinus tracts, and mycotic grains (granules) are extruded. 3. Mycetoma is caused by infection with several fungi (eumycotic mycetoma) or by higher bacteria (actinomycotic mycetoma). 4. Diagnosis is made by obtaining granules and staining with KOH for fungi and with Gram stain and modified acid fast for the presence of Nocardia organisms.

344

meClical

Mycology

SYSTEMIC MYCOSES

In a Nutshell

Systemic mycoses are also called deep mycoses. They can invade organs and are potentially lifethreatening.

Dimorphic Fungi

A. Histoplasmosis (Darling disease) is caused by Histoplasma capsulatum, which is found in

bird and bat droppings. Endemic areas in the central U.S. include the Ohio, Mississippi, and Missouri River valleys. 1. The diphasic pathogen H. capsulatum exists in soil in the mycelial phase and converts to

• Histoplasma • Blastomycoses • Coccidioides • Sporothrix

the yeast phase at 37.0°C, a. The mycelial form has septate branching hyphae-bearing spores at the lateral or terminal positions. b. Microconidia spores are the infectious particles. c. Macroconidia (8-14 mm) develop finger-like appendages over their surfaces (tuberculate macro conidia spores), which is a morphology used to identify this species. 2. Yeast forms are found within macrophages. Methenamine silver stains the cell membrane of this organism. 3. Transmission is mediated by airborne inhalation of microconidia spores that get deposited in alveoli and spread through lymphatics to the regional lymph nodes. Hematogenous spread may lead to metastatic foci of infection in the liver, spleen, etc. Human-to-human spread is infrequent. Within 7-21 days of primary exposure, cellmediated immunity develops with resulting granulomas at infected sites. 4. Clinical manifestations include acute and chronic pulmonary infections that very rarely progress to a disseminated histoplasmosis. (Ninety-five percent of infections are asymptomatic.) a. Acute pulmonary histoplasmosis arises 5-21 days after exposure and is expressed as headache and fever in most or all cases. (1) Chills, cough, and chest pain occur in two thirds of cases, with weakness, weight

loss, myalgia, and fatigue as less common symptoms. (2) Diagnosis is based on clinical suspicion and confirmed by culture and examination of sputum. Complement fixation tests are diagnostic. (3) No treatment is usually required since it is a benign and self-limited disease. b. Chronic pulmonary histoplasmosis occurs in patients with chronic obstructive lung disease. It begins as a benign segmental interstitial pneumonitis with 20% of cases progressing to a chronic cavitary disease. Cavitary disease is treated with a full course of amphotericin B. In some cases, surgical resection is necessary. c. Disseminated progressive histoplasmosis is uncommon, occurring in persons with deficient cell-mediated immunity, chronic underlying debilitating disease, or in patients undergoing corticosteroid or immunosuppressive therapy. (1) Clinical manifestations. A variety of clinical manifestations may arise, including systemic symptoms (fever, chills, anorexia, malaise, weight loss), hepatosplenomegaly (abnormal liver function tests), interstitial pneumonitis, and adrenal or renal involvement. (2) Diagnosis is confirmed by complement fixation and by cultures from blood, bone marrow, CSF, and urine. Tissue may be acquired for histopathology and culture of organisms. (3) Treatment consists of amphotericin B.

KAPLAIr . ._ I me lea

345

Microbiology

B. Coccidioidomycosis (San Joaquin fever; Valley fever) is due to infection with another dimorphic fungus, Coccidioides immitis. The organism is indigenous to deserts of the Southwestern U.S. and northern Mexico. C. immitis is inhaled as airborne arthroconidia (associated with fresh diggings, dust storms, etc.). Wind causes the spores to become airborne (infections increase during the dry dusty season). 1. C. immitis exists in soil in a mycelial phase. As it matures, alternate cells along a hypha become barrel-shaped (termed arthroconidia). These forms become airborne and are inhaled.

2. In the host, the spore swells up to 50 Il in diameter, becomes spherical (termed spherule), and reproduces to form internal spherical spores called endospores. Each endospore may form a new spherule or, if returned to soil, will revert to the mycelial form. 3. Preparations to identify organisms from tissues include KOH preparations, lactophenol cotton blue stain, and Gomori methenamine-silver stain. 4. Clinical manifestations arise as acute and/or potentially chronic and disseminated infection. a. Acute pneumonitis is subclinical in 60% of cases and is detected only by skin testing. 40% develop an influenza-like syndrome 7-28 days following exposure. (1) These symptoms include fever, malaise, dry cough, eosinophilia, toxic erythema (a fine, generalized erythematous macular rash), and erythema nodosum (particularly in females). (2) Diagnosis is confirmed by sputum culture. Skin tests are not helpful; however, serology for the presence of specific antibodies may be helpful. (3) Treatment is typically not required in adults since the pneumonitis will resolve spontaneously. Amphotericin B is given to infants, debilitated patients, or those at risk for dissemination; miconazole is given as an alternative. b. Disseminated coccidioidomycosis occurs in less than 1% of infected patients. ( 1) Patients at risk include leukopenic or immunosuppressed individuals, those with Hodgkin or AIDS, and certain racial/ethnic groups (African Americans, Filipinos). (2) Disseminated coccidioidomycosis may spread to any organ, causing a toxic state and high fever. (3) Treatment consists of amphotericin B. Fluconazole may also be effective. C. Blastomycosis is caused by Blastomyces dermatitidis, a dimorphic fungus growing as a mycelial form at room temperature and as a yeast form at 37.0°C.1t is endemic in states east of the Mississippi River. Conidia spores are thought to be infectious for humans, converting to the yeast form after inhalation into the lungs. Infections are limited to North America. Infection occurs in normal hosts, usually in occupations associated with soil contact. 1. Acute blastomycosis (pneumonitis) can be asymptomatic or a severe, often fatal, illness. The clinical presentation is marked by an influenza-like syndrome with fever, chills, productive cough, and pleuritic chest pain.

a. Diagnosis is made by sputum smear and cultures that are usually positive. Chest x-ray shows multiple parenchymal nodules or localized consolidation. b. Therapy may not be required since acute blastomycosis is usually a benign, self-limited disease.

346

meClical

Mycology

2. Chronic blastomycosis has a variable course of progressive illness. Lungs and skin are most commonly involved. Treatment consists of amphotericin B or ketoconazole combined with surgical excision or drainage of the local lesion. D. Paracoccidioidomycosis is sometimes called South American blastomycosis because It IS

endemic in Latin America (especially Brazil). In infected tissues, the yeast cells have multiple buds sprouting from a single parent cell, giving it a characteristic "pilot's wheel" appearance. Typically, the clinical manifestations are primarily pulmonary; often times it is asymptomatic.

OPPORTUNISTIC MYCOSES The opportunistic fungi are those that cause disease in immunocompromised patients (AIDS, chemotherapy, transplants). Unlike the systemic mycoses, these fungi are sometimes normal flora and are not limited to certain geographic regions.

In a Nutshell Fungal Disease Geography • Ohio, Mississippi, Missouri River valleys

~

Histoplasmosis

• Southwestern ~ Coccidioidomycosis deserts, California • States east of ~ Blastomycosis Mississippi River • Latin America

~

Paracoccidioidomycosis

A. Candida. Candidal infections may be cutaneous or systemic.

1. C. albicans is the major pathogenic species. 2. Candida are normal inhabitants of mucocutaneous body surfaces, soil, hospital environments, and some foods. C. albicans colonizes normal skin and most diseases are endogenous in origin. Occasional person-to-person spread occurs (e.g., newborn thrush). 3. Invasive disease results from host alterations, leading to a change in the commensal status of the organism. Factors important in the invasiveness of Candida include predisposing illnesses (such as diabetes mellitus), damaged mucosal surfaces (such as those caused by indwelling catheters), depression of the immune status (as in immunocompromised individuals), and the use of steroids or antibiotics. 4. Clinical manifestations depend on the site of infection. a. Oropharyngeal infection (includes thrush) is observed as discrete or confluent white patches on the tongue and buccal mucosa. Microscopic examination of scraping reveals pseudohyphae. Therapy consists of nystatin suspension, oral ketoconazole, or mycelex troches. b. Vaginal infection is frequently seen in diabetes mellitus, antibiotic therapy, and in pregnancy. It is associated with thick yellow-white discharge and intense pruritus. Nystatin suppositories are beneficial. Imidazole drugs (topical or oral) are also therapeutic. c. Gastrointestinal colonization causes disease in malnourished or immunocompromised persons, or in persons undergoing prolonged intra-abdominal surgical procedures. Clinical presentation may include diffuse ulcerative and erosive esophagitis, gastritis, or multiple superficial ulcerations of the small and large intestine. Stool and throat cultures may not be diagnostic because of frequent colonization by Candida. Therapy consists of nystatin, ketoconazole, and low-dose amphotericin B. d. Invasive, disseminated infection occurs, particularly in patients with leukemia, lymphoma, and AIDS. The gastrointestinal tract is probably the most common portal of entry. Symptoms include fever, shock, hypotension, and prostration. Renal infection occurs usually from hematogenous spread and may result in renal failure. Endocarditis occurs in drug abusers. e. Chronic mucocutaneous candidiasis represents extensive cutaneous disease that is refractory to treatment and can be disfiguring. 5. Diagnosis is made by demonstration of fungal pseudohyphae in the tissue or by culture and biochemical identification (e.g., urease positive).

metlical

347

Microbiology

6. Treatment. Systemic treatment includes ketoconazole or fluconazole; amphotericin B is used less frequently. Flucytosine is also valuable in systemic disease. Cutaneous involvement usually responds to topical miconazole. B. Cryptococcosis is due to infection with Cryptococcus neoformans. C. neoformans is an encapsulated yeast that reproduces by budding. C. neoformans is identifiable on Wright stain or India ink (especially helpful due to presence of capsule). C. neoformans is found worldwide in avian feces (particularly pigeon droppings), in soil, fruits, milk, and wood products. Immunosuppression due to malignancy or AIDS predisposes to this disease. 1. Pulmonary disease is common since this is the primary portal of entry. Disease is usually

transient and not severe if the patient is otherwise healthy. 2. Disseminated disease arises most often in immunocompromised individuals.

Note (ryptococcal meningitis has been a Boards' favorite. Think cryptococcus if: • Immunocompromised patient • Meningeal signs • Encapsulated organisms on India ink stain of (SF

a. Central nervous system involvement includes meningitis or lesions that occupy the cerebral white and gray matter space. Diagnosis is made by India ink stain. CSF cryptococcal antigen is found in the CSF and is also diagnostic. b. Treatment consists of amphotericin B alone or in combination with 5-fluorocytosine. During treatment, CSF must be monitored at weekly intervals by culture, India ink, cell count, glucose, and protein. CSF must be monitored every 3-4 weeks for antigen titer. C. Aspergillosis is a disease arising from several species of ubiquitous molds. A. fumigatus is the most common species. Organisms are normal inhabitants of the soil, and spores are readily disseminated in the air.

1. Allergic bronchopulmonary aspergillosis is marked by a hypersensitivity reaction to the fungal antigens. Inhalation of conidia or mycelial fragments may elicit an IgE-mediated hypersensitivity reaction causing bronchospasm. a. Clinical manifestations include episodic wheezing, fixed or transient pulmonary infiltrates, fever, and peripheral eosinophilia. Elevated IgE is usually seen. Positive· skin test to Aspergillus antigenic extract and positive sputum cultures are found in most infectious states. b. Treatment. No therapy is required if the disease is mild. Corticosteroids, however, are helpful in reducing symptoms. 2. Aspergillomas (fungus balls) are the result of colonization of pulmonary cavities (usually secondary to tuberculosis or sarcoidosis). Patients may be asymptomatic, but hemoptysis occurs in the majority of cases. Diagnosis is confirmed by chest x-ray, sputum culture, and/or positive serum precipitin. Surgery is indicated for massive hemoptysis. 3. Invasive aspergillosis usually occurs as an opportunistic infection in immunocompromised patients, with iatrogenic neutropenia. a. Pulmonary involvement is present in 90-95% of cases. Invasive aspergillosis presents as an unremitting fever and pulmonary infiltrate despite therapy with broad-spectrum antibiotics. Necrotizing bronchopneumonia is common. b. Extrapulmonary dissemination to the esophagus, brain, or GI tract (with GI bleeding) occurs in about 25% of cases. c. Diagnosis must demonstrate tissue invasion. Blood cultures are typically negative, whereas sputum culture is positive in only 10% of cases. Serologic diagnosis by serum precipitins (CIE), ELISA, or passive hemagglutination assays may by confirmatory if seroconversion occurs. Chest x-ray and lung biopsy may be necessary to make the definitive diagnosis. Septate branching hyphae will be seen in biopsy specimens.

348

meCtical

Mycology

d. Treatment. Surgical removal of the aspergilloma may be necessary. Amphotericin B is the therapeutic standard, although itraconazole may be a valuable alternative. D. Zygomycosis (mucormycosis) is most often caused by organisms from the genera Rhizopus and Mucor. These molds are ubiquitous on decaying vegetable matter in soil. They have nonseptate hyphae. 1. Individuals predisposed to invasive zygomycosis disease include those with diabetic

ketoacidosis, leukemia, and lymphoma, antibiotic and steroid use, and infants and children with malnutrition. 2. Clinical manifestations a. Rhinocerebral disease is the most common presentation. It typically occurs in diabetics with ketoacidosis and is an infection of the nasal mucosa, palate, sinuses, and/or orbit, whereby progressive neurologic deficits ensue as the organism invades to the base of the brain. b. Pulmonary disease is usually the consequence of inhalation of spores in a patient with leukemia or lymphoma. 3. Treatment involves control of underlying disease such as diabetes, surgical debridement, and amphotericin B.

In a Nutshell Fungi Forms In Vivo - Coccidioides

---7

spherules

-Histoplasma

---7

intracellular yeast

-Blastomyces buds

---7

broad-based

-Cryptococcus -Candida

---7

large capsule

---7

pseudo hyphae

-Aspergillus

---7

branching septate hyphae

-Mucor! Rhizopus

---7

nonseptate hyphae

E. Pneumocystis jiroveci (formerly carinii) was originally thought to be a protozoa because of its morphologic stages and sensitivity to antiprotozoal drugs. However, rRNA homologies suggest that it is a fungus. 1. Clinical manifestations. In individuals with normal cell-mediated immunity, infection is

asymptomatic. Defects in cell-mediated immunity, such as AIDS, cause trophozoites to invade the alveoli and cause interstitial pneumonia. 2. Diagnosis is made by the demonstration of organisms in endobronchial biopsy or bronchial lavage with methenamine-silver or immunofluorescent stain. 3. Treatment for pneumonia consists of trimethoprim-sulfamethoxazole or pentamidine. Steroids are indicated for severe pneumonitis. Prophylactic and suppressive therapy consists of trimethoprimsulfamethoxazole, with dapsone or pentamidine as a second choice.

meCtical

349

Protozoa

Protozoa are unicellular eukaryotic organisms. They have a true nucleus, mUltiple chromosomes, and organelles. All protozoa have endoplasmic reticulum and Iysosomes, whereas some have mitochondria and Golgi apparatus. They usually reproduce asexually in the human host.

INTRODUCTION Protozoa are classified into four groups.

Note Table V-13-l. Protozoa. Classification

Common Name

Examples

Sarcodina

Amoeba

Acanthamoeba, Entamoeba, Naegleria

Sporozoa

Sporozoans

Babesia, Cryptosporidium, Isospora, Plasmodium, Pneumocystis, Toxoplasma

Mastigophora

Flagellates

Dientamoeba, Giardia, Leishmania, Trichomonas, Trypanosoma

Ciliata

Ciliates

Balantidium

Although it behaves like a protozoa, Pneumocystis is now classified as a fungus. It was discussed in Mycology, the previous chapter.

Note

INTESTINAL AND MUCOCUTANEOUS PROTOZOA Intestinal and mucocutaneous protozoa include Giardia lamblia, Entamoeba histolytica, Trichomonas vaginalis, Isospora belli, and Cryptosporidium parvum. Of these organisms, Giardia lamblia and Entamoeba histolytica have the simplest life cycles. The environmental form is called the cyst. Transmission of these organisms is fecal-oral, and results from the ingestion of contaminated water or food. Once in the intestine, the organisms excyst and the intestine is colonized by the motile, feeding trophozoite. This form is responsible for the pathology associated with disease. Trichomonas vaginalis is a sexually transmitted flagellated organism that undergoes binary fission. It has no cyst stage. Isospora belli and Cryptosporidium parvum both have complex life cycles that include sexual and asexual stages. A. Giardia lamblia is the infectious agent of giardiasis, acquired by the ingestion of fecalcontaminated water or food. 1. Clinical manifestations. Disease is caused by trophozoite attachment to the wall of the

small intestine.

Most lumen dwelling parasites infect the gastrointestinal tract and cause diarrhea. Blood and tissue protozoa cause a variety of symptoms and may be fatal.

Note You should suspect Giardia in campers or hikers who present with diarrhea, bloating, flatulence, etc. KAPLA~.

meulCa

I

351

Microbiology

a. Manifestation of G. lamblia colonization ranges from asymptomatic infection to inflammation with diarrhea, cramps, bloating, flatulence, malaise, weight loss, and occasional steatorrhea from impaired fat absorption.

Note

b. Disease usually resolves within 3-4 weeks, although a minority of patients may have a subclinical course and remain chronic carriers.

Giardia trophozoites

c. Infection is usually more severe in children and in immunosuppressed adults.

have a characteristic "face-like" appearance.

2. Diagnosis is confirmed by the presence of cysts in formed stools of the patient. Trophozoites appear in diarrheal stools. Trophozoites from jejunal biopsy or duodenal secretions are usually superior for diagnosis. 3. Treatment consists of metronidazole or furazolidone.

B. Entamoeba histolytica is the etiologic agent for amebiasis. E. histolytica is spread by the fecal-oral route and is more common in areas with poor sanitation.

Clinical Correlate The liver abscess is characterized by hepatomegaly, right upper quadrant pain, fever, and weight loss.

1. Clinical manifestations. The organisms infect the colon of humans, invading and lysing intestinal epithelium with subsequent ulceration. a. Clinical disease may be mild with diarrhea, abdominal cramps, nausea, vomiting, and flatulence. b. More severe (invasive) disease results in dysentery, severe abdominal pain, dehydration, and bloody stools. c. Severe infection can also result in perforation of the bowel wall, resultant peritonitis, and liver abscess formation.

2. Diagnosis is made by the presence of E. histolytica cysts with four or less nuclei in stool specimens; E. coli has up to eight nuclei. Colonoscopy reveals punctate hemorrhagic areas on colonic mucosa rich with E. histolytica. Serologic methods are useful in invasive intestinal disease and in extraintestinal disease. 3. Treatment. Symptomatic amebiasis is treated with metronidazole (Flagyl) followed by iodoguinol. C. Trichomonas vaginalis causes vaginitis in women. Infection in men is frequently asymp-

tomatic but may lead to prostatitis or urethritis. 1. Clinical manifestations in women are dependent upon the physiologic status of vaginal

pH, flora, and the intensity of the infection. Symptoms include dyspareunia, dysuria, pruritis, and a copious discharge that is yellow and frothy. Symptoms worsen when vaginal pH is more alkaline. 2. Diagnosis is made by the identification of motile organisms from wet preparation of genital secretions. 3. Treatment consists of metronidazole for both the patient and her partner. D. Isospora belli has a complex life cycle. Oocysts are ingested from human fecally contaminated water or food. In the duodenum and small intestine, sporozoites are released that invade epithelial cells and multiply asexually to produce merozoites. Merozoites can reinfect the small intestine or undergo sexual differentiation into macrogametocytes and microgametocytes. Fertilization leads to the formation of oocysts, which are expelled in feces. 1. Clinical manifestations. Isospora may also be isolated in the stools of asymptomatic persons. Clinical disease is usually self-limiting after 1-2 weeks in an immunocompetent

host. Symptoms include abdominal pain, low-grade fever, diarrhea, and malabsorption. Increasing incidence of symptomatic infection with Isospora belli is seen in AIDS patients.

352

meCtical

Protozoa

2. Diagnosis is made by the identification of oocysts shed in stool. 3. Treatment consists of folate antagonists-trimethoprim-sulfamethoxazole or pyrimethamine and folinic acid. Immunocompromised patients require maintenance therapy. E. Cryptosporidium parvum is acquired by ingestion of oocysts. The oocysts release sporozoites that invade epithelial cells and multiply asexually to produce merozoites. Merozoites may reinfect cells in the jejunum and ileum and a sexual process results in the formation of oocysts, which are found in the stool. 1. Clinical manifestations. Cryptosporidium is increasingly recognized as a cause of traveler's and seasonal diarrhea. Disease presents with watery diarrhea, anorexia, and weight loss that clears in 10 days to 2 weeks. However, in immunodeficient patients, especially those with AIDS, it can be a life-threatening chronic disease. 2. Diagnosis is made by finding the oocyst in stools using acid fast stain, or through antigen detection.

Note On the exam, you are most likely to see Isospora and Cryptosporidium as causes of diarrhea in AIDS patients.

3. Treatment. Currently, no effective treatment exists for Cryptosporidium.

BLOOD AND TISSUE PROTOZOA Blood and tissue protozoa include Plasmodium species, Babesia species, Leishmania species, Trypanosoma species, and Toxoplasma gondii. A. Plasmodium species are intracellular parasites with a complex life cycle. The sexual phase occurs in the Anopheles mosquito and is transmitted to humans by the bite of the female; the asexual phase occurs in humans. Malaria affects 200 to 400 million people per year and kills approximately 1% of them. It is mainly a tropical disease. However, in the United States, it is the most common imported acute febrile illness. 1. Malaria in humans is caused by one of four species of Plasmodium.

a. Plasmodium falciparum causes malignant tertian malaria. Plasmodium falciparum causes the most deaths from malaria. It can infect all ages of red blood cells, leading to a higher level of parasitemia. Also, infected erythrocytes adhere to vascular endothelium, clogging the capillaries. This leads to thrombosis and tissue ischemia. (Fever spikes are irregular.) b. Plasmodium vivax causes benign tertian malaria. It is found widely distributed in the tropics and some temperate areas. There are 48-hour fever spikes. (1) The form acquired from the mosquito, the sporozoite, can remain dormant in the liver for extended periods. (2) Treatment requires both a blood schizonticide and a tissue schizonticide to eradicate the exoerythrocytic stage. (3) The form released from liver cells, the merozoite, uses the Duffy blood group antigen to enter reticulocytes.

Note P. vivax infects only young RBCs (reticulocytes), whereas

P. malariae infects only old erythrocytes. P. falciparum species are less discriminating and infect RBCs of all ages.

c. Plasmodium ovale causes benign tertian malaria, and is found only in West Africa. It can infect populations that are Duffy negative because its merozoites use a different receptor. Like Plasmodium vivax, it may remain dormant in the liver as well. This necessitates treatment with both tissue and blood schizonticides. There are 48-hour fever spikes.

d. Plasmodium malariae has rare focal distribution in the tropics. It causes quartan malaria, and without treatment can persist for decades, although the primary attack

iiiectical

353

Microbiology

In a Nutshell

resolves in 3 weeks to six months. It invades old erythrocytes only. There are 72-hour fever spikes.

Plasmodium life Cycle Infected mosquito injects sporozoites

~

Sporozoites taken up by hepatocytes

+

Merozoites form and infect RBCs

/\

Form gametocytes

Schizonts develop into merozoites

Taken up by mosquito where fertilized to form zygote

1

a. An infected mosquito bites a human, taking a blood meal and injecting sporozoites from its salivary gland into the blood of the host. b. Sporozoites travel through the blood to the liver and multiply asexually in hepatic cells to form merozoites (exoerythrocytic stage). Upon the rupture of infected liver cells, organisms are released into the bloodstream to infect erythrocytes. ( 1) P. ovale and P. vivax may persist in the liver. (2) They may remain quiescent in the liver for up to 5 years, converting to merozoites, and can then invade RBCs and cause an acute malaria attack.

Form trophozoites that become schizonts

Merozoites are released and infect other RBCs

2. The life cycle involves a sexual stage (sporogony) in the mosquito and an asexual stage (schizogony) in humans.

1

+

Form haploid sporozoites that can migrate to salivary glands and are injected during blood meal

(3) The incubation period is about two erythrocytic cycles and is dependent upon the malaria type. c. Merozoites enter RBCs and develop into ring trophozoites. The ring trophozoites further develop into amoeboid trophozoites that become schizonts. d. Schizonts contain merozoite daughter cells. Release of the merozoites leads to periodic paroxysms of disease due to the resultant parasitemia. e. Some merozoites develop into macrogametocytes (females) and microgametocytes (males). The mosquito ingests gametocytes with a blood meal from the infected host. f. Gametocytes enter the mosquito gut, where the male initiates exflagellation followed by fertilization. The zygote invades the gut wall of the mosquito to form an oocyst within 24 hours following ingestion. Sporozoites are formed and then released into the stomach. Sporozoites then migrate to salivary glands and are injected into the human during a blood meal. 3. Clinical manifestations. All species can cause anemia, dehydration, electrolyte abnormalities, splenomegaly, and acute splenic rupture. a. Periodic fever and chills arise and last up to one hour followed by diaphoresis. Nausea, anorexia, vomiting, and malaise are clinical complications that follow. b. P. falciparum causes the greatest morbidity and mortality due to the greater degree of parasitemia and adherence to vascular endothelium. Complications can include the following: (1) Blackwater fever characterized by intravascular hemolysis, icterus, hemoglobinuria, and acute renal failure.

Note

(2) Cerebral malaria characterized by headache, confusion, rapid coma, and convulsions. Mortality from cerebral malaria is between 20 and 30%.

The gametocytes of P. falciparum have a

characteristic "banana-shaped" appearance; other species have spherical gametocytes.

4. Diagnosis is made by blood smears stained with Giemsa. Early treatment is critical and depends on the species. 5. Treatment depends on the stage of the illness and the infecting organism. a. Chloroquine is the treatment of choice unless the infecting organism is chloroquineresistant P. falciparum. b. Alternate drugs for chloroquine-resistant P. falciparum include quinine, quinidine, mefloquine, pyrimethamine, and artemisinin. Tetracycline is usually added to the above drugs.

354

meClical

Protozoa

c. Primaquine is a tissue schizonticide required to eliminate tissue merozoites in Plasmodium vivax and Plasmodium ovale infection. Ingestion by individuals with a glucose-6-phosphate dehydrogenase (G6PD) deficiency leads to hemolysis. 6. Risk factors. Genetic factors influence host susceptibility to malarial infection. a. In West Africa, heterozygotes for the sickle cell trait have a selective advantage because the growth of the trophozoite is inhibited by low oxygen tension. b. Individuals lacking the Duffy blood group antigen are not susceptible to Plasmodium vivax infection. c. Glucose-6-phosphate dehydrogenase (G6PD) deficiency increases resistance to Plasmodium falciparum infection. B.

Babesia microti is an erythrocytic protozoan that is responsible for the human disease babesiosis.

Note If you see "Nantucket," think babesiosis.

1. Transmission and epidemiology. Babesia microti is transmitted via Ixodes dammini, the

same tick vector as in Lyme disease, so coinfections are likely. Babesia primarily infects animals; humans are incidental hosts. The species has a rodent reservoir. Babesiosis is common in the Northeastern United States, especially on islands off the northeast coast (e.g., Nantucket). Babesiosis causes severe disease or death if the patient is asplenic. 2. Clinical manifestations. B. microti elicits symptoms 1 to 3 weeks after the tick bite. Typical symptoms include malaise, fatigue, fever, generalized myalgia, nausea, sweats, arthralgia, and possibly depression. Symptoms may wax and wane for several weeks and may be mistaken for malaria. Travel history may be important diagnostically. 3. Diagnosis is made by an appropriate clinical history and by analysis of blood smears for the presence of Babesia within erythrocytes. The life cycle occurs exclusively in red blood cells, and there are no schizonts. Immunoblot of anti-Babesia serum antibodies is also diagnostic. 4. Treatment. B. microti infection is usually self-limited. Therefore, therapy is not indicated except for symptom abatement (e.g., aspirin given for fever reduction). When infection is severe or the patient is known to be asplenic, the drugs of choice are clindamycin and quinine.

In a Nutshell Review of the Tick-Borne Diseases • Babesiosis (Babesia) • Lyme disease (Borrelia) • Endemic relapsing fever (Borrelia) • Rocky Mountain spotted fever (Rickettsia) • Ehrlichosis (Ehrlichia) • Tularemia (Froncisella tularensis)

C. Leishmania species cause zoonotic infections that are transmitted by the bite of the phle-

botomine sandfly. Leishmaniasis occurs primarily in India, Africa (Old World), and Central and South America (New World). The parasite invades the host's reticuloendothelial cells and resides in the phagolysosomes. 1. Clinical disease may be expressed as cutaneous, mucocutaneous, or visceral forms.

a. Cutaneous disease is usually self-limited. Infectious species for cutaneous disease include L. mexicana, L. tropica, L. major, and L. braziliensis. b. Mucocutaneous disease (espundia) is a chronic syndrome that attacks the nasal cartilage. Mucocutaneous disease is difficult to treat and is caused by L. braziliensis. This infection may lead to death from asphyxiation or superinfection. c. Visceral disease (kala azar) principally affects the liver, spleen, lymph nodes, bone marrow, and entire reticuloendothelial system. L. donovani is the etiologic agent. 2. Diagnosis is made by clinical findings, finding parasites in slit-skin smears taken from nonulcerated parts of a cutaneous lesion, or in the case of visceral leishmaniasis, by isolation of L. donovani from biopsy samples from spleen, bone marrow, liver, or lymph nodes.

meClical

355

Microbiology

3. Treatment of cutaneous leishmaniasis depends upon the location and extent of the lesion. Pentavalent antimonials can be used to treat large, disfiguring lesions. Visceral and mucocutaneous leishmaniasis are treated with the pentavalent antimonials sodium stibogluconate (Pentostam) and meglumine antimoniate (Glucantime), amphotericin B, or pentamidine. D. Trypanosoma is the genus of protozoan responsible for African and American trypanosomiasis. 1. African trypanosomiasis, African sleeping sickness, is caused by Trypanosoma brucei gambiense in Western and Central Africa and by Trypanosoma brucei rhodiense in Eastern Africa. Both species are transmitted by the bite of an infected tsetse fly, and lead to CNS involvement and death if not treated. a. Clinical manifestions (1) Disease is first expressed as a chancre appearing at the site of inoculation. (2) Parasitemia arises 2-3 weeks later, invading the lymphoid-macrophage system and leading to fever, rash, headache, lymphadenopathy, and mental status changes. (3) Once the organism spreads to the CNS, the disease progresses with anorexia, lassitude, fatigue, wasting, and eventually stupor, coma, and death. b. Diagnosis. African sleeping sickness is diagnosed by a combination of history and clinical findings. Giemsa-stained smears of peripheral blood can detect parasites, especially in the hemolymphatic stage. The CSF should be examined for abnormalities involving cell count and protein concentration. Sediment from the centrifuged CSF can yield organisms in individuals with CNS involvement. c. Treatment. Infection is difficult to cure. Suramin or pentamidine are the drugs of choice for treatment of patients with hemolymphatic disease, but with normal CSF. The drug of choice for disease with CNS involvement is melarsoprol. 2. Chagas disease, or American trypanosomiasis, is caused Trypansoma cruzi. It is a zoonotic infection transmitted by the reduviid bug or "kissing bug." The organism enters through the mucous membranes or breaks in the skin.

Clinical Correlate Worldwide, Chagas disease is one of the most common causes of heart disease.

a. Clinical disease is caused by the invasion of the lymphoid-macrophage system, endocrine glands, myocardium, and neural tissue. b. Diagnosis of Chagas disease is made by peripheral blood smear demonstrating the presence of trypomastigotes. Xenodiagnosis is sometimes used, where laboratory triatomine bugs are first allowed to feed on the patient. The stools of the bugs are then examined for the presence of parasites. c. Treatment of acute disease consists of nifurtimox, a derivative of nitrofuran. No effective therapy exists for chronic disease.

E. Toxoplasma gondii, the etiologic agent of toxoplasmosis, occurs worldwide. 1. There are three forms of the organism: the trophozoite, which is the invasive form; the tissue cyst, which contains intracystic organisms; and the oocyst in which the sporozoites are formed.

2. Transmission to humans usually occurs via secondary hosts. a. The organism undergoes the sexual cycle in the intestine of cats, the definitive host, to form oocysts that are passed in the feces. b. Ingested oocysts invade the intestine of intermediate hosts and disseminate hematogenously to form pseudocysts in tissue.

356

meClical

Protozoa

c. Cysts can occur in all tissues, including muscle, brain, and eye.

d. Tissue cysts can then be ingested by humans in raw or undercooked meat from intermediate hosts.

Note

e. Alternatively, infection can be acquired from ingestion of the oocysts in cat feces.

If you see an HIVjAIDS patient with "ring enhancing lesions" on MRI, think toxoplasmosis. (fhese same lesions in a 72-year-old smoker, think metastatic lung cancer.)

3. Clinical manifestations. Intact cell-mediated immunity usually restricts infection to the asymptomatic pseudocyst stage in adults. a. Primary infection may be associated with a mild mononucleosis-like illness. b. In immunodeficient patients (AIDS, treated cancer patients), disease usually arises as an acute infection originating from a chronic, quiescent infection that is activated due to altered immunity. CNS disease may be expressed as a meningoencephalitis or as a mass lesion with seizures. c. Congenital infection may result if the mother acquires primary infection during early pregnancy. Chorioretinitis, diffuse intracranial calcifications, hydrocephaly, anemia, and seizures are associated with congenital disease. 4. Diagnosis is confirmed by the observation of trophozoites in tissue by Giemsa stain. Serology of blood and CSF is also diagnostic. S. Treatment consists of pyrimethamine and sulfadiazine (if the disease is severe) or spiramycin or clindamycin.

Note Toxoplasma is the 'T' in the ToRCHeS acronym for congenital infections: • Toxoplasmosis • Rubella • CMV • HSV, HIV • Syphilis

KAPLAlf I medlea 357

Helminths

Helminths, or worms, are eukaryotic, multicellular organisms. Most do not multiply in their host. This characteristic affects the disease state in the host. If a host has a few worms, there is little disease. In the small proportion that have a high worm burden, there is severe disease. Parasitic helminths can be divided into two phyla: Platyhelminthes, the flatworms, and Nemathelminthes, the roundworms.

PLATYHELMINTHES Flatworms are dorsoventrally flattened, and have no body cavity. Plathyhelminthes share several features. All are hermaphrodites except for schistosomes. They have complex life cycles with development in different hosts. Finally, severe disease occurs only in a small percent of infected patients with large worm burdens or forms of the parasite in ectopic sites. Plathyhelminthes can be subdivided into cestodes (tapeworms) and trematodes (flukes). A. Cestodes (tapeworms) are long worms composed of strobila (ribbons of composed segments). The scolex (head) with sucking disks allows the worm to burrow into the intestinal wall of the definitive host. Posterior to the scolex are multiple segments called proglottids, each with both male and female reproductive organs. Each proglottid will develop into a sack of eggs that eventually breaks off from the main worm and opens, releasing eggs that will be passed in the feces of the definitive host. Humans may serve as either a definitive or intermediate host. A definitive host harbors the adult form of the worm. An intermediate host does not support the adult form. When an intermediate host is infected fecal-orally with cestode eggs, the larvae that hatch will migrate to various tissues and encyst. This can result in severe disease.

Note In the U.s., worm diseases are rare. On the USMLE, worm questions are equally rare. So, don't spend too much time on this chapter! Enterobius vermiculoris is the most common helminth infection in the United States.

1. Taenia saginata (beef tapeworm) and Taenia solium (pork tapeworm) are transmitted

to humans by the ingestion of larvae in poorly cooked beef and pork, respectively. Adults attach and live in the small intestine. Infection is usually asymptomatic or produces mild symptoms. a. Clinical manifestations. Severe infection is marked by abdominal pain, nausea, diarrhea, and weight loss. Coiled organisms may obstruct the appendix, pancreatic duct, or biliary duct. b. Diagnosis is made by the identification of eggs or proglottids in the stool. c. Treatment consists of niclosamide or praziquantel.

2. Cysticercosis is due to infection with the eggs of Taenia solium, most likely acquired from other humans. a. Clinical manifestations. When humans act as an intermediate host for Taenia soiium, severe disease often results.

mellical

359

Miaobiology

(1) Larvae penetrate the intestine to the circulation and form cysts anywhere in the body, especially the brain.

(2) The encysted form, called the cysticercus, is viable for up to 5 years. When alive, cysticerci cause virtually no inflammatory response, but may compress adjacent structures, causing symptoms especially in the eye or the base of the brain. (3) When they degenerate, a granulomatous reaction develops, and there is calcification of the organism that can be seen on x-ray. (4) Symptoms depend on the number and location of the cysts, and may not appear until years after infection. CNS symptoms include vomiting, headache, visual changes, seizures, and psychiatric disorders. b. Diagnosis of cysticercosis is through biopsy of subcutaneous nodules, brain biopsy, serology, and characteristic CT scan. c. Treatment. Symptomatic CNS disease is treated with praziquantel and steroids. 3. Diphyllobothrium latum (fish tapeworm) is transmitted by ingestion of the larvae in freshwater fish. D. latum is found primarily in the Great Lakes area and Scandinavia.

Note

a. Clinical manifestations. It is usually asymptomatic, but can cause diarrhea and vitamin BI2 deficiency with megaloblastic anemia.

Niclosamide has traditionally been the drug of choice in treating cestodes. However, praziquantel is being used with increasing frequency.

b. Diagnosis is made by the presence of eggs or proglottids in the feces. c. Treatment consists of nic10samide or praziquantel. 4. Echinococcus granulosus infection results when humans inadvertently ingest the eggs of the canine tapeworm.

a. Clinical manifestations occur when the ingested eggs hatch and the larvae migrate to multiple tissues, forming cysts. Cysts are usually asymptomatic but can enlarge and cause mechanical problems. Sixty percent of cysts occur in the liver. b. Diagnosis is made from characteristic x-ray or serology. c. Treatment consists of surgical removal. Care must be taken not to burst the cyst because anaphylaxis can result.

B. Trematodes (flukes) are flatworms that are not segmented. They have a primitive digestive tract and often have suckers for attachment to the host. Flukes are divided into two categories: tissue flukes and blood flukes. 1. Tissue flukes. Tissue flukes are leaf-shaped and have at least two intermediate hosts. The

first host of a tissue fluke is a snail. Infection is acquired by ingestion of immature forms. a. Clonorchis sinensis, the liver fluke, is endemic in Asia. Following ingestion of infected fish, the organism excysts in the duodenum and journeys to the bile ducts. (1) Clinical manifestations. Acute infection produces epigastric pain. Chronic

infection predisposes the host to cholangitis and cholangiocarcinoma. (2) Diagnosis is made by finding the characteristic eggs in the stool. (3) Treatment consists of praziquantel. b. Paragonimus westermani, the lung fluke, is transmitted by the ingestion of cysts in crabs or crayfish. The lung fluke is primarily found in Asia, South Central America, Africa, and India. When ingested, the larvae excyst in the small intestine and pass through the diaphragm into the lung.

360

mdical

Helminths

(1) Clinical manifestations. During migration, cough and abdominal pain may be experienced. Chronic infection is characterized by lung abscesses with hemoptysis. Migration to the brain may induce vomiting and seizures. (2) Diagnosis is made by finding eggs in the sputum or in feces. (3) Treatment consists of praziquantel. 2. Blood flukes. Blood flukes are composed of Schistosoma species. Infection is caused by the penetration of intact skin by a larval form. a. Schistosoma species. Over 200 million people are infected worldwide; 5 to 10% of these are symptomatic. Three species of schistosomes infect humans: Schistosoma mansoni, Schistosoma japonicum, and Schistosoma haematobium. All schistosomes require a developmental cycle in a snail. Humans become infected when they come in contact with contaminated water. (1) Transmission occurs when a form called the cercariae penetrates the skin. The organism migrates through lymphatic or venous channels until adult worms lodge in the portal (Schistosoma mansoni and Schistosoma japonicum) or pelvic veins (S. haematobium). Eggs secrete enzymes to work their way out of blood vessels and into the lumen of the bladder or colon. (2) Clinical manifestations. Acute schistosomiasis due to S. mansoni causes fever, anorexia, weight loss, abdominal pain, and lymphadenopathy. Chronic disease due to S. mansoni is characterized by portal hypertension, splenomegaly, and variceal bleeding. After a heavy infection with S. haematobium, the host develops hematuria. S. haematobium infection has been linked to bladder cancer. (3) Diagnosis is made by finding eggs in the urine (S. haematobium) or stool (other species). (4) Treatment consists of praziquantel.

In a Nutshell Platyhelminthes (Ratworms)

I Cestodes (Tapeworms) Segmented

~

'\ Trematodes (Flukes) Nonsegmented

~

• Taenia saginata Tissue flukes • Clonorchis • Taenia so!ium sinensis • Diphyllobothrium • Paragonimus tatum westermani • Echinococcus Blood flukes • Schistosoma species

C. Nematodes are roundworms that are not segmented and have an outer proteinaceous cuti-

cle. Parasitic nematodes may be grouped into the intestinal nematodes and filarial worms. Diagnosis of nematode infection is usually made by visualizing the characteristic eggs in the stool. Treatment is usually mebendazole. 1. Intestinal nematodes a. Ascaris lumbricoides, the cause of ascariasis, infects up to 1 billion people worldwide. Infection is spread by ingestion of infective eggs from contaminated soil or food. Larvae are released in the intestine and penetrate intestinal blood vessels to the lung, where they move through alveolar capillaries and are coughed up and swallowed. They mature in the small intestine, where the adult can live for up to 2 years. Eggs are passed in the feces and need 10 to 15 days in moist soil or water to develop into the infective form. Eggs are very resistant to the environment and can survive up to 7 years. (1) Clinical manifestations. Eighty-five percent of disease is asymptomatic. (a) During the migration of larvae through the lungs, Ascaris can cause pneumonia with eosinophilia. (b) Intestinal manifestations range from vague abdominal pain to obstruction. (2) Diagnosis is made by finding eggs in the stool. (3) Treatment consists of pyrantel pamoate or mebendazole.

KAPLAlf _ I medlea

361

Microbiology

b. Trichuris trichiura (whipworm). Humans are the principal host, and infection is spread by the ingestion of embryonated ova. Larvae hatch in the small bowel, penetrate the villi, then move into the lumen of the cecum. (1) Clinical manifestations. In very heavy infection, usually in children, the mucosa becomes edematous and friable. Diarrhea and even rectal prolapse may develop. (2) Diagnosis is made by finding the characteristic eggs in the stooL (3) Treatment consists of mebendazole. c. Ancylostoma duodenale, the Old World hookworm, and Necator american us, the New World hookworm, infect almost one quarter of the world's population. Eggs from an infected individual hatch in soil or water and become free-living larvae. Within 1 week, they molt and become infective larvae that can survive for up to one year in soil. Infective larvae penetrate the skin and travel to the lungs via the blood. Once in the lungs they are coughed up and swallowed and establish residence in the small intestine. There, they can survive for 2 to 10 years.

(1) Clinical manifestations. Adult worms receive 25 to 30% of their nutrition from blood. This can lead to anemia, depending on the infecting species and the number of organisms. Pruritis can occur at the site of penetration. During migration through the lungs, hookworms can cause pneumonia with eosinophilia. (2) Diagnosis is made by finding the characteristic eggs in the stooL (3) Treatment consists of mebendazole. d. Enterobius vermicularis, the pinworm, causes infections that are equally prevalent in developed and developing countries. Humans are the only known host. Transmission is through ingestion of an infective egg that hatches and develops into an adult. Gravid females migrate at night to the perianal or perineal regions and deposit eggs. (1) Clinical manifestations. In sensitized individuals, the outer layer of the egg can cause severe perianal and perineal pruritis. (2) Diagnosis is made by finding the characteristic eggs on cellophane tape ("Scotch tape" technique). (3) Treatment consists of a single dose ofmebendazole. e. Strongyloides stercoralis, the threadworm, has a more complex life cycle than the other nematodes. Eggs hatch before leaving the intestine, so larvae are passed in feces. In soil, they develop into the infective stage. Infection occurs when larvae penetrate the skin and migrate to the intestine via the lungs. Unlike all other nematodes, autoinfection is possible. The change to the infective form can occur in the intestine or on perianal skin; reinfection ensues. (1) Clinical manifestations. During acute infection, when the larvae enter through the skin, pruritis to urticaria may occur. When larvae are migrating through the lungs, a mild cough may be present. Upon reaching the intestine, there may be intermittent watery diarrhea and abdominal pain. Disseminated infection, which can be fatal, occurs in the immunosuppressed and in those on steroids or chemotherapy. (2) Diagnosis is made by finding the larvae in stool or through duodenal aspiration. (3) Treatment consists of thiabendazole.

362

meClical

Helminths

f. Trichinella spiralis, the agent of trichinosis, is acquired from the ingestion of living organisms in inadequately cooked pork or wild carnivore meat, such as certain species of bears. (1) Clinical manifestations. Disease is initially expressed as abdominal pain and diarrhea. A second phase occurs when larvae invade muscle, resulting in fever, periorbital edema, eosinophilia, myalgia, and conjunctival petechial hemorrhages. (2) Diagnosis is confirmed by the presence of cysts in biopsy tissue. (3) Treatment consists of mebendazole. Corticosteroids can be added in patients with severe symptoms. 2. Tissue nematodes a. Wuchereria bancrofti is the main etiologic agent of filariasis, also known as elephantiasis. Filariasis is transmitted by mosquito bite. The larvae are deposited on the skin and enter the bite wound and then the lymphatics, where they mature into adults. Filariasis is found primarily in Africa, Southeast Asia, South America, and the South Pacific. (1) Clinical manifestations are marked by inflammatory responses accompanying the death of the adult worms and the molting of younger worms. Lymphatic obstruction occurs from granuloma formation and fibrosis, leading to marked lymphedema, especially of the lower extremities and scrotum. Cellulitis, dermatitis, and lymphangitis also arise and are associated with fever.

In a Nutshell Nematodes (Roundworms)

/~ Intestinal

Tissue

•Ascaris • Trichuris •Ancylostoma • Necator • Enterobius • Strongyloides • Trichinella

• Wuchereria • Onchocerca • Toxocara

In a Nutshell Worm Clues

(2) Diagnosis is confirmed by finding microfilariae in the blood or lymphatics.

• Brain cysts, seizures

---7

Taenia solium

(3) Treatment consists of diethylcarbamazine for micro fIlariae and adult worms. Corticosteroids may be necessary during marked inflammatory responses.

• Liver cysts

---7

• Megaloblastic anemia/B 12 deficiency

---7

Echinococcus Diphyllobothrium latum

• Biliary trad disease, cholangiocarcinoma

---7

Clonorchis sinensis

• Hemoptysis

---7

• Portal hypertension, esophageal vances

---7

Paragonimus westermani Schistosoma mansoni

• Hematuria, bladder cancer

---7

• Redal prolapse

---7

b. Onchocerca volvulus causes onchocerciasis or "river blindness:' It is transmitted by the bite of the blackfiy, which resides alongside flowing streams or rivers in Africa, and Central and South America. Onchocerciasis is second only to trachoma as the main cause of infectious blindness. (1) Clinical manifestations are expressed as subcutaneous nodules, a papular ery-

thematous pruritic rash, and blindness. (2) Diagnosis is made by the observation of microfIlaria in a skin biopsy and anterior chamber of the eye. (3) Therapy consists of ivermectin and surgical excision of nodules. c. Toxocara canis causes toxocariasis (visceral larva migrans). It is found in dogs, particularly puppies, and is transmitted by the ingestion of eggs in dirt soiled with feces. (1) Clinical manifestations are characterized by visceral involvement of liver, lung, brain and eyes, hemorrhage, necrosis, and eosinophilic granulomas. (2) Diagnosis is usually based on clinical evidence unless a biopsy of viscera (e.g., liver) reveals larvae. (3) Treatment consists of corticosteroids and antihelminthic agents such as diethylcarbamazine and albendazole.

• Microcytic anemia (blood loss)

Schistosoma haematobium

Trichuris trichiura ---7 Ancylostoma and Necator

• Perianal pruritis (Scotch tape)

---7

Enterobius vermicularis

• Autoinfedion

---7

• Elephantiasis, lymphedema

---7

• Blindness

---7

Strongyloides stercoralis Wuchereria bancrofti Onchocerca volvulus

KAPLA~. I meulCa

363

Antimicrobial Agents

This comprehensive chapter reviews the drugs used to treat the pathogenic microorganisms described in the previous chapters. The outline is divided into antibacterials (antibiotics), antimycobacterials, antifungals, antihelminthics, and antiviral agents. For each drug, the mechanism of action, indications for use, and side effects and toxicity are reviewed.

ANTIBACTERIAL AGENTS Antibiotics are chemicals that may be produced entirely by microorganisms or that may be modified (semisynthetic) to broaden the spectrum of activity, increase the chemical stability, or improve the pharmacokinetic properties. Some antibiotics inhibit bacterial growth (bacteriostatic); others kill organisms (bactericidal); and some possess both properties in a dose-dependent manner. Antibiotics are usually classified according to bacterial specificity or mechanism of action. A. Overview of classes of antibiotics 1. Cell wall synthesis inhibitors. Bactericidal agents that interfere with the synthesis of

bacterial cell walls make microorganisms vulnerable to changes in the osmolarity of the environment. The cell wall contains complex, cross-linked peptidoglycans, which conveys rigidity. The cross-linking occurs across peptide chains as a result of transpeptidation and is catalyzed by enzymes inhibited by antibiotic. a. AlII3-lactam antibiotics include

~-lactam

rings:

In a Nutshell Cell Wall Synthesis Inhibitors

• Penicillins • Cephalosporins

13-ladams

• Carbapenems

(1) Penicillins andcephalosporins. These drugs act as analogs ofD-alanyl-D-alanine.

They prevent the final step in cell wall synthesis by inhibiting the transpeptidase enzyme responsible for cross-linking and peptidoglycan synthesis. The penicillins and cephalosporins differ primarily in that the penicillins are derivatives of 6-aminopenicillanic acid, whereas the cephalosporins are characterized by substituent groups added to 7-aminocephalosporanic acid. Differences in susceptibility of Gram-positive and Gram-negative organisms depend on structural differences in cell walls, the presence of different binding proteins, the nature of peptidoglycans, and the activity of autolytic enzymes.

• Monobadams • Vancomycin • Bacitracin • Cycloserine

(2) Carbapenems (3) Monobactams b. The drugs listed below inhibit cell wall synthesis at early stages within cell cytoplasm; drugs must penetrate the cell membrane to be effective. (1) Vancomycin blocks the growing end of peptidoglycan. (2) Bacitracin blocks dephosphorylation of lipid carrier.

meClical

365

Microbiology

(3) Cycloserine prevents D-alanine additions to form pentapeptides. 2. Protein synthesis inhibitors. These drugs affect the function of bacterial ribosomes, thereby inhibiting protein synthesis. a. The protein synthesis inhibitors that interact with the 30S ribosomal subunit include the following: (1) Aminoglycosides are bactericidal agents that block initiation of protein synthesis, causing accumulation of protein synthetic initiation complexes. Because tetracyclines do not inhibit bacterial cell wall synthesis, they are effective against cell wall-deficient organisms and bacterial variants that may develop during treatment with cell wall-inhibiting antibiotics. (2) Tetracyclines act as bacteriostatic agents that inhibit binding of aminoacyltransfer RNA (tRNA) to mRNA-ribosome complex.

In a Nutshell Protein Synthesis Inhibitors • Aminoglycosides • Tetracyclines • Spectinomycin • Erythromycin • Lincomycin • Clindamycin • Chloramphenicol

(3) Spectinomycin blocks initiation. b. The protein synthesis inhibitors that interact with the 50S ribosomal subunit are: (1) Erythromycin is a macrolide antibiotic that contains a large lactone ring to which sugars are attached. Erythromycin inhibits bacterial protein synthesis by blocking release of the uncharged tRNA from the 50S ribosomal subunit. It is prescribed for patients allergic to penicillins. (2) Clindamycin is a lincosamide that has an unknown mechanism thought to be similar to that of erythromycin (inhibits 50S subunit of ribosome). Whereas clindamycin's Gram-positive antibacterial spectrum is similar to that of erythromycin, it has broader coverage, including anaerobes. (3) Chloramphenicol is a bacteriostatic agent that acts by binding the 50S subunit of the bacterial ribosome to inhibit peptidyltransferase action. This binding is readily reversible and is inhibited by the macrolide and lincosamide antibiotics. 3. Antimetabolites. Folate antagonists are bacteriostatic agents that interfere with bacterial synthesis or reduction of folate. a. Sulfonamides arrest cell growth by inhibiting the bacterial synthesis of folic acid. Sulfonamides are structural analogs of the folic acid precursor, para-aminobenzoic acid (PABA). They competitively inhibit dihydropterate synthetase, the enzyme that directs the incorporation of PABA and a pteridine moiety into dihydropteroic acid. Organisms that do not synthesize folic acid because they obtain it from other sources (e.g., humans) are unaffected. b. Trimethoprim is a structural analog of the pteridine portion of dihydrofolate reductase and acts as a competitive inhibitor of this enzyme, which converts dihydrofolate to tetrahydrofolate (active form offolic acid). Tetrahydrofolate is required as a methyl donor in the synthesis of adenine, guanine, and thymine.

366

meCtical

Antimicrobial Agents

Penicillins

Amidase H

\

I

H

H

i

1/ S ,......- CH 3

R-N-C-C

C ..... I ,CH 3 C-N-C......- H

I B I A

t

O.?'

...

COOH

Lactamase Substituted 6-aminopenicillanic acid Cephalosporins

o II

H

H

H

I

1/S,

-C-N-989

R1

t

A

......-H

Y'H

C-N C O.?' '9~

. . . CH 2-R2

COOH Substituted 7-aminocephalosporanic acid Monobactams

o

H

H

H

II

I

.

1

R-C-N-C-C- CH3

IB I

C-N O.?'

'

R:-C-Cr ~ N~NH2 I O-C(CH3h

S03H

I

COOH Substituted 3-amino-4-methylmonobactamic acid (aztreonam)

HO I

H

H

,

1

Carbapenems

HC-C-C~ /" I B I ~ S-R H3C

.?'C-N

o

NH

II

R:-CH 2-CH 2-NH-CH

COOH Substituted 3-hydroxyethylcarbapenemic acid (imipenem) Clavulanic acid

Figure V-1S-1. Structure of four [3-lactam antibiotics and clavulanic acid. The ring in each structure is the [3-lactam ring. (Reprinted with permission from Katzung BG (ed): Basic and Clinical Pharmacology 6th ed. East Norwalk, Connecticut, Appleton and Lange, 1995, p 681.)

meCtical

367

Microbiology

4. Cell membrane inhibitors act directly on the cell membrane to affect permeability and lead to leakage of intracellular compounds. a. Cell membrane inhibitors that disrupt cell membranes of Gram-negative bacteria (bactericidal) include: (1) Polymyxin, which binds to phospholipids and alters cell permeability

(2) Colistin b. Cell membrane inhibitors that interact with membrane sterols in fungal cells include: (1) Amphotericin B, which binds to ergosterol-altering cell membranes. (2) Nystatin (same mechanism as amphotericin) (3) Fluconazole, clotrimazole, and ketoconazole inhibit ergosterol synthesis. 5. Nucleic acid synthesis inhibitors a. Fluoroquinolones (e.g., ciprofloxacin) and nalidixic acid inhibit DNA gyrases (topoisomerases) necessary for supercoiling of DNA. b. Rifampin binds to and inhibits DNA-dependent RNA polymerase present in bacteria.

(4)

(1 )

Cell-wall synthesis Cycloserine Vancomycin teichoplanin Bacitracin Penicillins Cephalosporins Monobactams

(4)

"'" ,_.----:= (5)

-'C3(Jl'-{3Ur---\3U~

Erythromycin (macrolides) Chloramphenicol Clindamycin

Protein synthesis (30S inhibitors)

PABA

Tetracycline Spectinomycin Streptomycin Gentamicin tobramycin (aminoglycoside) Amikacin

(2)

Figure V-15-2. Antimicrobial sites of bactericidal action on microorganisms. The five mechanisms are: (1) inhibit cell wall synthesis; (2) damage cell membrane; (3) modify nucleic acid/DNA synthesis; (4) modify protein synthesis; and (5) modify metabolism within the cytoplasm. (THFA = tetrahydrofolic acid; DHFA = dihydrofolic acid; and PABA = para-aminobenzoic acid.) (Modified with permission from BrodyTM, Larner J, Minneman KP, Neu He: Human Pharmacology: Molecular to Clinical, 2nd ed. St. Louis, Missouri, Mosby-YearBook, 1994, p 618.)

368

meClical

Antimicrobial Agents

B. Efficacy 1. Successful antimicrobial therapy depends on achieving inhibitory or bactericidal activity

at the site of infection without significant toxicity to the host. 2. In most infections, the normal local and systemic host defense mechanisms playa crucial role in the final elimination of the pathogen. 3. The degree of in vitro susceptibility of bacterial strains to particular antibacterial agents is estimated by determining the minimum inhibitory concentration (MIC), the lowest concentration of antibiotic that prevents growth, and the minimal bactericidal concentration (MBC) , the lowest concentration of antibiotic that kills all the organisms in an in vitro MIC assay. C. Resistance. Microorganisms are capable of acquiring resistance to antimicrobial agents by

genetic changes that are passed on from generation to generation. 1. Spontaneous chromosomal mutations produce a genetically altered bacterial population that is resistant to the drug action, survives, and gives rise to a new drug-resistant population. 2. Drug resistance is usually acquired, not by chromosomal change, but by R-factors from other bacteria in the form of extrachromosomal DNA pieces that contain resistancemechanism information. R-factors are plasmids that carry genes for resistance to one or more antibiotics. Plasmid transfer accounts for over 90% of antibiotic resistance.

Note The Kirby-Bauer disk diffusion method is commonly used to determine antibiotic sensitivity. The organism is exposed to disks saturated with different antibiotics. After growth, the degree of growth inhibition around each disk is measured and the susceptibility/resistance to the antibiotic is determined.

3. Changes in drug permeability. Tetracyclines are able to accumulate in susceptible bacteria; in resistant bacteria, resistance occurs by increasing the energy-dependent efflux of tetracyclines. 4. Drug deactivation. The principal mechanism of resistance to the penicillins and cephalosporins is by B-Iactamase action. Aminoglycosides and chloramphenicol are inactivated by acetylation or other enzymatic modification. 5. Decreased drug conversion to active compound. The antifungal drug, flucytosine, must be converted in vivo to fluorouracil, which is further metabolized to the active form of the drug. Fungi become resistant to flucytosine by losing enzyme activity along the activation pathway. 6. Altered metabolic pathway may occur in bacteria resistant to sulfonamides and in fungi resistant to flucytosine. Some sulfonamide-resistant bacteria can use preformed folic acid (e.g., from mammalian cells). 7. Altered amount of drug receptor. Some organisms become resistant to penicillins and cephalosporins by synthesizing altered penicillin-binding proteins and to fluoroquinolones by altered DNA gyrase activity. 8. Decreased receptor affinity for drug. Resistance to erythromycin may be associated with alteration of a specific protein on the 50S subunit of the bacterial ribosome necessary for drug binding. 9. Evaluations of the effectiveness of antimicrobial agents must use the results of clinical trials as well as the in vitro activity of the drug against potential pathogens. Combinations of antibiotics are often used to broaden coverage with mixed or unknown types of infection, to prevent or delay the emergence of bacterial resistance to the drugs, and to achieve therapeutic synergy.

KAPLA~.

meulCa

I 369

Microbiology

BETA-LACTAMS AND OTHER CELL WALL SYNTHESIS INHIBITORS A. Penicillins (Table V-IS-I) are ~-lactam antibiotics similar to the cephalosporins. The development of large-scale production has led to structural modifications of the original penicillin G, with formation of derivatives with greater effectiveness against a variety of infections. 1. Penicillin G (benzyl penicillin)

a. Pharmacologic properties ( I) The cyclic amide structure, called a ~-lactam, binds to a transpeptidase and prevents peptidoglycan cross-linkage essential for completion of cell wall synthesis. Enzymatic or acid hydrolysis of the ~-lactam results in loss of antibiotic activity. (2) Penicillins also increase bacterial cell wall breakdown by activating bacterial autolysins. (3) They possess bactericidal action that affects only growing cells during cell wall synthesis. (4) Gastric acid hydrolyzes penicillin G and only 30% of the active drug is absorbed.

Table V-IS-I. Penicillins. Drug Acid labile Benzyl penicillin (penicillin G)

Route

Bacterial Specificity

Given orally; poor absorption when given intramuscularly and intravenously

Used against most Gram-positive cocci and some Gram-negative; effective against Neisseria; hydrolyzed by acid and ~-lactamase

Phenoxymethyl penicillin (penicillin V) Acid stable Ampicillin

Penicillinaseresistant penicillins Methicillin Oxacillin, cloxacillin, and dicloxacillin Nafcillin Broad-spectrum activity Carbenicillin Ticarcillin Piperacillin

370

meClical

Given orally only

Similar lability but less effective than penicillin G; preferred for upper respiratory tract infections

Given orally and parenterally

Used against all staphylococci, many E. coli, and H. injluenzae strains (others now resistant); lactamase-vulnerable but stable in combination with clavulanic acid (Augmentin); similar to ampicillin but higher blood levels and fewer gastrointestinal problems

Poor orally; must be given parenterally Fair oral absorption; given intramuscularly and intravenously Poor orally; given parenterally

Spectrum is similar to penicillin G but less potent. Used against ~-lactamase-containing staphylococcal infections Most active against resistant S. aureus

Poor orally; given intramuscularly Lactamase sensitive; active against and intravenously Pseudomonads, Proteus Given intravenously Given intravenously

Available with clavulanic acid; more potent against Gram-negative organisms

Antimicrobial Agents

(5) Crystalline penicillin G given intramuscularly results in therapeutic peak plasma levels that last for 2-3 hours. It may be given intravenously when large doses are required. (6) Repository forms are used to prolong antibiotic levels by delaying the rate of systemic absorption from parenteral sites. Procaine salt of penicillin G is absorbed more slowly after intramuscular administration, attaining therapeutic peak plasma levels lasting 24 hours. Benzathine penicillin G maintains low drug levels for up to 4 weeks after a single intramuscular injection; it is used for prophylaxis of group A streptoccocal infection and for treatment of syphilis. (7) Penicillin is 55% bound to serum proteins and is widely distributed throughout the body. Significant cerebrospinal fluid penetration is seen only with meningeal inflammation. (8) Seventy to eighty percent excreted in urine (i.e., 10% by glomerular filtration, 90% by renal tubular secretion); renal tubular secretion can be blocked by concurrent administration of the weak organic acid, probenecid. Probenecid depresses the tubular secretion of penicillin, allowing it to have a longer half-life. b. Indications for use (1) Gram-positive cocci, including Streptococcus pneumoniae and Streptococcus pyo-

genes (2) Gram-negative cocci, including Neisseria meningitidis (3) Gram-positive bacilli, including Bacillus anthracis, Clostridium perfringens, Listeria monocytogenes, and Corynebacterium diphtheriae

(4) Treponema pallidum, the causative agent of syphilis c. Side effects and toxicity of penicillin are generally few, but in some patients, allergic

reactions may occur. (1) Hypersensitivity reactions occur in up to 10% of patients. Symptoms include

rashes, urticaria, fever, serum sickness, Stevens-Johnson syndrome, and anaphylaxis due to antibody formation to degradation products. Cross-sensitivity with cephalosporins also exists. (2) Diarrhea (3) Jarisch-Herxheimer reaction (flu-like symptoms, including fever, chills, and myalgia) can occur in secondary syphilis within the first few hours following penicillin G therapy. d. Resistance mechanisms (1) Bacteria containing penicillinase enzymes (~-lactamases) in their periplasmic space can inactivate penicillin G by opening the ~-lactam ring. It is a common

resistance mechanism among staphylococci and Gram-negative bacteria. Methicillin-resistant organisms (e.g., many Staphylococcus epidermidis organisms) are resistant even to penicillins not sensitive to penicillinase; these organisms are usually treated with vancomycin. (2) Penicillin penetrates the bacterial cell envelope and attaches to a number of penicillin-binding proteins (PBPs) on the bacterial cytoplasmic membrane. Bacterial resistance may also result from altered affinity and number PBPs.

KAPLAN' ii-leaI me

371

Microbiology

2. Penicillin V a. Pharmacologic properties. It is effective against oral bacteria; most used penicillin in dentistry. It is available only in oral form. It is more resistant to gastric acid destruction and has greater gastrointestinal absorption than penicillin G. b. Indications for use (1) Penicillin V is less active than penicillin G, especially against Gram-negative bacteria (e.g., Neisseria, meningococcal meningitis). (2) It is used only when an oral form is desired for susceptible organisms. 3. Broad-spectrum penicillins a. Ampicillin (1) Pharmacologic properties are similar to penicillin G (both are destroyed by ~­ lactamase), but ampicillin is acid stable and has increased activity against Gramnegative organisms (e.g., Haemophilus injluenzae, Escherichia coli, Proteus, Salmonella.) Ampicillin is less active than penicillin G is against Gram-positive cocci. It is available for oral and parenteral administration. Resistance mechanisms include inactivation by penicillinase or altered properties of PBPs. (2) Indications for use include some gonococcal infections, upper respiratory infections (e.g., H. injluenzae, S. pneumoniae, S. pyogenes), urinary tract infections (e.g., E. coli, Enterococcus, Proteus mirabilis), meningitis (e.g., H. injluenzae, S. pneumoniae, N. meningitidis), and Salmonella and Shigella infections. It is also preferred in Listeria infections. (3) Side effects and toxicity are similar to penicillin G, but rashes are more common and an idiosyncratic reaction occurs in patients with mononucleosis that causes a maculopapular rash. It can also cause pseudomembranous colitis. b. Amoxicillin is a parahydroxyl derivative of ampicillin. (1) Pharmacologic properties are similar to ampicillin, but there is better intestinal absorption and less gastrointestinal disturbance. Amoxicillin is hydrolyzed by ~­ lactamases but is stable in combination with ~-lactamase inhibitor, davulanic acid (Augmentin). Amoxicillin is resistant to gastric acid destruction and can be taken orally. It attains higher peak serum levels than ampicillin after similar oral dosage; this is the major difference between the two agents. The in vitro antibacterial spectrum of amoxicillin is identical to ampicillin except it is less effective against Shigella. (2) Indications for use include infections of the skin, soft tissue, and lower urinary and respiratory tracts, caused by nonpenicillinase-producing strains of staphylococci, streptococci, H. injluenzae, E. coli, and P. mirabilis; uncomplicated anogenital and urethral gonococcal infections; and otitis media. (3) Side effects and toxicity include lower incidence of nausea, vomiting, and diarrhea than with ampicillin, and hypersensitivity reactions. c. Amoxicillin plus davulanic acid (Augmentin) (1) Pharmacologic properties are similar to amoxicillin, but the combination with

clavulanic acid broadens coverage to include ~-lactamase-producing organisms, including H. injluenzae. Clavulanic acid covalently reacts with ~-lactamase and prevents the enzyme from hydrolyzing the ~-lactam ring. Augmentin is administered orally only.

372

meClical

Antimicrobial Agents

(2) Indications for use include severe otitis media, sinusitis, pneumonia (e.g., H. inJluenzae, Moraxella catarrhalis), and animal bites (Pasteurella multocida). (3) Side effects and toxicity are similar to amoxicillin given alone except that there is a greater incidence of nausea and vomiting. d. Ampicillin with sulbactam (a penicillinase inhibitor) (1) Pharmacologic properties are similar to ampicillin. Sulbactam broadens coverage to include ~-lactamase-positive organisms and some anaerobes. There is parenteral administration only.

Note Clavulanic acid and sulbactam are inhibitors of [3-lactamase that can greatly broaden the spectrum of penicillins.

(2) Indications for use include intra-abdominal infections where anaerobic coverage is desired and severe urinary tract infections (UTIs), including those caused by enterococci. (3) Side effects and toxicity are similar to ampicillin. 4. Antipseudomonal penicillins a. Carbenicillin ( 1) Pharmacologic properties. Carbenicillin is administered parenterally or orally. It is excreted unchanged by the kidney. There is increased antibacterial activity against Pseudomonas species and other Gram-negative organisms such as Enterobacter and Proteus. (2) Indications for use. The main indication is treatment for systemic infection with Pseudomonas aeruginosa. Resistance develops rapidly; thus, it should almost always be used with an aminoglycoside. (3) Side effects and toxicity. Carbenicillin is generally well-tolerated, but occasional hypersensitivity reactions occur, including hypokalemia, sodium overload, and platelet dysfunction. b. Ticarcillin (1) Pharmacologic properties are similar to carbenicillin. It can be given in lower doses by parenteral route only. It is excreted by the kidney. Resistance involves inactivation by penicillinases.

(2) Indications for use. It is more active than carbenicillin against E. coli, Enterobacter, and Proteus. (3) Side effects and toxicity are similar to carbenicillin. c. Ticarcillin with c1avulanic acid (Timentin) ( 1) Pharmacologic properties are similar to ticarcillin; clavulanic acid broadens coverage to include ~-lactamase-producing organisms. The drug is used only parenterally. (2) Indications for use include hospital-acquired infections (nosocomial infections) requiring Pseudomonas coverage and broad Gram-positive protection. (3) Side effects and toxicity are the same as with ticarcillin. d. Piperacillin (1) Pharmacologic properties. Piperacillin is more active than ticarcillin or carbenicillin against Pseudomonas. It is also active against Proteus, Enterobacter, and

mec.ical

373

Microbiology

In a Nutshell Carbenicillin, ticarcillin, and piperacillin are penicillins with broad-spectrum coverage used primarily in the treatment of pseudomonal infections (along with an aminoglycoside).

Klebsiella. Administration is by the parenteral route. Toxicity and resistance mechanism are similar to ticarcillin and carbenicillin, but these drugs are more resistant to penicillinase. (2) Indications for use. These agents should be used with an aminoglycoside against serious Pseudomonas infections. 5. Penicillinase-resistant penicillins (antistaphylococcal penicillins) a. Methicillin (1) Pharmacologic properties. Methicillin is penicillinase-resistant; it provides

increased resistance to hydrolysis of the ~-lactam ring by staphylococcal penicillinase and other ~-lactamases. It is used only parenterally because of poor oral absorption and sensitivity to gastric acid. Fifty percent of the drug is excreted unaltered in urine. It is not effective against Gram-negative organisms. (2) Indications for use include penicillin-resistant S. au reus infections. Methicillinresistant Staphylococcus infections should be treated with vancomycin. (3) Side effects and toxicity include allergic reactions, reversible bone-marrow depression, phlebitis, and nephrotoxicity. Because the incidence of interstitial nephritis is greater than with other penicillins, methicillin is not the agent of choice in most cases. b. Oxacillin, dicloxacillin, and cloxacillin are used mainly against infections with lactamase-producing staphylococci.

~­

(1) Pharmacologic properties. Spectrum is similar to penicillin G but less potent. Because these drugs are resistant to gastric acid destruction, they may be administered orally or parenterally. High concentrations of these drugs are found in bile and in pleural and amniotic fluids. There is rapid renal excretion. These drugs are less effective than ampicillin against Gram-negatives. Dicloxacillin is the most active of the oxacillins against S. aureus, but it is less active than nafcillin. (2) Indications for use involve penicillin G-resistant staphylococci. (3) Side effects and toxicity include rash, urticaria, pruritus, hypersensitivity reactions, and hepatitis. c. Nafcillin (1) Pharmacologic properties. Nafcillin is administered parenterally (variable oral

absorption). There is variable inactivation by gastric acid and biliary excretion. (2) Indications for use. Nafcillin is useful in treating staphylococcal infections and mixed infections with penicillin-resistant staphylococci and streptococci. (3) Side effects and toxicity. Nafcillin is generally well-tolerated, but allergic reactions, gastrointestinal discomfort, and mild phlebitis may occur. B. Cephalosporins originated from a Cephalosporium fungus and have been found to inhibit

Staphylococcus aureus. As with the penicillins, semisynthetic cephalosporins now exist for clinical use and are classified as first-, second-, third-, or fourth-generation drugs. 1. Mechanism of action is similar to that of the penicillins; cephalosporins block terminal cross-linking of the bacterial cell wall peptidoglycan and activate cell wall autolytic enzymes.

2. The nucleus of the cephalosporins consist of a ~-lactam ring fused to a six-membered ring similar in structure to the nucleus of the penicillins.

374

meClical

Antimicrobial Agents

3. Resistance to the cephalosporins is by ~-lactamases and by lack of antibiotic penetration in Gram-negative organisms. 4. The first-generation cephalosporins are broad-spectrum agents with activity against Gram-positive organisms (except Enterococcus and methicillin-resistant Staphylococcus) and Gram-negative bacteria (including E. coli, Klebsiella, Proteus). 5. Anaerobic bacteria, including Bacteroides fragilis, are sensitive to the second-generation agents (e.g., cefoxitin), which are less potent than first-generation cephalosporins but have broader Gram-negative coverage. Haemophilus, Neisseria, E. coli, Klebsiella, and Proteus are sensitive to the second-generation drugs. Gram-positive infections are generally treated with this drug class. 6. The third-generation drugs can penetrate the central nervous system (CNS) and, with the exception of cefoperazone, are active against bacterial meningitis. These third-generation agents are resistant to j3-lactamases. Gram-negative organisms are susceptible, whereas Gram-positives are poorly treated by third-generation agents. 7. Fourth-generation cephalosporins, much like third-generation drugs, have good activity against Gram-negative bacteria. However, this generation has strong activity against Gram-positives, similar to first-generation drugs. 8. Pharmacologic properties are similar to the penicillins but somewhat less potent, requiring higher dosages. a. Most cephalosporins are excreted by the kidney (glomerular fIltration and tubular secretion). b. Those with acetylated residues (cephalothin, cephapirin) are metabolized in the liver, and their metabolites are renally excreted. c. Some are highly protein bound (e.g., cephalothin, cefamandole, cefoxitin, and

cephapirin). d. Variable susceptibilities to

~-lactamases

9. Indications for use include upper respiratory infections, acute otitis media, urinary tract infections, skin and soft tissue infections with S. aureus, prophylaxis before surgery, systemic infections with bacteria sensitive to these agents, and life-threatening infections before specific organisms are identified (due to broadness of antimicrobial spectrum). 10. Side effects and toxicity

a. Allergic reactions may occur, including urticaria, rash, fever, and eosinophilia. There is a 5-10% cross-reactivity between cephalosporins and penicillins in those hypersensitive to penicillins. b. There may also be nausea, vomiting, diarrhea, and elevated liver function test values (pseudomembranous colitis). c. Hypoprothrombinemia (with cefamandole, cefoperazone, moxalactam)

d. Disulfiram-like reaction with alcohol moxalactam)

(with cefamandole, cefoperazone,

e. Nephrotoxicity f. Positive direct Coombs test 11. Specific agents (Table V-15-2) KAPLA~.

meulCa

I

375

Microbiology

In a Nutshell • First generation

Table V-15-2. Cephalosporins.* Similar to penicillin and ampicillin

• Second generation Extended Gram-negative coverage, including anaerobes • Third generation

Excellent Gram-negative coverage; cross blood-brain barrier

Drug First generation Cephalothin Cefazolin Cephalexin Cephradine Cefaclor Second generation Cefamandole Cefuroxime

Cefoxitin

Third generation Cefotaxime Ceftizoxime Ceftriaxone Cefixime (oral agent)

Cefoperazone Fourth generation Cepirome Cefepine

Route and Bacterial Specificity These drugs inhibit most Gram-positive organisms except enterococci. They are used to treat respiratory infections in children.

This drug is more active than first-generation drugs against Haemophilus species, some E. coli, Klebsiella, and other Enterobacteriaceae. This drug inhibits Gram-positive organisms, is excellent against Haemophilus and Neisseria species, and has greater resistance to ~-lactamase than does cefamandole. This drug is less active against Gram-positive organisms, but its high ~-lactamase stability and inhibitability to Enterobacteriaceae and 85% of anaerobic bacteria make it useful for aspiration pneumonitis and intra-abdominal and intrapelvic infections. These drugs are excellent against Gram-positive streptococci, including S. pneumoniae and Haemophilus and Neisseria species. This drug is similar in action to cefotaxime and ceftizoxime. It is used to treat nosocomial infections, Lyme disease, and gonorrhea. It precipitates in the bladder and may cause diarrhea. CefIxime inhibits streptococci, Haemophilus species, Neisseria, Moraxella, and many Enterobacteriaceae. It is used to treat respiratory infections. Cefoperazone has an Antabuse-like action and it changes prothrombin activity. It is less ~-lactamase stable but is active against Pseudomonas. These drugs have increased activity against Grampositive bacteria, inhibit Pseudomonas species, and are not labile to some ~-lactamases.

*Note: Cephalosporins are similar to the penicillins in structure; they also have similar activities, which have increased with each "generational" modification in structure.

C. Monobactams: aztreonam. As the name implies, monobactams have a single ~-lactam ring. L Pharmacologic properties. Aztreonam interferes with cell wall synthesis. Excretion is

mostly urinary. Tissue levels are excellent. 2. Indications for use. Aztreonam is used to treat Gram-negative infections; it provides no Gram-positive or anaerobic coverage (narrow spectrum). 3. Side effects and toxicity include swelling and local irritation, mild LFT abnormalities, and anaphylactic reactions (but little cross-reactivity with other ~-lactams).

376

meClical

-

-----

---

--

- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

Antimicrobial Agents

D. Carbapenems: imipenem

1. Pharmacologic properties a. Imipenem is a synthetic ~-lactam antibiotic. b. It inhibits cell wall synthesis. c. Imipenem is marketed in combination with cilastatin, a renal dipeptidase inhibitor, which prevents renal tubular metabolism and accumulation of nephrotoxic metabolites. d. Imipenem has broad-spectrum coverage, including penicillinase-producing Grampositives, Gram-negatives, P. aeruginosa, and anaerobes. It is the most potent and broadest-spectrum ~-lactam currently on the market. 2. Indications for use. Use is essentially limited to nosocomial infections. 3. Side effects and toxicity include nausea, vomiting, and diarrhea; allergic reactions; and seizures. E. Other cell wall inhibitors 1. Vancomycin a. Pharmacologic properties (1) Vancomycin is a bactericidal agent that inhibits cell wall synthesis by binding D-

Ala-D-Ala, thus preventing its incorporation into peptidoglycan. (2) It is poorly absorbed after oral administration. (3) It is active against Gram-positive bacteria but not against Gram-negative organisms. b. Indications for use (1) Vancomycin is given orally to treat pseudomembranous colitis caused by the

anaerobe Clostridium difficile. (2) It is given intravenously for treatment of methicillin-resistant staphylococci (MRSA) and penicillin-resistant pneumococci. (3) It is useful in patients allergic to penicillins and cephalosporins in the treatment of Gram-positive infections. (4) Finally, vancomycin is used with gentamicin for Streptococcus faecalis or Streptococcus viridans endocarditis or for serious infections in patients with penicillin allergy. c. Side effects and toxicity. Rapid infusion can cause facial and neck erythema ("red man" syndrome). There may also be ototoxicity (rare), phlebitis, and nephrotoxicity. 2. Bacitracin is a cyclic polypeptide that inhibits cell wall synthesis by blocking the dephosphorylation of the phospholipid that transports peptidoglycan subunits across the cytoplasmic membrane. Because of nephrotoxicity, use is limited to topical application.

In a Nutshell Uses of Vancomycin

• Methicillin-resistant staphylococci • Enterococcal infections in the penicillin-allergic patient • Pseudomembranous colitis (C difficile) • Penicillin-resistant S. pneumoniae

3. Cycloserine is a broad-spectrum antibiotic that inhibits D-alanine incorporation into peptidoglycan. Its use is limited to treatment of tuberculosis (TB).

IlAPLA~. I meulca

377

Microbiology

PROTEIN SYNTHESIS INHIBITORS A. Aminoglycosides 1. Overview. Binds the 30S ribosomal subunit and causes change in the codon:anticodon

recognition sites. Bactericidal action results from the irreversible inhibition of the initiation of protein synthesis. a. Pharmacologic properties. All aminoglycosides are administered parenterally because of poor intestinal absorption and are used against aerobic Gram-negative bacteria. b. Side effects and toxicity (1) Ototoxicity and renal impairment are side effects of all aminoglycosides and are usually dose dependent, whereas hypersensitivity reactions are idiosyncratic. (2) Increased nephrotoxicity is seen when arninoglycosides are given concurrently with amphotericin, furosemide, and cephalosporins. In addition, elderly patients or patients with shock, dehydration, or pre-existing renal disease are more susceptible to the nephrotoxicity. (3) Aminoglycoside doses must be reduced in renal insufficiency. (4) Resistance develops rapidly to all aminoglycosides mainly by plasmid-encoded enzyme inactivation via acetylation, phosphorylation, or adenylation. 2. Gentamicin a. Pharmacologic properties (1) Gentamicin is active against Enterobacter, E. coli, Klebsiella, Proteus,

Pseudomonas, Neisseria, Serratia, and Shigella. (2) It is active against Streptococcus viridans and Streptococcus faecalis when combined with penicillin or ampicillin. b. Indications for use ( 1) Gentamicin is used for serious infections with susceptible Gram-negative bacteria. (2) It is also used topically in burns infected with Pseudomonas and for ocular infections. 3. Tobramycin

Note Tobramycin is the most active aminogJycoside against Pseudomonas.

a. Pharmacologic properties. Tobramycin demonstrates an antibacterial spectrum including aerobic Gram-negative bacilli such as Pseudomonas, Klebsiella, Enterobacter, Proteus, Citrobacter, and Providencia. b. Indications for use. Tobramycin is used with penicillins, cephalosporins, and alone to treat infections in all sites except the CSF. 4. Streptomycin a. Indications for use (1) Although primarily reserved for treatment of TB (Mycobacterium tuberculosis), it is active against many other microbes and is often used in combination with a [3lactam antibiotic in life-threatening diseases such as K. pneumoniae pneumonia. (2) It may be used with penicillin to treat streptococcal endocarditis. (3) Streptomycin is ineffective against anaerobic infections because there is an oxygen-dependent transport step required for its penetration into the cell.

378

mectical --------------

Antimicrobial Agents

b. Side effects and toxicity. Ototoxicity and nephrotoxicity are more common than with most other aminoglycosides. 5. Neomycin a. Pharmacologic properties. Neomycin is active against E. coli, Enterobacter, Klebsiella, Proteus, and some S. aureus species. b. Indications for use (1) Neomycin is used as a topical application for superficial skin infections. (2) It is also given orally to patients with hepatic coma because it reduces the number of ammonia-producing bacteria in the gastrointestinal tract. (3) Finally, it is given as an oral prophylaxis to prepare the bowel prior to intestinal surgery. c.

Side effects and toxicity. Neomycin is the most toxic aminoglycoside with doserelated ototoxicity and nephrotoxicity; it is not for parenteral administration. There may be diarrhea and malabsorption following oral administration.

6. Amikacin has the widest antibacterial spectrum of the aminoglycosides. a. Pharmacologic properties include resistance to most bacterial enzymes that inactivate other aminoglycosides. It is susceptible to inactivation by acetylation. Amikacin is active against E. coli, P. aeruginosa, Proteus, Klebsiella, Enterobacter, Serratia, Acinetobacter, Citrobacter, Providencia, and M. tuberculosis. b. Indications for use. Amikacin is used mainly for treatment of organisms resistant to other aminoglycosides. It is the drug of choice for burns infected with resistant Pseudomonas. B. Tetracyclines. Tetracyclines (four rings) are broad-spectrum bacteriostatic antibiotics. They bind at the 30S ribosomal subunit and block protein synthesis by interfering with the interaction of aminoacyl-tRNA and the mRNA-ribosome complex.

1. Tetracycline a. Pharmacologic properties (1) Variable gastrointestinal absorption after oral administration occurs by forming insoluble complexes in the gut with calcium or other ions. The presence of food, milk, metallic salts, or antacids results in poor absorption. (2) Intravenous administration is used for serious infections, malabsorption syndromes, and critically ill patients. (3) Tetracycline diffuses readily into most tissues and fluids. 4) The drug's broad antibacterial spectrum includes a wide variety of Gram-positive and Gram-negative bacteria. (5) Resistance to tetracycline develops in direct proportion to usage. Resistance mechanisms include tetracycline-resistant ribosomes, bacterial production of enzymes that degrade the antibiotic, and decreased permeability of the bacterial cell surface to the drug (plasmid mediated). b. Indications for use (1) Tetracycline is useful for the following infections: Rickettsia (Rocky Mountain spotted fever, Q fever), Chlamydia (lymphogranuloma venereum; psittacosis, Chlamydophila pneumoniae), Francisella tularensis, Vibrio cholerae (cholera), Borrelia (Lyme disease), Ureaplasma, and Mycoplasma pneumoniae.

In a Nutshell Tetracyclines can be used to treat the following organisms:

• Rickettsia • Chlamydia • Francisella tularensis • Vibrio cholerae • Mycoplasma pneumoniae (second choice after erythromycin)

• Ureaplasma • Borrelia • Severe acne

meclical

379

Microbiology

(2) Tetracycline is an alternative treatment for infections with L. monocytogenes and N. gonorrhoeae.

(3) It is also used as a treatment for chronic severe acne (topical or oral administration). c. Side effects and toxicity (1) Intravenous administration can produce thrombophlebitis and hepatotoxicity. (2) Fetal and neonatal tooth discoloration make it contraindicated during pregnancy, nursing, and in children under 8. (3) Gastrointestinal disturbances, including esophageal ulceration, occur in 10% of patients. (4) Dry mouth, hoarseness, stomatitis, glossitis, pharyngitis, enterocolitis, and proctitis can occur. (5) There can be hepatotoxicity with prolonged administration of high doses; pregnant women are more susceptible. (6) Pseudotumor cerebri, elevation of blood urea nitrogen (BUN), and photosensitivity also occur. 2. Demedocycline demonstrates variable gastrointestinal absorption affected by food and milk ingestion. This antibiotic is similar to tetracycline, but photosensitivity limits its use. It has been used to treat inappropriate secretion of antidiuretic hormone (SIADH) because it renders kidney tubules insensitive to ADH. 3. Doxycycline and minocycline a. Pharmacologic properties are like those of the naturally occurring tetracyclines. Resistance mechanisms are similar to other tetracyclines. (1) Doxycycline and minocycline are more fat soluble and penetrate bacteria better

than other tetracyclines when changes in drug penetration and resistance develop. (2) They are completely absorbed from the gastrointestinal tract; their absorption is not as affected by food or milk as with tetracycline. (3) Some species of B. fragilis are more susceptible to doxycycline and minocycline than to tetracycline. (4) Unlike other tetracyclines, doxycycline is primarily excreted in feces and can be used in patients with renal insufficiency. b. Indications for use (1) Doxycycline can be administered to patients with renal insufficiency without

exacerbating azotemia. (2) Similar to tetracycline, minocycline is more active and more toxic than doxycycline. (3) These drugs are commonly used to treat sexually transmitted diseases; they are very effective against both Chlamydia infections and gonorrhea. They are also used in the treatment of Lyme disease. c. Side effects and toxicity involve vestibular disturbances, including dizziness and nausea. C. Spectinomycin. Spectinomycin inhibits protein synthesis by interacting with the 30S ribo-

somal subunit. Clinical use is limited to the treatment of N. gonorrhoeae in patients allergic to penicillin.

380

iileClical

-------------

Antimicrobial Agents

D. Macrolides and lincosamides. The antibacterial action of these agents is through binding to the 50S subunit of the bacterial ribosome and interfering with protein synthesis. 1. Erythromycin a. Pharmacologic properties (1) Because most of the drug is inactivated by acid, it is administered orally with an enteric coating that dissolves in the duodenum. Most of drug is concentrated in the liver and excreted in bile. (2) Erythromycin diffuses readily into all body fluids except the brain or CSF. (3) Erythromycin has an antimicrobial spectrum similar to penicillin G and is active against Gram-positive bacteria including Listeria, Staphylococcus aureus, S. pneumoniae, S. viridans, S. faecalis, Clostridium, Corynebacterium, and Actinomyces. (4) Erythromycin is active against Mycoplasma pneumoniae, Treponema, Chlamydia, Rickettsia, and Legionella pneumoniae. (5) Resistance is now becoming more of a problem as most nosocomial staphylococcal infections are now resistant. Mechanisms include failure of the organism to take up antibiotic and plasmid-encoded decreased binding to the 50S ribosomal subunit. b. Indications for use (1) Erythromycin is used in patients with penicillin allergy; it is an alternative to penicillin for susceptible pathogens. (2) It is the drug of choice for the treatment of Legionnaires disease and M. pneumoniae. (3) It is a prophylaxis for endocarditis and recurrent rheumatic fever. (4) It is an alternative to penicillin in treating syphilis. (5) Long-acting macrolides such as azithramycin and clarithramycin are replacing erythromycin as preferred therapeutics. (6) It is an alternative to tetracycline in treating chlamydial infections. (7) A topical preparation of erythromycin is used in the treatment of acne.

In a Nutshell Erythromycin is the first choice for treatment of Legione//a and M. pneumoniae. It is a backup choice in the treatment of syphilis (penicillin is first choice) and Chlamydia (tetracycline is first choice). It is also used widely as an alternative to penicillin in penicillin-allergic patients.

c. Side effects and toxicity

(1) Gastrointestinal disturbances are common. (2) There may be cholestatic jaundice. (3) Like the aminoglycosides, sensorineural hearing loss may occur with large doses. (4) Drug interactions interfere with the drug-metabolizing cytochrome P-450 enzyme system, producing increased digoxin effect (decreased metabolism, increased absorption) and increased theophylline effect (decreased metabolism). Drug interactions also increase the effects of carbamazepine, warfarin, and cyclosporine. 2. Clindamycin a. Pharmacologic properties (1) It is widely distributed to bones, fluids, and tissues but shows poor CNS pene-

tration. The spectrum of activity is similar to that of erythromycin.

meclical

381

Microbiology

(2) It is the drug of choice for serious infections caused by the anaerobic organisms, B. fragilis, Fusobacterium, and Peptococcus. (3) It is active against common Gram-positive pathogens, including staphylococci and streptococci, but it is not active against most Gram-negative organisms. (4) Clindamycin is excreted mainly through the liver, bile, and urine. (5) Clindamycin reacts with the 50S ribosomal subunit and interferes with amino acid transfer to the growing peptide chain (mechanism unknown). b. Indications for use. Clindamycin's most important use is in the treatment of severe anaerobic infections caused by Bacteroides and other anaerobes. (1) Primary lung abscesses with susceptible pathogens and aspiration pneumonia (2) Intra-abdominal sepsis and intrapelvic infections (3) Orthopedic infections with susceptible pathogens (4) Acne (topically)

In a Nutshell Clindamycin • Used to treat anaerobic infections (e.g., Bacteroides) • Causes pseudomembranous colitis

c. Side effects and toxicity (1) Clindamycin can produce pseudomembranous enterocolitis, now described as antibiotic-associated colitis (AAC), resulting from suppression of intestinal organisms and proliferation of the anaerobe, Clostridium difficile. C. difficile is treatable with vancomycin.

(2) Like the aminoglycosides, it increases neuromuscular blockade in the presence of neuromuscular blocking agents . E. Chloramphenicol was isolated from cultures of Streptomyces and is the first completely synthetic antibiotic.

1. Pharmacologic properties a. A potent inhibitor of microbial protein synthesis, chloramphenicol binds to the 50S subunit of bacterial ribosome to block the action of peptidyltransferase. b. Chloramphenicol is bacteriostatic for many bacteria and rickettsiae. c. It can be administered parenterally or orally with good CNS penetration. d. It is inactivated by conjugation to glucuronide in the liver (90%), and its metabolites are rapidly excreted in the urine. e. It is clinically active against many strains of Gram-positive and Gram-negative bacteria, Rickettsia, anaerobes, and Mycoplasma. f. Gram-negative bacteria develop resistance via a factor acquired by conjugation, which induces acetyltransferase enzyme to inactivate chloramphenicol by acetylation. Resistant staphylococci contain an inducible form of chloramphenicol acetyltransferase for this purpose. 2. Indications for use a. Chloramphenicol is used as a treatment for acute typhoid fever and other serious Salmonella infections, particularly in developing nations; infections with ampicillinresistant H. influenzae, especially meningitis (some third-generation cephalosporins are also effective); and meningitis caused by N. meningitidis and Streptococcus pneumoniae in patients hypersensitive to penicillin. b. The drug is used as an alternative treatment of rickettsial infections when sulfonamides and tetracyclines cannot be used.

382

meClical

Antimicrobial Agents

c. In general, use is limited by toxicity; it is used only for serious infections when other agents are not viable alternatives. 3. Side effects and toxicity a. Pancytopenia, which is dose-related and often reversible, may occur.

In a Nutshell Protein Synthesis Inhibitors Bind to 30S ribosomal subunit: • Aminoglycosides

b. Aplastic anemia may occur in 1/30,000 patients. It is not dose-related and may occur months after drug use.

• Spectinomycin

e. Hemolytic anemia may occur in patients with glucose-6-phosphate (GGPD) deficiency.

• Tetracyclines

d. Nausea, vomiting, glossitis, stomatitis, diarrhea, and enterocolitis may occur.

Bind to 50S ribosomal subunit:

e. "Gray baby syndrome" occurs, especially in premature infants of mothers on chloramphenicol due to an infant's immature liver function (lacking glucuronide synthetase); it is potentially fatal. f. Chloramphenicol inhibits cytochrome P-450 and prolongs drug action when normally metabolized by this system.

• Erythromycin • C1indamycin • Chloramphenicol

FOLATE ANTAGONISTS A. Sulfonamides are rarely used alone because a large number of other drugs covering the same bacterial spectrum are available. However, the presence of certain opportunistic infections with AIDS has renewed interest in the sulfonamides for use against organisms that must synthesize folic acid. 1. Pharmacologic properties a. All are structurally similar to PABA and compete with PABA for enzyme sites on dihydropteroate synthetase to inhibit folic acid synthesis. b. Their action is bacteriostatic. e. A large fraction is bound to plasma proteins and distributed throughout body water. It penetrates CSF and crosses the placenta.

d. Sulfonamides are acetylated in liver and eliminated by glomerular filtration. e. Resistance mechanisms include: (1) Increased bacterial synthesis of PABA to overcome competitive inhibition or

through structural changes in the enzyme. (2) Inactivation of the drug by acetylation (3) Acquired ability to use preformed folate 2. Indications for use a. Usage has diminished due to resistance and the availability of penicillins. However, in combination with the antimalarial drug, trimethoprim, sulfonamide inhibits Pneumocystis jiroveci and Isospora belli, opportunistic infections of AIDS patients. b. Sulfonamides are used to treat acute urinary tract infections caused by susceptible strains of E. coli, Klebsiella, Enterobacter, Staphylococcus aureus, Proteus, and Staphylococcus saprophyticus. e. They are the drugs of choice for treatment of Nocardia.

d. Sulfadiazine is used with pyrimethamine for the treatment of toxoplasmosis and chloroquine-resistant falciparum malaria. KAPLA~.

meulCa

I 383

Microbiology

e. They are alternative drugs for the treatment of chancroid, trachoma, and inclusion conjunctivitis (tetracycline or erythromycin is first choice). f. They are used as an alternative treatment for lymphogranuloma venereum (tetracycline is first choice). 3. Side effects and toxicity a. Hypersensitivity rashes, eosinophilia, and angioedema; kernicterus (displaces bilirubin from albumin) and increased serum bilirubin levels in neonates; Stevens-Johnson syndrome (rare); and hematologic disorders such as agranulocytosis, aplastic anemia, thrombocytopenia, and hemolytic anemia in G6PD deficiency. b. Drug interactions can occur, resulting in potentiation of oral hypoglycemics, warfarin, and phenytoin by displacing them from albumin. 4. Individual agents (Table V-lS-3) Table V-lS-3. Sulfonamides. Agent

Important Features

Clinical Usage

Sulfisoxazole

Short-acting agent; rapidly absorbed and eliminated; low incidence of crystalluria

Sulfadiazine

Short-acting agent; high incidence of crystalluria; must hydrate patient

Sulfamethoxazole

Intermediate-acting agents; similar to sulfisoxazole but slower absorption and excretion; high risk of crystalluria

Sulfasalazine

Very poor gastrointestinal absorption

Main usage for urinary tract infections; also active against lymphogranuloma venereum and chancroid; can be used to treat Nocardia Best sulfonamide for treatment of meningitis; used for treatment of nocardiosis; used in combination with pyrimethamine for treatment of toxoplasmosis Usage similar to sulfisoxazole; marketed in combination with trimethoprim (Bactrim, Septra) for treatment of urinary tract infections, shigellosis, prostatitis; combination also used for treatment of P. jiroveci Used for treatment of ulcerative colitis and regional enteritis Used in ophthalmic infections Used to prevent colonization and infection of wounds and burns Same as silver sulfadiazine

Sulfacetamide Most often used as a topical solution Silver sulfadiazine Topical solution Mafenide Sulfadoxine

Topical solution; inhibits carbonic anhydrase; may cause metabolic acidosis Very long-acting; risk of severe reactions, such as Stevens-Johnson syndrome, limits use

Used in combination with pyrimethamine for treatment and prophylaxis of chloroquineresistant Plasmodium falciparum malaria; has also been used in prophylaxis of AIDS-related pneumonia caused by P. jiroveci

B. Trimethoprim-sulfamethoxazole 1. Pharmacologic properties. As indicated, these drugs are a combination of a sulfa drug

with an antimalarial drug, also known as co-trimoxazole, consisting of five parts sulfamethizole to one part trimethoprim. a. The synergistic combination acts as an inhibitor of two sequential steps in the synthesis of folic acid. b. Sulfamethoxazole inhibits PABA incorporation into folic acid, and trimethoprim inhibits the reduction of dihydrofolic acid to tetrahydrofolic acid.

384

meClical

Antimicrobial Agents

2. Indications for use. These drugs are used in the treatment of acute and chronic urinary tract infections, including prostatitis, shigellosis, salmonellosis carriers, respiratory infections caused by H. infiuenzae and Streptococcus pneumoniae, Pneumocystis jiroveci (treatment and prophylaxis) in immunosuppressed hosts, and as alternatives to chloramphenicol and ampicillin in treating typhoid and paratyphoid fevers. It is also used for the treatment of Isospora belli. 3. Side effects and toxicity a. These include hypersensitivity reactions, rarely, Stevens-Johnson syndrome; gastrointestinal disturbances with glossitis and stomatitis; and nephrotoxicity. b. Blood dyscrasias include megaloblastic anemia, leukopenia, and thrombocytopenia. e. Patients with AIDS being treated for P. jiroveci often have reactions that include fever, rash, and, sometimes, pancytopenia.

NUCLEIC ACID INHIBITORS A. Fluoroquinolones: ciprofloxacin and norfloxacin are newer antibiotics with a unique action on enzymes that catalyze the direction and extent of DNA supercoiling. 1. Pharmacologic properties

In a Nutshell Uses of Sulfonamides • Urinary tract infections (+/trimethoprim depending on severity) • Pneumocystis jiroved pneumonia (with trimethoprim) • Isospora belli (with trimethoprim) • Nocardia infections • Toxoplasmosis (with pyrimethamine) Chloroquine-resistant falciparum malaria (with pyrimethamine)

a. Fluoroquinolones are structurally related to nalidixic acid, an older quinolone. b. They possess rapid oral absorption and are mostly excreted in urine. e. Mechanism involves inhibition of bacterial DNA gyrases.

d. They are the only oral agents effective against Pseudomonas. 2. Indications for use. Ciprofloxacin and norfloxacin are used in Gram-negative infections. a. Norfloxacin is effective in UTls, including resistant and recurrent organisms, and in prostatitis resulting from E. coli infections. b. Ciprofloxacin is effective for treating UTls, gonorrhea, diarrheal diseases, and soft tissue infections. It has been shown to be effective against respiratory infections (resulting from Haemophilus or Streptococcus pneumoniae), in bronchitis, and in Pseudomonas infections in cystic fibrosis patients. 3. Side effects and toxicity. Adverse reactions are mostly gastrointestinal with rash and crystalluria (rare). B. Nitrofurantoin 1. Pharmacologic properties a. Nitrofurantoin is effective against many Gram-positive and Gram-negative bacteria, but not against Pseudomonas. b. Its mechanism of action is through the damage of bacterial DNA. e. It is rapidly absorbed and undergoes urinary excretion. It shows no systemic antibacterial activity.

2. Indications for use. Nitrofantoin is limited solely as a urinary antiseptic for acute or chronic UTIs. 3. Side effects and toxicity a. Gastrointestinal upset KAPLAlf I me dlea 185

Microbiology

Clinical Correlate

b. Pulmonary inftltrates and fibrosis (may be fatal)

Antimicrobials to Avoid

c. Peripheral neuropathy (especially in patients with renal impairment)

in Patients with G6PD Deficiency

d. Hemolytic anemia (should not be used in patients with G6PD deficiency)

• Nitrofurantoin • Primaquine • Sulfa Drugs • Chloramphenicol • Dapsone

Note Mycobacteria were discussed in detail in the Mycobacteria and Actinomycetes chapter of this section.

ANTIMVCOBACTERIAL AGENTS M.tuberculosis invades many organs and requires prolonged therapy. Because single-drug therapy for a long period (weeks) allows the growth of resistant mutants, TB must be treated simultaneously with two or more drugs. Standard therapy for pulmonary and extrapulmonary TB may be either isoniazid and rifampin for 9 months or isoniazid and ethambutol for 8 months. In cases of infection with drug-resistant TB, overwhelming disseminated TB (miliary), or TB meningitis, three-drug regimens are often used. Atypical Mycobacterium infections (e.g., Mycobacterium avium-intracellulare) are usually treated with multidrug regimens. A summary of these agents is provided in Table V-lS-4. A. Treatment of tuberculosis. The number of cases in the U.S. has increased dramatically in part due to immigration, AIDS, and the number of homeless individuals.

Table V-lS-4. Drugs used to treat tuberculosis and leprosy. Drug

Adverse Effects

Isoniazid

Elevation of hepatic enzymes, peripheral neuropathy, hepatitis, CNS effects, and increased phenytoin concentration Orange-red discoloration of secretions, urine, tears, and contact lenses; hepatitis, drug fever, flu-like symptoms, and thrombocytopenia; interferes with methadone, warfarin, medroxyprogesterone, theophylline, dapsone, and ketoconazole Gastrointestinal upset, elevation of liver enzymes, rash, arthralgia, and hyperuricemia Optic neuritis (everything appears green), decreased visual acuity, and skin rash Ototoxicity, nephrotoxicity, hypokalemia, and hypomagnesemia Abdominal cramps, gastrointestinal upset, insomnia, headache, photosensitivity, and hypersensitivity reactions; drug interactions with warfarin and theophylline Auditory and renal toxicity, vestibular toxicity (rare), hypokalemia, and hypomagnesemia Gastrointestinal upset, bloating, liver enzyme elevation, metallic taste, and hypothyroidism (especially if on para-aminosalicylic acid [PAS]) Psychosis, depression, seizures, rash, headache, and increased phenytoin concentrations Gastrointestinal upset, elevated liver enzymes, sodium loading, decreased digoxin, and increased phenytoin levels Orange-brown skin discoloration, gastrointestinal complaints, and rare visual disturbances Anemia, rash, and methemoglobinemia

Rifampin

Pyrazinamide Ethambutol Streptomycin Ciprofloxacin

Amikacin Ethionamide Cycloserine Para-aminosalicylic acid Clofazimine Dapsone

386

iileilical

Antimicrobial Agents

1. Isoniazid (INH) is a primary agent in all treatment measures. a. Pharmacologic properties (1) Inhibits biosynthesis of mycolic acids (mycobacterial cell wall components).

(2) INH is absorbed well after oral administration and demonstrates 20% CSF penetration. (3) The drug is metabolized in the liver by acetylation, and the speed of acetylation-and consequently INH's half-life-is genetically determined (fast versus slowacetylators). (4) There is no activity against atypical mycobacteria other than M. kansasii. b. Side effects and toxicity (1) Peripheral neuropathy is a most common side effect, but it can be prevented by the administration of pyridoxine (vitamin B6 ). (2) Hepatotoxicity usually occurs in a mild transient form during the initial 1-2

months of therapy. Risk increases with age and pre-existing liver disease. (3) Rash, fever, and eosinophilia may occur. (4) Induction of antinuclear antibody (ANA) and lupus-like syndrome may occur. (5) Bone-marrow depression and arthritis are rare effects. 2. Rifampin

Clinical Correlate Drug-induced, lupus-like syndrome with a positive ANA is seen with INH, hydralazine (antihypertensive), and procainamide (antiarrhythmic). Symptoms abate with drug withdrawal. This is a USMLE favorite side effect.

a. Pharmacologic properties (l) Rifampin is a bactericidal agent with activity against intra- and extracellular TB organisms. It is also active against certain Gram-positive bacteria (e.g., S. au reus) and certain Gram-negative bacteria (e.g., N. meningitidis). It is used prophylactically for people exposed to meningitis caused by H. inJluenzae or meningococci. It eliminates nasal carriage of methicillin-resistant S. aureus and N. meningitidis.

(2) The mechanism of action involves the inhibition of DNA-dependent RNA polymerase, thereby blocking RNA synthesis. (3) There is oral absorption with metabolism in the liver and then enterohepatic circulation. (4) Resistance emerges if rifampin is used alone against TB. (5) Rifampin is also active against atypical mycobacteria and M. leprae. b. Side effects and toxicity rarely occur. (l) Rifampin may color urine, saliva, sputum, and tears orange-red, but this discoloration is harmless.

(2) Rash, fever, nausea, vomiting, jaundice, and hepatotoxicity may occur. (3) Blood dyscrasias include leukopenia, thrombocytopenia, and anemia. (4) There is stimulatory interaction with the cytochrome P-450 system. This decreases the oral anticoagulant effect, may render oral contraceptives ineffective, decreases the glucocorticoid effect, decreases digitoxin levels, decreases quinidine levels, and decreases oral hypoglycemic and barbiturate effects. This also decreases methadone levels, which may precipitate withdrawal.

In a Nutshell Rifampin is secreted into many body fluids and may discolor them (orange-red). It also stimulates the cytochrome P450 system, resulting in increased elimination of anticoagulants, digitoxin, oral contraceptives, methadone, barbiturates, etc.

meClical

387

Microbiology

B. Treatment of leprosy. Mycobacterium leprae is responsible for the development of leprosy. 1. Dapsone is an organic sulfone but is unlike the sulfonamides.

a. Pharmacologic properties (1) Dapsone inhibits folate synthesis with a mechanism like that of the sulfonamides; it is bacteriostatic for M. leprae. (2) Gastrointestinal absorption from the upper gastrointestinal tract is 90% complete. It is widely distributed and active in the skin. It is acetylated in the liver like isoniazid, and it is excreted as the glucuronide and sulfate conjugates. b. Indications for use. Dapsone is the preferred drug in the treatment of leprosy, usually in combination with either rifampin or clofazimine. Because leprosy is a chronic disease, therapy is usually continued for several years. c. Side effects and toxicity (1) Hemolysis occurs and can be severe with G6PD deficiency. (2) Methemoglobinemia can occur. (3) Nausea, vomiting, anorexia, and headache can occur. (4) Dapsone can cause fatal mononucleosis-like syndrome. 2. Rifampin (see above) 3. Clofazimine a. Clofazimine is a phenazine dye that inhibits DNA template function. b. Clofazimine is bactericidal against M. leprae; it is also active against M. avium intra-

cellulare. c. It is absorbed gastrointestinally and accumulates in tissues.

ANTIVIRAL AGENTS Viruses are obligate intracellular parasites that depend on the metabolism of the host cell. Therefore, agents that inhibit or kill viruses are also likely to injure the host cells that harbor them. Developmental approaches in the design of antiviral agents have focused on the selective inhibition of enzyme systems unique to virus-infected cells. However, at this time, very few agents have been found to be both effective and safe. A. Treatment of herpesviruses 1. Acyclovir requires phosphorylation to be effective. It is phosphorylated efficiently by a virus-specific thymidine kinase and becomes trapped within virus-infected cells. Guanine analog (acycloguanosine) is converted to the triphosphate, the active form of the drug, which inhibits virus-specific DNA polymerase.

a. Pharmacologic properties (1) Acyclovir can be used orally or intravenously.

(2) It is only slightly protein bound. It is well distributed, including in the CNS. (3) Acyclovir inhibits multiplication of various herpesviruses, including varicellazoster, herpes simplex types I and II, and Epstein-Barr virus. It is not effective against cytomegalovirus (CMV).

388

meClical - - - - - - - - - - -

----

Antimicrobial Agents

b. Indications for use (1) It is used as a topical treatment for herpes simplex infections with little skin penetration. (2) Intravenous treatment is required for mucosal, cutaneous, and disseminated herpes simplex infections and treatment of herpes zoster or chickenpox in immunocompromised hosts. (3) Oral administration can lessen the severity of genital herpes if administered early in the attack, but intravenous therapy is necessary for severe infections. c. Side effects and toxicity

(1) Nephrotoxicity may occur. (2) Encephalopathy, bone marrow depression, and abnormal hepatic function may occur. 2. Vidarabine, also called ara-A, is an adenosine arabanoside that is phosporylated within cells and inhibits viral DNA polymerases. a. Pharmacologic properties (1) Vidarabine is available for intravenous and topical administration.

(2) It inhibits viral DNA polymerase. (3) It should be administered intravenously with a lot of fluid. b. Indications for use (1) It is used in the treatment of ocular herpes simplex keratoconjunctivitis. (2) It is used in the parenteral treatment of herpes simplex encephalitis (if acyclovir resistant). (3) It is used in the treatment of herpes zoster infections in patients with suppressed immunologic response. c. Side effects and toxicity

(1) Ophthalmic preparation is less irritating than idoxuridine, but it may cause lacrimation, burning, conjunctival and corneal edema, and photophobia. (2) Systemic infusion may result in nausea, vomiting, diarrhea, hallucinations, ataxia, psychoses, and tremor. 3. Ganciclovir a. Pharmacologic properties. Ganciclovir is a synthetic analog of guanosine whose mode of action is similar to that of acyclovir; i.e., it inhibits viral DNA polymerase after being converted to the triphosphate form in the infected cell.

In a Nutshell

b. Indications for use (1) Available only in intravenous form, ganiclovir is active against CMV and other herpesviruses.

Ganciclovir ~ CMV

(2) It is primarily used for CMV infections in immunocompromised patients. (3) It is more toxic than acyclovir, so use is restricted to CMV.

KAPLAN· . I med lea

389

Microbiology

c. Side effects and toxicity

(1) Bone marrow suppression with granulocytopenia occurs in up to 40% of patients and thrombocytopenia occurs in 20%. (2) Hepatotoxicity may occur. 4. Foscarnet a. Pharmacologic properties. Foscarnet inhibits viral DNA polymerase. Unlike acyclovir and ganciclovir, foscarnet does not need to be activated by a viral kinase. b. Indications for use. It is effective in CMV retinitis and herpesvirus infections that are resistant to ganciclovir and acyclovir, respectively. c. Side effects and toxicity are more common with foscarnet than ganciclovir.

(1) Nephrotoxicity may occur. (2) Bone marrow suppression may occur but is less than that seen with ganciclovir. 5. Idoxuridine (IdUR) is used only topically. a. Pharmacologic properties (1) As a thymidine analog, it can be used to treat herpes simplex keratitis and dendritic ulcers. (2) It is phosphorylated in cells; triphosphate derivative is incorporated into both viral and mammalian DNA. (3) It renders DNA more susceptible to breakage with altered viral proteins secondary to faulty transcription. (4) It is restricted to topical application because of liver and bone marrow toxicity.

In a Nutshell Neuraminidase inhibitors (zanamivir, oseltamavir) are now available for the treatment of influenza and are active against influenza A and B.

b. Indications for use. Idoxuridine is used in the treatment of herpes simplex infections of cornea, conjunctiva, and eyelids, including herpes keratitis. c. Side effects and toxicity include conjunctival irritation, photophobia, edema of the eyelids and cornea, and corneal epithelium punctuate defects. B. Treatment of respiratory viral infections 1. Amantadine and rimantadine are used for the treatment and prophylaxis of influenza A. They are ineffective against influenza B. a. Pharmacologic properties (1) Amantadine is completely absorbed from the gastrointestinal tract after oral

administration, and 90% is excreted unmetabolized in urine. Rimatadine is metabolized extensively in the liver and only 25% is excreted unchanged in urine. (2) The drugs inhibit replication of orthomyxoviruses (specifically influenza A) by interfering with viral protein uncoating. They are basic amines that elevate the pH of the cytoplasmic vesicle in which the virus resides and thus inhibit fusion of viral peptomeres with the vesicle membrane, the prelude to uncoating. (3) Rimantadine has a longer plasma half-life than amantadine. b. Indications for use (1) Prophylactic administration of the drug to high-risk patients in the presence of an influenza A virus epidemic

390

meCtical

Antimicrobial Agents

(2) Treatment shortens the duration of influenza symptoms in patients with influenza A if administered within the first 12-24 hours. (3) Amantadine is used in treating symptoms of Parkinson disease because it causes the release of dopamine and other catecholamines from neuronal storage sites and delays reuptake of these neurotransmitters into synaptic vesicles. c. Side effects and toxicity (1) Common CNS reactions include irritability, tremor, slurred speech, ataxia,

depression, insomnia, lethargy, and dizziness. (2) Drug interactions between amantadine and anticholinergic drugs such as benztropine mesylate (Cogentin) may cause confusion and hallucinations in patients. (3) Rimantadine has fewer side effects than amantadine.

In a Nutshell Current HIV therapy consists of two nucleoside reversetranscriptase inhibitors (AZT) and one protease inhibitor. This highly active retroviral therapy (HAART) results in a J- in viral RNA, an i in C04+ count, and a J- in opportunistic infections.

In a Nutshell

2. Ribavirin is a synthetic purine nucleoside analog active against respiratory syncytial virus. a. Pharmacologic properties. Ribavirin undergoes phosphorylation, interfering with nucleotide synthesis, viral mRNA production, and viral protein production (mechanism unknown). b. Indications for use. Aerosols are used to treat respiratory syncytial virus in infants and children.

Antivirals • Acyclovir

-7

Inhibits viral DNA polymerase. Used to treat HSV, VZV, and EBV.

• Vidarabine (ara·A)

-7

Inhibits viral DNA polymerase. Used primarily in invasive HSV infections resistant to acyclovir (e.g., encephalitis).

• Ganciclovir

-7

Inhibits viral DNA polymerase. Used to treat (MV infections.

Foscarnet

-7

Inhibits viral DNA polymerase. Used to treat ganciclovirresistant (MV retinitis in imrnunocornprornised patients.

Idoxuridine (ldUR)

-7

Inhibits enzymes involved in DNA synthesis. Used topically for treatment of herpetic keratitis.

Amantadine, Rimantadine

-7

Interfere with influenza A virus penetration and uncoating. Used in treatment and prophylaxis of influenza A.

c. Side effects and toxicity include anemia and conjunctivitis. C. Treatment of human immunodeficiency virus (HIV). Because antiviral drugs inhibit virus

replication after infection has occurred, they are most effective when given at an early stage of infection. Current practice is to use these drugs in asymptomatic or mildly symptomatic persons in whom the CD4+ cell number drops below SOO/Ill or to use them as prophylaxis in individuals accidentally exposed to HIV or born of HIV + mothers. 1. Zidovudine (AZT) a. Pharmacologic properties (1) AZT is a thymidine analog that terminates DNA transcription by viral reverse transcriptase, preventing viral replication. (2) AZT penetrates the CNS and undergoes hepatic and renal excretion. b. Indications for use. AZT is used in the treatment of AIDS and various stages of HIV infection (studies suggest prolonged survival in HIV-positive patients). c. Side effects and toxicity. Side effects are numerous, but toxicity associated with bone marrow suppression is the most serious problem. ( 1) Anemia, which may be severe and require transfusions, can occur. (2) Megaloblastic erythrocyte changes and granulocytopenia may occur within weeks of therapy, but using lower doses of AZT decreases side effects. (3) Liver function abnormalities are exacerbated by acetaminophen, and bone marrow toxicity is exacerbated by ganciclovir. (4) Less severe adverse effects of AZT include headache, insomnia, diarrhea, rashes, and fever.

(Continued)

2. Dideoxycytidine (ddC) and dideoxyinosine (ddI) also inhibit reverse transcriptase. They have activity against AZT-resistant HIV. Both drugs cause peripheral neuropathies.

I KAPLAlf _ medlea

391

Microbiology

3. 3-Thiacytidine (3TC) is an inhibitor of reverse transcriptase. It is most often used in conjunction with AZT. Pancreatitis is the major adverse side effect, particularly in infants. Ribavirin

~

Interferes with nucleic acid synthesis (DNA and RNA). Used to treat infections caused by respiratory syncytial virus. Also effective against Lassa fever.

Zidovudine (All)

~

Inhibits reverse transcriptase. Used in treatment of HIV.

ddC, ddl, and

~

Same mechanism as AIr. Used in AZT-resistant cases.

3TC Protease inhibitors (ritonavir, saquinovir, indinavir)

Interferons (ot, ~, 'I)

~

~

Newer agents used in HIV treatment. Block viral protease required for normal viral protein synthesis. Interfere with viral protein synthesis. Used in treatment of hairy cell leukemia, Kaposi sarcoma, and condylomata acuminatum.

4. Protease inhibitors such as ritonavir, saquinovir, and indinavir are the newest drugs used in the treatment of HIV. They interfere with the viral protease that cleaves the polypeptide chain translated from the polycistronic viral mRNA, thus, the gag-pol polyprotein is not cleaved and functional reverse transcriptase is not produced. These drugs are usually used in conjunction with reverse transcriptase inhibitors. Adverse reactions most commonly seen are diarrhea, abdominal discomfort, and nausea. D. Human interferons are naturally occurring glycoproteins produced in response to viral infection. They inhibit viral replication and promote antiviral responses. Three types of interferon are produced: interferon a. (produced by monocytes), interferon ~ (produced by fibroblasts), and interferon y (produced by T lymphocytes). 1. Pharmacologic properties

a. Human interferons (IFNs) are small glycoproteins produced by nucleated cells of the body in response to viruses (especially double-stranded nucleotides). b. Once released, IFNs inhibit viral multiplication in other cells by inducing cellular enzymes that block protein synthesis and therefore viral protein synthesis. (I) The induction of a protein kinase phosphorylates elongtion factor 2, thereby preventing the initiation of protein synthesis. (2) A kinase induced in the cell synthesizes an adenine trinucleotide that activates an endonuclease that cleaves mRNA. c. The interferons have been produced by recombinant DNA techniques and can be generated in large amounts. d. IFNs are administered intramuscularly, subcutaneously, or intravenously. They are not effective orally. 2. Indications for use. IFNs are used in the treatment of hairy cell leukemia, Kaposi sarcoma in patients with AIDS; condylomata acuminatum (genital warts); and herpes keratoconjuctivitis, in combination with other antiviral agents. 3. Side effects and toxicity a. A flu-like syndrome is experienced during the first few days of treatment, including fever, chills, headache, nausea, and vomiting. b. There may also be bone marrow suppression, neurotoxicity, elevated liver function tests, and nephrotoxicity.

ANTIFUNGAL AGENTS A. Polyene antibiotics. Polyene antibiotics are effective against both filamentous and yeast-like fungi, including Histoplasma, Blastomyces, Coccidioides, Cryptococcus, Candida, Aspergillus, Mucor, and Rhizopus species. The polyenes have no activity against dermatophytes or bacteria. Polyenes interact with ergosterols in the cytoplasmic membrane of fungi, leading to rapid leakage of small molecules and fungal death. 1. Amphotericin B

a. Pharmacologic properties (1) Systemic infections are treated by slow intravenous infusion; amphotericin B

cannot be administered orally.

392

meClical

Antimicrobial Agents

(2) The drug binds to serum

~-lipoproteins

and tissue membranes.

(3) There is poor eNS penetration; intrathecal administration may be required for fungal meningitis. (4) The drug re-enters the circulation slowly from tissue depots with a small portion excreted in bile (major route) or urine over weeks. b. Indications for use (1) Amphotericin B is a broad-spectrum antifungal agent used for treatment of sys-

temic fungal infections. It is active against Histoplasma, Cryptococcus, Candida, Blastomyces, and Aspergillus. (2) The drug is also used in the treatment of mucocutaneous leishmaniasis and amebic meningoencephalitis (freshwater amebae). c. Side effects and toxicity (1) Low therapeutic index (small test dose usually given)

(2) Fever, chills, nausea, vomiting, headache, diffuse pain, and hypotension (3) Nephrotoxicity, including decreased glomerular fIltration rate, azotemia, renal tubular acidosis, and hypokalemia (4) Normochromic, normocytic anemia from bone-marrow suppression 2. Nystatin (mycostatin) a. Pharmacologic properties (1) Its structure and mechanism of action are similar to amphotericin B, but it is more toxic than amphotericin B. (2) It is used primarily in topical preparations but can be taken orally for oral and esophageal candidiasis. It is not for parenteral administration. (3) It is not absorbed from skin, mucous membranes, or the gastrointestinal tract. b. Indications for use include candidal infections of skin, mucous membranes, and vagina; and prophylaxis to prevent intestinal fungal overgrowth in patients on chemotherapy. c. Side effects and toxicity include nausea and vomiting (rare). B. Imidazoles. These agents inhibit 14-alpha-demethylase and block the synthesis of fungal

cell membrane ergosterol, leading to increased membrane permeability and loss of essential nutrients. 1. Miconazole and clotrimawle

Note

a. Pharmacologic properties. Miconazole and clotrimazole are topically active antifungals.

Aspergillus is very resistant to

b. Indications for use. The drugs are used in the treatment of ringworm and vulvovaginal candidiasis. Intravenous miconazole is rarely used because of toxicity.

these imidazole antifungals. Aspergillosis is treated with amphotericin B.

c. Side effects and toxicity

(1) Intravenous miconazole most commonly causes nausea, phlebitis, anemia, thrombocytopenia, and pruritus. (2) Hyponatremia occurs in 50% of patients. (3) Arthralgias, anaphylaxis, acute psychosis, and hyperlipidemia may also occur.

I KAPLAlf medlea

393

Microbiology

Note

2. Ketoconazole, itraconazole, and fluconazole

Ketoconazole absorption is favored by low pH; absorption is therefore decreased by antacids and H2 blockers. Fluconazole is unaffected by pH.

a. Pharmacologic properties (1) Administered orally

(2) Ketoconazole is metabolized in the liver. b. Indications for use (1) Ketoconazole is used to treat coccidioidomycosis, histoplasmosis, blastomycosis, paracoccidioidomycosis, and mucocutaneous candidiasis. (2) Itraconazole is used in blastomycosis, histoplasmosis, and aspergillosis.

(3) Fluconazole is used in treatment of cryptococcal meningitis in AIDS patients and in severe cases of candidiasis.

c. Side effects and toxicity are rare. C. Miscellaneous antifungal agents

1. Flucytosine a. Pharmacologic properties (1) The mechanism of action involves conversion to 5-fluorouracil in fungal cells, then metabolism to 5-fluorodeoxyuridylic acid, an inhibitor of thymidylate synthetase (pyrimidine antimetabolite). (2) It is well absorbed orally. (3) CSF levels are 80% of serum levels. (4) Eighty percent of the drug is excreted in urine by glomerular filtration.

b. Indications for use (1) Flucytosine is administered with amphotericin B in the treatment of cryptococcal meningitis and systemic candidiasis.

(2) It is rarely used alone because of rapid development of resistance.

c. Side effects and toxicity (1) Hematologic effects include anemia, leukopenia, and thrombocytopenia. (2) Nausea, vomiting, diarrhea, and enterocolitis can occur.

2. Griseofulvin a. Pharmacologic properties (1) Griseofulvin inhibits fungal mitosis by interacting with microtubules to cause mitotic spindle disruption. (2) It must be taken orally to be effective. (3) It binds to keratin and is deposited in skin, hair, and nails (keratin-precursor cells), where it is taken up by the fungus. b. Indications for use. Griseofulvin is used in the treatment of dermatophytic infections with Epidermophyton, Microsporum, and Trichophyton species.

394

meClical

Antimicrobial Agents

c.

Side effects and toxicity (1) CNS effects include headache, lethargy, confusion, fatigue, syncope or vertigo

(less often), gastrointestinal disturbances, and hepatotoxicity. (2) Hematologic effects include leukopenia and granulocytopenia with long-term and high-dose therapy. (3) Drug interactions involve reduction in warfarin activity (increased metabolism) and decreased barbiturate absorption from the alimentary tract. 3. Trimethoprim-sulfamethoxazole and pentamidine are used in the treatment of Pneumocystis jiroveci pneumonia. a. Trimethoprim-sulfamethoxazole is a first-line drug used in both prophylaxis and therapy in immunocompromised patients. b. Pentamidine is administered by aerosol for treatment of P. jiroveci pneumonia. It can be used intramuscularly for the treatment of leishmaniasis and trypanosomiasis.

ANTI PROTOZOAL AGENTS Therapy for protozoal infections include chemotherapeutic techniques, control of the insect vector, eradication of the infection reservoir, and improvement of sanitation and living conditions. Antihelminthic agents expel worms from the gastrointestinal tract and eliminate helminths that have migrated into body tissues. A. Antimalarial drugs are usually classified according to the stage of the life cycle that they affect. They include treatment of the acute attack, drugs to effect radical cure, drugs for prophylaxis, and those used to prevent transmission. 1. Chloroquine is very effective against erythrocytic forms but not against the liver stages. a. Pharmacologic properties (1) Chloroquine inhibits the ability of parasites to digest hemoglobin and thus interferes with their viability. (2) Chloroquine also intercalates into the DNA of the parasite and causes cessation of DNA synthesis. b. Indications for use (1) Chloroquine is used in prophylaxis and treatment of susceptible Plasmodium organisms. (2) It is the drug of choice for treatment of malaria, although the increasing incidence of resistant P. falciparum strains limits its use. (3) It is used as a treatment for extraintestinal amebiasis. (4) It is occasionally used in rheumatoid arthritis and SLE because of its marked anti-inflammatory action. c.

Side effects and toxicity (1) Mild pruritus, nausea, headache, and vomiting may occur. (2) Hematologic abnormalities may occur. (3) There may be quinidine-like effects and fatal arrhythmias with high doses. (4) There may be retinopathy with long-term use. KAPLA~. I meulca 395

Microbiology

2. Primaquine a. Pharmacologic properties (1) Primaquine is an 8-aminoquinoline compound that causes hemolysis in patients

with "primaquine sensitivity:' a genetic deficiency of erythrocyte G6PD. It may cause methemoglobinemia with cyanosis. (2) It binds to DNA and interferes with cell division. (3) Primaquine is a liver schizonticide and is also effective against the gametocytic form. b. Indications for use. Primaquine is used for prophylaxis. It is also the only drug known to provide a radical cure of malaria caused by P. vivax or P. ovale; it kills the exoerythrocytic form of the parasite. 3. Quinine a. Pharmacologic properties (1) Quinine is an alkaloid with local anesthetic and irritant actions. Its mechanism of action is unknown, but it binds to malarial pigment, hemozoin, and may intercalate in the DNA. (2) It is effective against the erythrocytic stage (blood schizonticide) of all species of Plasmodium. (3) It undergoes hepatic metabolism with renal excretion of metabolites. (4) A weak analgesic, antipyretic agent, it has a depressant action on the heart and an oxytoxic effect on the gravid uterus. (5) It has a curare-like effect on skeletal muscle. b. Indications for use (1) It is an alternative to chloroquine when chloroquine resistance occurs. (2) It is used alone or with tetracycline, pyrimethamine, and a sulfonamide to treat chloroquine-resistant strains of P. falciparum. (3) It is used for the treatment of nocturnal leg cramps and myotonia congenita. (4) It is used for the treatment of babesiosis. c. Side effects and toxicity (1) Cinchonism includes tinnitus, headache, blurred vision, nausea, and diarrhea.

Urticaria and pruritus often occur with use. (2) "Blackwater fever" results from acute hemolysis, hypoprothrombinemia, thrombocytopenic purpura, and agranulocytosis with kidney failure. (3) Because of a curare-like effect, it may worsen symptoms of myasthenia gravis. (4) Excessive levels of quinine may cause hypotension, cardiac arrhythmias, deliriurn, and coma. 4. Mefloquine a. Pharmacologic properties (1) A chloroquine derivative, it is a blood schizonticide active against P. falciparum

and P. vivax. (2) Well absorbed orally, its long half-life is probably due to enterohepatic recycling.

396

meClical

Antimicrobial Agents

(3) It is thought to interfere with transport of nutrients from the host. (4) It is excreted through feces. b. Indications for use (1) It is the only antimalarial agent that is effective alone against multiresistant strains. (2) It is used prophylactically for travelers to areas where chloroquine-resistant strains are endemic (e.g., Southeast Asia, South America, and sub-Saharan Africa). c. Side effects and toxicity

(1) Gastrointestinal side effects are most common. (2) Use is contraindicated in pregnancy and for patients on channel blockers, and other antiarrhythmics.

~-blockers,

calcium-

5. Pyrimethamine a. Pharmacologic properties (1) Pyrimethamine is similar in structure to trimethoprim. Both are potent dihydrofolate reductase inhibitors of folic acid synthesis. (2) When used with a sulfonamide, it inhibits two consecutive steps in the formation of folic acid from PABA in parasites. b. Indications for use. Pyrimethamine is given in combination with quinine in the treatment of acute malarial attacks with P. falciparum and in the treatment of toxoplasmosis (in combination with a sulfonamide). c. Side effects and toxicity. There may be significant hematologic abnormalities at increased dosages. 6. Tetracycline and doxycycline may be used in the prophylaxis and treatment of malaria. B. Agents used in the treatment of amebiasis. Amebiasis refers to an infection caused by

Entamoeba histolytica. Dysentery with diarrhea and abdominal pain are common features, although infected individuals may remain asymptomatic while continuing to spread the disease. The cecum and colon are sites of primary involvement, but liver abscesses may develop. 1. Metronidazole

a. Pharmacologic properties (1) A reduced nitro group acts as an electron acceptor to deprive cells of required

reducing equivalents; it may also cause DNA strand breakage with subsequent functional impairment. (2) There is good absorption after oral administration; it is given intravenously or orally. (3) It has both gastrointestinal luminal and systemic activity; it is used for both intestinal amebiasis and amebic liver abscesses (with chloroquinine). (4) It is usually given with a drug acting luminally, such as diloxanide furoate. (5) It undergoes hepatic metabolism. (6) There is good CSF penetration. b. Indications for use (1) It is used in the treatment of all amebic infections except asymptomatic intestinal amebiasis.

Note Metronidazole, in addition to its antiparasitic activity, is also useful for treating anaerobic bacterial infections.

I KAPLAlf medlea

397

Microbiology

(2) It is the agent of choice in the treatment of trichomoniasis and giardiasis. (3) There is general anaerobic coverage, such as in postsurgical abdominal and pelvic infections with Bacteroides fragilis and in flare-ups of intestinal diverticulitis. (4) It is a good agent for treating most brain abscesses. c. Side effects and toxicity ( 1) Gastrointestinal effects include nausea, vomiting, anorexia, and epigastric distress. (2) CNS effects are rare but include seizures, neuropathy, and ataxia. (3) A disulfiram-like reaction may occur; thus patients should not consume alcohol. (4) Because of metabolic interactions, metronidazole effects are diminished when given with phenobarbital, but it can decrease warfarin metabolism. (5) It should be avoided in pregnant women, especially during the first trimester. 2. Diloxanide furoate a. Pharmacologic properties (1) Diloxanide is an insoluble ester whose mechanism of action is unknown. (2) It exerts a luminal amebicidal action directly in the bowel lumen, but it has no systemic effects. (3) It is absorbed after oral administration. b. Indications for use (1) Diloxanide is the drug of choice for the treatment of asymptomatic cyst carriers. (2) It is not effective against hepatic abscesses. c. Side effects and toxicity. Diloxanide is generally well-tolerated; flatulence is the most common side effect. 3. Chloroquine a. Pharmacologic properties (1) Liver concentration is 100 times that of plasma concentration.

(2) It achieves only low concentration in the intestinal wall, so it is not useful in treating intestinal amebiasis. b. Indications for use. Chloroquine is used in the treatment of extraintestinal amebiasis, especially hepatic amebiasis, and malaria. C. Agents used in the treatment of giardiasis. Giardiasis is a gastrointestinal tract infection caused by the flagellate Giardia lamblia. Most people with giardiasis are asymptomatic and excrete cyst forms in stool; those with acute disease usually pass trophozoites with frank diarrhea.

1. Metronidazole a. Pharmacologic properties. Metronidazole is favored in the treatment of urogenital trichomoniasis and intestinal and extraintestinal amebiasis infections. (1) It is readily absorbed and penetrates all tissues. (2) The mechanism of action involves entry into cells of the organism, where it is chemically reduced, and these products cause death by interacting with DNA and interfering with cell division.

398

meilical ~

------ - - - - -

Antimicrobial Agents

b. Side effects and toxicity include nausea, headache, dry mouth, and a disulfiram-like effect if alcohol is consumed. 2. Quinacrine a. Pharmacologic properties (1) Quinacrine is an acridine derivative.

(2) It may accumulate in tissues with chronic administration. (3) Its mechanism of action is unknown.

b. Indication for use. It is used in the treatment of giardiasis. c. Side effects and toxicity include nausea and vomiting; headache and dizziness; rash,

urticaria, or yellow discoloration of the skin; optic nerve damage. It is contraindicated in pregnancy. D. Agents used in the treatment of leishmaniasis. Leishmaniasis results from invasion of the reticuloendothelial system by Leishmania transmitted to humans by sandtlies. Few drugs are uniformly effective in leishmaniasis. Drugs employed include sodium stibogluconate and pentamidine. E. Agents used in the treatment of trypanosomiasis. The two types of trypanosomiasis are African sleeping sickness, caused by the bite of a tsetse fly infected with T. gambiense; and

South American Chagas disease, caused by the bite of reduviid bugs infected by Trypanosoma cruzi. 1. Melarsoprol

a. Pharmacologic properties (1) Melarsoprol is a trivalent arsenic compound that reacts with sulfhydryl groups to

inactivate enzymes. (2) It is administered by slow intravenous infusion. (3) It enters the CNS.

b. Indications for use include African trypanosomiasis meningoencephalitis. c. Side effects and toxicity include: gastrointestinal and abdominal pain and vomiting,

encephalopathy (may be fatal), hypotension, albuminuria, rash and angioedema, arthralgia, peripheral neuropathy, and myocardial damage. 2. Nifurtimox

a. Pharmacologic properties (1) It is a nitrofuran derivative.

(2) It is orally administered. (3) It is rapidly metabolized and excreted by the kidney. b. Indications for use. It reduces parasitemia in the acute stage of Chagas disease (T. cruzi infections). c. Side effects and toxicity include generally mild anorexia, nausea, vomiting, abdominal

pain, myalgia, and skin rashes. 3. Pentamidine is an ethionate and is effective against tissue forms of Leishmania.

KAPLA~. I meulca

399

Microbiology

4. Suramin a. Indications for use (1) It is used in prophylaxis against African trypanosomiasis. (2) It is used in the treatment of early African trypanosomiasis (without eNS involvement). b. Side effects and toxicity include nausea, vomiting, and colic; photophobia and paresthesias; rash and urticaria; albuminuria and hematuria. 5. Other drugs used in treatment include melarsoprol and nifurtimox. F. Agents used in the treatment of trichomoniasis. Trichomoniasis most commonly refers to vaginal infections caused by Trichomonas vaginalis. Trichomonads may persist in extravaginal foci, particularly the urethra, and diagnosis is easily obtained by microscopic examination of a hanging drop preparation containing fresh exudate from the vagina, semen, or prostatic fluid. Metronidazole is the drug of choice in trichomoniasis.

G. Agents used in the treatment of toxoplasmosis. Toxoplasmosis is a common infectious disease caused by the protozoan Toxoplasma gondii. This organism is usually transmitted to humans by ingestion of raw or inadequately cooked meat. Treatment of toxoplasmosis includes pyrimethamine and a sulfonamide.

ANTIHELMINTHIC AGENTS A summary of agents is presented in Table V-IS-S. A. Nematodes (roundworms) 1. Mebendazole

a. Pharmacologic properties (1) A broad-spectrum antihelminthic agent, it is effective only against intestinal

nematodes. (2) It interferes with the microtubule synthesis of the parasite. (3) It has poor gastrointestinal absorption. b. Indications for use. It is used in treatment of Trichuris trichiura and Enterobius vermicularis. It is also effective against Ascaris lumbricoides, Ancylostoma duodenale, and

Necator american us. c. Side effects and toxicity (1) Transient abdominal pain and diarrhea occur. (2) Because it is poorly absorbed, there is no systemic toxicity. (3) Teratogenic effects are possible. 2. Thiabendazole a. Pharmacologic properties (1) Thiabendazole is readily absorbed after oral administration.

(2) It has broad-spectrum activity against nematodes. (3) It inhibits the helminth-specific enzyme, fumarate reductase, although this may not be the primary mode of action.

400

meclical

(4) It may interfere with microtubule synthesis.

Antimicrobial Agents

b. Indications for use (1) Thiabendazole is the drug of choice in treating Strongyloides stercoralis and

cutaneous larva migrans. (2) It is the primary therapy for trichinosis. e. Side effects and toxicity. The most common side effects are dizziness, anorexia, nau-

sea, and vomiting; occasionally, fever, erythema multiforme, and the Stevens-Johnson syndrome are seen. 3. Ivermectin a. Pharmacologic properties (1) Causes parasitic immobilization by increasing GABA-mediated transmission (2) Does not cross the blood-brain barrier b. Indications for use. It is the drug of choice for treatment of onchocerciasis (Onchocerca volvulus) and is being used in several nematode infections. c. Side effects and toxicity include fever, rashes, headache, dizziness, and pruritis. B. Trematodes (flukes)

1. Praziquantel a. Pharmacologic properties (1) It is rapidly absorbed with oral administration; there is extensive first-pass

metabolism. (2) It penetrates the CSF. (3) It increases the membrane permeability of susceptible worms to calcium, resulting in paralysis of the parasite. b. Indications for use (1) Praziquantel is the agent of choice for all trematode infections, including all forms of schistosomiasis. (2) It is also active in cestode infections, such as Taenia saginata, Taenia solium, Diphyllobothrium latum, and Hymenolepis nana. e. Side effects and toxicity include abdominal pain, nausea, headache, dizziness, occa-

sional fever, rashes, and eosinophilia.

KAPLA~. I meulca

401

Microbiology

Table V-I5-5. Antihelminthic drugs. Helminth

Preferred Drug

Roundworms (nematodes)

Mebendazole Thiabendazole Mebendazole Thiabendazole Mebendazole Mebendazole Praziquantel

Enterobius vermicularis Strongyloides stercoralis Ascaris lumbricoides Trichinella spiralis Tapeworm (cestodes) (Taenia saginata, Taenia solium) Hookworm (Necator americanus) Whipworm (Trichuris trichiura) Flukes (trematodes) Schistosoma haematobium Schistosoma mansoni Schistosoma japonicum

Pyrantel Mebendazole

Praziquantel Praziquantel Praziquantel

2. Oxamniquine

a. Pharmacologic properties. Oxaminiquine has schistosomicidal activity against both mature and immature worms. b. Indications for use. It is used primarily for Schistosoma mansoni infections. c. Side effects and toxicity include dizziness and somnolence. C. Cestodes (tapeworms)

1. Nidosamide a. Pharmacologic properties (1) There is very little gastrointestinal absorption following oral administration.

(2) It has prominent activity against most cestodes, by inhibiting anaerobic metabolism, parasitic oxygen, and glucose uptake. b. Indications for use (1) Niclosamide is used in treating tapeworm infections from T. saginata, D. latum,

andH. nana. (2) It has some effectiveness in treating T. solium infections; however, there is some risk of cysticercosis, as the drug does not kill ova. Praziquantel is the drug of choice for cysticercosis. c. Side effects and toxicity are mild malaise, abdominal pain, and nausea.

2. Praziquantel is used in the treatment of cysticercosis.

402

meclical

SECTION VI

Immunology

----

----------

Basic Immunology

The immune system is an intricate collection of organs, tissues, cells, and soluble factors that allow individuals to defend against harmful agents such as viruses, bacteria, and tumor cells. The immune system includes the primary or central lymphoid organs in which the leukocytes develop, the secondary or peripheral lymphoid organs and tissues in which immune responses occur, and the leukocytes circulating in the blood. This chapter reviews the cells and organs that compose the immune system and the basic characteristics of immune responses, including antigens, antibodies and T-cell receptors, and mechanisms of regulation. Further, T-cell and B-cell activation, the cellular interactions necessary for cell-mediated (e.g., T-cell) and humoral (e.g., antibodies) responses will be examined, as well as the cytokines that influence these processes. Finally, complement and inflammation will be reviewed, along with important immunologic laboratory methods.

GENERAL PROPERTIES OF IMMUNE RESPONSES The immune system is composed of innate and adaptive immune responses. The innate system is thought of as a nonspecific protection against infection and includes barriers such as the skin and epithelial lining of the gut, as well as cell surface receptors for bacterial proteins termed "toll-like" receptors. Adaptive immunity consists of all responses that lead to the development of specific antibodies and antigen-specific T lymphocytes.

In a Nutshell Primary lymphoid Organs

LYMPHORETICULAR SYSTEM

• Bone marrow

The lymphoreticular system (Figure VI -1-1) is composed of the primary lymphoid organs, in which hematopoiesis and lymphopoiesis occur, and secondary lymphoid organs, in which immune responses occur. Primary lymphoid organs in children and adults include the bone marrow and thymus; in the fetus, the spleen and liver are also primary organs. Hematopoiesis occurs in the bone marrow, producing mature erythrocytes, platelets, monocytes, granulocytes, and B cells, as well as precursors for T cells, NK cells, dendritic cells, and mast cells. The T cells finish maturation in the thymus, and all other cells finish maturation in the periphery. The major secondary or "peripheral" lymphoid organs and tissues include the lymph nodes, spleen, and the mucosa-associated lymphoid tissue system (MALTS). MALTS includes the gut-associated lymphoid tissue (GALT), bronchus-associated lymphoid tissue (BALT), and submucosal lymphoid tissues of the genitourinary tract. The purpose of secondary lymphoid organs is to trap and present antigen to circulating lymphocytes and then stimulate adaptive immune responses. These secondary tissues or organs protect all surfaces and fluids of the body. Extracellular fluid, or lymph, is filtered through lymph nodes and MALT, and the primary filter for the blood is the spleen and to some extent the liver.

• Thymus

Secondary lymphoid Organs • Lymph nodes • Spleen • Tonsils • Mucosa-associated lymphoid tissue (MALT) - Gut-associated lymphoid tissue (GALT) - Bronchus-associated lymphoid tissue (BALT)

iiie&ical

405

Immunology

Waldeyer ring lymph nodes, } - tonsils, and adenoids (BALT) Thymus ---F-----'-?.,....,.,r.-_+--- Lung X~...-\--- Lymph nodes

Bone marrow

>-"~~a-+--+-- Spleen :~I*'\--+--t--

Lamina propria

Mesenteric -+-I-+~~..6'r--T---T-t- Peyer patch (GALT) lymph nodes Lymph --"o,;1~t--:-.... nodes

b--'--+---"6~

Urogenital

Figure VI-1-1. The Iymphoreticular system.

Note Bone marrow is the source of pluripotent stem cells (precursors of all white blood cell types as well as RBCs) and the site of B-cell maturation.

Note Early contact with antigen in primary lymphoid organs tends to produce tolerance. Later contact in peripheral lymphoid organs tends to produce activation of both T and B cells.

406

meClical

A. Bone marrow structure and function. Bone marrow is a primary lymphoid organ because it is the site of hematopoiesis and B-cell maturation, as well as the site of origin of the stem cells involved in T-cell production. 1. Bone marrow structure. The bone marrow is a very large tissue comprising 3-5% of body mass in humans. It is found in the axial skeleton, cranium, ribs, and long bones.

There are two functional components of approximately equal size: the vascular and adipose portion, and the hematopoietic portion. The latter is involved in the formation of blood cells, which are all derived from a single hematopoietic stem cell. 2. Hematopoietic cell differentiation (Figure VI-1-2). All blood cells are derived from pluripotent hematopoietic stem cells that differentiate into myeloid and lymphoid progenitor cells. a. Progenitor cells differentiate after the by appropriate stimuli in certain anatomic sites, such as primary lymphoid organs (thymus and bone marrow) and secondary lymphoid organs (lymph nodes). Stimuli include colony-stimulating factors, erythropoietin, thymosin, and antigen (both self and foreign). Polycolony-stimulating factors include stem cell factor, IL-3, GMCSF, and IL-6. b. After maturation in the thymus or bone marrow, lymphocytes leave these sites and migrate to the spleen, lymph nodes, and MALT (secondary lymphoid organs), where further development occurs under the influence of antigens and cytokines.

Basic Immunology

PRE-B-CELL

PRE-B-CELL

INITIATES H CHAIN GENE REARRANGEMENT

EXPRESSES CYTOPLASMIC IMMUNOGLOBULIN HEAVY CHAINS OF M CLASS; LACKS MEMBRANE Ig

IMMATURE B LYMPHOCYTE

EXPRESSES MEMBRANE IgM AND IgD, HIGH DENSITY OF RECEPTORS FOR COMPLEMENT COMPONENTS TOLERIZABLE

MATURE B LYMPHOCYTE

EXPRESSES MEMBRANE IgM AND IgD, HIGH DENSITY OF RECEPTORS FOR COMPLEMENT COMPONENTS INDUCEABLE

PLASMA CELL

ANTIBODYSECRETING

HELPER T CELL (CD4)

CYTOTOXIC T CELL (CDS) PRE-T-CELL ENTERS THYMUS CORTEX-MEDULLA NK CELL PLURIPOTENTIAL STEM CELL

NEUTROPHIL

MACROPHAGE MONOCYTE EOSINOPHIL MYELOID STEM CELL

BASOPHIL

PLATELET MEGAKARYOCYTE ERYTHROCYTE COLONY-FORMING PRECURSORS

ERYTHROBLAST

Figure VI-1-2. Hematopoietic cell differentiation.

B. Thymus structure and function. Precursors of thymic (T) lymphocytes travel from the bone marrow to the thymus, where early differentiation and maturation take place, including T-cell receptor expression and clonal deletion of autoreactive T cells. Mature T lymphocytes leave the thymus to seed the secondary lymphoid organs in thymic-dependent regions, where they can be activated and undergo their final maturation into effector T cells that function in immune responses. 1. Development of the thymus. The thymus is derived from the third and fourth pharyn-

geal pouches and is found in the mediastinum. a. The development of the thymus begins with epithelial (endodermal) outgrowths of the third and fourth pharyngeal pouches. b. Subsequently, this epithelial reticulum is infiltrated by pre-T cells and other mesodermal elements. c. The thymus reaches its maximum weight at puberty and then slowly involutes.

d. There is a significant amount of programmed cell death (apoptosis) that occurs in the thymus. This is a reflection of the elimination of autoreactive lymphocytes during differentiation in this organ (tolerance by clonal abortion). 2. Organization of the thymus. The stroma of the thymus consists of a prominent connective tissue capsule, which invaginates into the parenchyma as septa and divides the thymus into lobules. The parenchyma is organized into a cortex and medulla. a. Cortex is composed of tightly packed differentiating thymocytes surrounded by a meshwork of epithelial cells and macrophages. KAPLA!!._

I 407

meulCa

Immunology

In a Nutshell Thymus Cortex • Darker peripheral zone • Extensive population of immature T cells, epithelial cells, and macrophages Thymus Medulla • Lighter central zone • Larger number of epithelial cells and mature large- and medium-sized T cells • Hassall corpuscles

Clinical Correlate DiGeorge syndrome is the absence of the thymus, resulting in a severe deficit of T cells. Afflicted individuals have no T-cell-mediated immunity. DiGeorge syndrome is often accompanied by hypoplasia of the parathyroid glands with resultant tetany. Early death from severe infection often occurs. Babies with DiGeorge syndrome have low-set ears and develop tetany shortly after birth due to a lack of parathormone. The immune deficiency becomes apparent a few months later when maternallgG disappears from the circulation.

Clinical Correlate Each node receives lymph from a defined and limited region of the body. Neoplasms can metastasize via these nodes. For example, a common metastatic site for breast cancer is the axillary nodes.

408

meCtical

b. Medulla consists of epithelial cells and mature T cells. The medulla exhibits a paler staining than the cortex as a result of the large reticular cells and the presence of larger lymphocytes that are not as densely packed. The medulla also contains the characteristic Hassall corpuscles, which are composed of concentrically arranged dead and dying reticular cells, macrophages, neutrophils, and nuclear material whose origin is unknown. 3. Blood supply. Arteries from the connective tissue capsule and septa enter the thymus at the level of the corticomedullary junction. These arteries branch into capillaries, which loop up to the periphery and turn back to the medulla to form venules, which leave the septa. a. Thymic capillaries have a nonporous endothelium with a thick basement membrane. b. Epithelial cells surround thymic vessels and constitute an incomplete barrier, which separates the blood from the thymocytes. The thymic reticulum is composed of branching epithelial cells that are joined one to another by desmosomes. 4. Thymus is large at birth in relation to other organs; it increases in size until puberty, when involution begins. Thymectomy in young animals results in poor development of the other lymphoid tissues and the absence of cell-mediated immunity. This can be reversed by a thymic graft. Congenital absence of the thymus (i.e., DiGeorge syndrome) results in poor development of peripheral lymphoid tissues and the absence of cell-mediated immunity. 5. Thymosin, a family of lymphokines that stimulate thymus-dependent zones in the peripheral lymphoid tissues, is produced by thymic epithelium. C. Lymphatics. More plasma is flitered from capillary beds into the tissues of the body than is

reabsorbed back into the venous end of those capillary beds. This excess fluid moves through the tissues or organs as interstitial fluid and is collected in small lymphatic vessels throughout the tissues as lymph. These small lymphatics fuse into larger afferent lymphatics that enter lymph nodes. The efferent lymphatics leaving one node may become the afferent lymphatics entering another node in a cluster. Eventually, most efferent lymphatics fuse into the large thoracic duct that extends the length of the thorax and empties into the left subclavian vein, returning the lymph fluid to the blood. D. Lymph nodes are highly organized secondary organs that are the most common site for an adaptive immune response because they fliter the lymph that washes through the body tissues. During an infection, lymph nodes may increase two to five times in size. They are encapsulated, kidney-shaped structures that have a concave side with a hilum. The blood vessels and nerves enter and exit at the hilum, and the efferent lymphatic exits from this region. Beneath the stromal capsule, the parenchyma is organized into a cortex, paracortex, and medulla. 1. The stroma consists of a dense connective tissue capsule that surrounds each lymph node

and sends collagenous trabeculae into the node to divide its parenchyma into incomplete compartments (Figure VI-1-3). a. Reticular cells produce reticular fibers that anastomose with the trabeculae and form an extensive network. b. The delicate reticulum filters the lymph and suspends the lymphocytes and macrophages. c. This stromal organization and function facilitates cell-to-cell and antigen-receptor interactions.

Basic Immunology

2. Cortex. Beneath the capsule (except at the hilum) lies a cortex, which is composed of B cells in a diffuse lymphatic tissue network that intermingles with the subcapsular and peritrabecular sinuses (macrophages). T cells are found predominantly in the paracortex. a. Lymphatic sinuses are the lymphatic passageways within lymph nodes. They are lined with flat endothelial cells in the area of the capsule and are partially lined by reticular fibers and macrophages elsewhere. The sinuses receive lymph brought by afferent vessels and transport it toward the medulla. b. Germinal centers may be present in the nodules of the cortex (see Figure VI-I-3). They are composed mostly of B cells, some T cells, and macrophages and are transient structures that are only present during an immune response. Antigen stimulation increases the number and development of germinal centers. In germinal centers, B cells develop into plasma cells in response to specific antigens. 3. The medulla of the lymph node occupies the center of the organ. It contains medullary cords composed of lymphoid tissue that extend from the cortex. Medullary sinuses, like those in the cortex, transmit the lymph toward the hilum, where it exits through the efferent lymphatic. 4. Function. Lymph nodes serve as filters trapping foreign particles from the lymph before circulating to other areas of the body.

In A Nutshell Germinal center of follicle (Clones dividing)

Cortex Paracortex (T-cell rich) Medulla

Primary follicle (8-cell rich)

• Lymph nodes filter tissue fluids. • Spleen filters blood-borne antigens.

Efferent lymphatic (Memory cells exit)

Afferent lymphatic (Ag enters)

Clinical Correlate Figure VI-1-3. Lymph node.

a. Lymph enters the node via afferent lymphatic vessels, which are located on the convex side of the organ (see Figure VI -1-3). b. From the afferent lymphatics, lymph passes through the subcapsular sinus to the peri trabecular sinuses. It then enters the medullary sinuses and exits the lymph node via the efferent lymphatic vessels at the hilum. E. The spleen is a peripheral lymphoid organ in the upper left quadrant of the abdominal cavity, which acts as a filter for blood, clears old and defective erythrocytes (RBCs), and provides protection from blood-borne pathogens.

Patients with sickle cell disease undergo gradual splenic infarction. This predisposes them to septicemia, particularly caused by Streptococcus pneumoniae. Other opportunists include Salmonella, meningococci, and H influenzae.

meClical

409

Immunology

1. The stroma of the spleen consists of the following:

a. A dense connective tissue capsule that contains smooth muscle cells b. Trabeculae that branch off of the capsule and partially partition the parenchyma of splenic pulp c. A delicate meshwork of reticular connective tissue that filters the blood 2. Splenic parenchyma consists of white and red pulp.

Note In the splenic white pulp, Band T lymphocytes are segregated. The central portion of PALS are rich in T cells, whereas the marginal zone are populated by B cells.

a. White pulp consists of lymphatic tissue arranged in sheaths around arterioles (periarteriolar lymphoid sheath) and in nodules (marginal zones). Antigenic stimulation increases the amount of white pulp. (1) The periarteriolar lymphocyte sheaths (PALS) are accumulations of diffuse

lymphatic tissues that are rich in T cells. The marginal zone is occupied mostly by B cells. (2) B lymphocytes cluster peripherally in the PALS to form primary follicles. After antigenic stimulation, these follicles develop into secondary follicles with germinal centers containing rapidly dividing B cells. (3) At the marginal zone, dendritic cells trap and process antigen and migrate to PALS to present it to antigen-specific cells of the immune system. b. Red pulp consists primarily of erythrocyte-filled sinusoids and macrophages in a reticular fiber network; most filtration occurs here. (1) The sinuses vary in size and are separated by pulp (i.e., Billroth) cords. (2) RBCs and platelets are exposed to macrophages in the pulp cords; macrophages phagocytize worn-out or damaged cells. (3) Splenic sinusoids are lined by loosely arranged endothelial cells, which have numerous fenestrations. These sinusoids are surrounded by a poorly developed basal lamina. (4) Phagocytosis is carried out primarily by macrophages located outside the sinusoids. The macrophages extend finger-like projections into the sinusoids, which push through the discontinuous basement membrane between the endothelial cells. 3. Blood supply. Arteries from the hilum of the spleen pass along trabeculae to enter the periarteriolar sheath. a. These vessels branch like brush bristles to form penicillar arteries, which continue in capillaries and open blindly in the red pulp, connecting directly to venous sinusoids. b. The venous sinusoids are specialized, large-caliber spaces that are bordered by elongated endothelial cells and held in place by circularly arranged reticular fibers. c. Venous sinusoids drain into veins that eventually exit at the hilum.

F. Gut-associated lymphoid tissue (GALT) is not encapsulated and is present in the submucosa and lamina propria in the gastrointestinal tract. It is the site of immune responses to injested microbes and some food antigens. 1. Structure. Lymphoid tissue in GALT includes the large follicular aggregates in the small

intestine called Peyer patches, the lamina propria beneath the mucosal epithelia in the villi, and the intraepitheliallymphocytes (IELs) found between mucosal epithelial cells along the surface of the villi.

410

meClical

Basic Immunology

2. Function a. The lymphoid tissues lining the intestinal tract are well exemplified by the Peyer patches. Structurally unique antigen-presenting cells, called M cells, are located in mucous membranes. They endocytose microbes and antigens and present specific epitopes to T lymphocytes located between follicles and in the lamina propria. With Tcell help, the B cells become activated and form germinal centers, where they differentiate into 19A-secreting plasma cells. b. The 19A dimers react with a polyimmunoglobulin receptor on intestinal epithelial cells. The dimer (held together by the J chain) is internalized and crosses the epithelial cell cytosol. It is then proteolytically cleaved from the poly-Ig receptor and excreted into the lumen of the intestine. A small portion of the Ig receptor remains attached to the dimer; this is called the secretory component. The function of this small peptide is to protect the antibody molecule from enzymatic hydrolysis in intestinal fluid.

Clinical Correlate Peyer patches and other lymphoid tissues will be hypoplastic in individuals with Bruton hypogammaglobulinemia. Lymphoid tissue is almost totally absent in patients with severe combined immunodeficiency.

G. Bronchus-associated lymphoid tissue (BALT) includes the lymphoid tissue beneath the respiratory mucosa and the aggregates of nodular lymphatic tissue called tonsils. These are structurally similar to lymph nodes and contain deep crypts that allow antigen to be trapped, degraded, and processed by antigen-presenting cells, such as macrophages and dendritic cells. 1. Organization of the tonsils. Tonsils are peripheral lymphoid organs composed of aggre-

gates of nodular and diffuse lymphatic tissues that protect epithelial surfaces. The lymphoid tissues are located beneath the epithelium in the underlying connective tissues. a. Lymphatic nodules contain aggregates of B lymphocytes, which differentiate into plasma cells to produce antibodies for the humoral immune response. b. T cells are found primarily in diffuse lymphatic tissues. 2. Types of tonsils. Three important examples of tonsillar tissue are the palatine tonsils, the lingual tonsils, and the pharyngeal tonsil. a. Palatine tonsils are located bilaterally in the oropharynx. (1) They are composed of dense lymphoid tissue, which forms a band oflymphatic nodules with germinal centers. These are intermingled with diffuse lymphatic tissue beneath the stratified squamous epithelium.

Clinical Correlate Tonsillitis involves the formation of abscesses in the crypts.

(2) A dense connective tissue capsule often separates the tonsil from subjacent tissues. (3) Each tonsil has numerous epithelial invaginations, or crypts, that contain desquamated epithelial cells, lymphocytes, and bacteria in their lumina. b. Lingual tonsils are smaller and more numerous aggregates of lymphoid tissues located at the base of the tongue. (1) They are covered by the stratified squamous epithelium on the dorsum of the tongue. (2) Each aggregate possesses a single crypt. c. Pharyngeal tonsil is an unpaired accumulation of lymphoid tissue located on the

posterior wall of the nasopharynx. (1) It is usually covered by a pseudostratified columnar epithelium with cilia and goblet cells; however, the epithelium can be obscured by an infiltration of lymphocytes or by metaplasia (seen in smokers). The pharyngeal tonsils, or hypertrophic pharyngeal tonsils, are often referred to as adenoids. (2) Instead of forming crypts, the overlying epithelium occurs in a series of folds.

meC:lical

411

Immunology

H. Lymphocyte recirculation 1. Lymphocytes are capable of high levels of recirculation, continuously moving from the blood out into the tissues and then returning via the lymphatics. This movement is facilitated by cell adhesion molecules (CAM).

2. The lymphocytes bear in their membranes specific CAMs, called selectins, which interact specifically with addressins (another type of CAM) that are found in the lymphatic vasculature, particularly in the postcapillary venules. It is here that specialized cells with a plump cuboidal shape are found; these are called high endothelial venules due to the height in the region caused by the cuboidal cells. 3. As T cells home to lymph nodes, they make contact with antigens presented in the groove of class II MHC molecules located on the surface of antigen-presenting cells. This dynamic leads to T-cell help and B-cell activation.

Note Lymphocyte homing is controlled by cell adhesion molecules (CAMs).

4. Once the activation has occurred, the lymphocytes stay trapped in peripheral lymphoid tissues, where they differentiate to mature effector cells in the case of T cells or plasma cells in the case of B cells. Lymph node swelling is thus a product both of lymphocyte trapping and division. S. Different populations oflymphocytes express different homing receptors. Naive and memory cells also express different receptors.

Note Cells of the immune system develop in the bone marrow.

Note Macrophages and neutrophils both phagocytose bacteria that are coated with antibody and/or complement. The C3b fragment of complement and/or IgG can bind to bacteria, then bind to receptors on phagocytic cells and signal them to engulf the organisms. The Fc receptors on macrophages are also useful for opsonization of bacteria by antibody. They react with the Fc region of IgG antibody molecules and hold the microbe close to the phagocytic cell membrane, thus facilitating the engulfment process.

CELLS OF THE IMMUNE SYSTEM Leukocytes include lymphocytes (B cells, T cells, and large granular lymphocytes or NK cells), mononuclear phagocytes (monocytes and macrophages), polymorphonuclear granulocytes (neutrophils, eosinophils, and basophils), mast cells, and dendritic cells. All leukocytes, as well as erythrocytes and platelets, initially develop in the adult bone marrow (described below); many complete maturation there, although T cells complete their maturation in the thymus. A. Monocytes and macrophages are derived as follows: stem cell-7 monoblast -7 promonocyte -7 circulating monocyte -7 tissue macrophage. Macrophages have a life span of months to years. 1. Function of monocytes and macrophages. These cells playa central role in cell-mediated immunity. They ingest particles via pinocytosis or phagocytosis and modulate inflammation via secretion of mediators. They also act as cells that process and present antigens to T lymphocytes (antigen-presenting cells).

a. Macrophages secrete over 100 mediators, including interleukins 1,8, and 12, collagenase, elastase, lipase, proteases, prostaglandins, leukotrienes, thromboxanes, lysozyme, and interferon. They also secrete complement component C2. b. Macrophages have Fc receptors and class II MHC molecules on the cell surface that mediate their biologic functions. The Fc receptors allow the uptake of immune complexes (Ig complexed to antigen), and class II molecules present antigenic pep tides to Th cells. c. Circulating monocytes differentiate into tissue macrophages with specific names. For example, tissue macrophages present within the sinusoids of the liver are called Kupffer cells. Macrophages found in the lung are termed alveolar macrophages; macrophages in the brain are called microglial cells. (1) Kupffer cells encounter antigens first from intestinal lumen absorption and serve

to clear particulate and soluble matter from portal circulation. They phagocytose bacterial endotoxin, soluble immune complexes, activated clotting factors, and microorganisms.

412

meclical

Basic Immunology

(2) Alveolar macrophages destroy inhaled antigens and microbes. 2. Morphology of macrophages a. Epithelioid cells, usually found in granulomas, are derived from blood monocytes and are activated by an immune response to antigen. b. Multinucleated giant cells are formed by the fusion of macrophages. 3. Macrophage activation. Macrophages are stimulated by lymphokines (mostly IFN-y) to kill microorganisms and tumor cells. a. Activated macrophages have increased lysosomal hydrolytic enzymes and an increased chemotactic response. C5a and various cytokines from lymphocytes, neutrophils, and fibroblasts are chemoattractants for activated macrophages. b. Antigen coated with appropriate complement proteins (e.g., C3b) and/or antibody is more readily phagocytosed. This process is called opsonization. The C3b receptor is called CRI or CD35. c. Morphologic changes occur during activation and include increases in size, number of

pseudopods, and pinocytotic vesicles. The process takes several days. d. Macrophages experience a respiratory burst during phagocytosis via the hexosemonophosphate shunt pathway. This is a source of energy needed for cell membrane synthesis, and it also generates toxic oxygen metabolites such as singlet oxygen, superoxide anion, and hydrogen peroxide.

IgG Immunoglobulin

~ Macrophage

Antibody and complement C3b

Bacterial organism

In a Nutshell Figure VI-1-4. Antibody and complement C3b.

4. Macrophages present extracellular antigens to T helper cells. Antigen undergoes phagocytosis or pinocytosis. Once in the cytoplasm of the macrophage, the antigen is degraded into small peptides. The pep tides are then noncovalently bound to class II MHC molecules in an endosomal vesicle. The complex is then transported to the cell surface, where it stimulates class II-restricted antigen-specific Th cells (CD4). B. Dendritic cells are present in peripheral blood and lymphoid organs. Their primary func-

tion is to digest and process antigen for presentation to Th cells. Dendritic cells include Langerhans cells of skin, veiled cells in afferent lymphatics, and interdigitating reticulum cells in spleen and lymph nodes.

Macrophages process exogenous antigens and present the epitopes in a groove of the class II MHC molecules. CD4 T cells bearing receptors specific for the epitope will react with the epitope/MHC complex and be triggered to release cytokines. Endogenous antigens are similarly presented to CDS cells on class I MHC, also present on macro phages and other APCs.

meclical

413

Immunology

C. Granulocytes are also called polymorphonuclear leukocytes. There are three types of granulocytes: neutrophils, eosinophils, and basophils.

1. Neutrophils (polymorphonuclear leukocytes, PMNs) represent 60% of circulating blood leukocytes. Circulating neutrophils have a receptor for the Fc region of IgG (FcyR) and C3b (CD35) (Figure VI-I-4).

In a Nutshell PMNs kill microbes via: • Toxic oxygen metabolites • Digestive enzymes present in lysosomal granules

Clinical Correlate Eosinophilia is a hallmark of: • Atopic allergies • Worm infections It can also be seen in collagen vascular diseases, neoplastic disorders, and any skin rash.

a. Neutrophils have a multilobulated nucleus with dense chromatin and cytoplasmic lysosomes containing peroxidases and acid hydro lases. b. They reach the tissues by diapedesis, inserting pseudopodia with cell adhesion molecules between the endothelial cells so that movement through blood vessel walls occurs. c. Neutrophils are the first cells to arrive at acute inflammatory sites. They actively kill bacteria; their half-life is about 10 hours in the blood and 3 days in tissues. d. Cytoplasmic granules contain digestive enzymes. These include azurophilic granules that contain myeloperoxidase and specific granules that contain lactoferrin. e. Neutrophils phagocytize and then kill the organism by generation of H 2 0 2 and toxic oxygen radicals and via the action of granule-derived enzymes. 2. Eosinophils represent 1-3% of circulating leukocytes. a. Approximately 50% of circulating eosinophils have receptors for complement. b. Eosinophils have a bilobed nucleus and contain crystalloid granules staining red with Giemsa. c. Eosinophil chemotactic factors include histamine, C5a, LTB 4 , PAF, and ECF-A (eosinophil chemotactic factor of anaphylaxis).

Note Eosinophil granule contents that help control allergic reactions include histaminase and arylsulfatase.

d. They are functionally important in late inflammatory reactions, particularly in parasitic infections and allergy. e. Some important contents of eosinophils and their functions include: ( 1) Histaminase degrades histamine. (2) Pyrogen produces fever. (3) Peroxidase kills microorganisms. (4) Arylsulfatase degrades leukotrienes C4' D 4 , and E4 • (5) Major basic proteins are toxic to worms. 3. Basophils represent 1% of circulating leukocytes and are the smallest type of granulocytes. a. They contain abundant granules with RNA, mucopolysaccharides, and hypersensitivity mediators, such as histamine. b. They have receptors for the Fc portion of IgE. c. IgE binding promotes degranulation as it does in the tissue mast cells. Histamine and other mediators are released during degranulation and are responsible for the symptoms seen in atopic allergies. This is discussed further in the Clinical Immunology chapter. D. Lymphocytes, which include Band T lymphocytes, represent 30% of circulating leukocytes. Lymphocytes have a high nucleus-to-cytoplasm ratio and are distinguished by their antigen receptors and cell surface markers. 1. B lymphocytes. B lymphocytes terminally differentiate into plasma cells, which secrete large amounts of immunoglobulin (antibodies).

414

iileilical

Basic Immunology

a. There are two major subsets of B lymphocytes: CDS-positive cells produce 19M antibody to soluble polysaccharides and self-antigens. They are stimulated by nonspecific cytokines from Th cells. CDS-negative cells produce 19G, 19A, or 19E antibody specific to individual to protein antigens, cellular antigens, and bacteriallipopolysaccharides. These cells require direct physical interaction with specific Th cells. b. Memory B cells, generated after the primary exposure to an antigen, produce antibody with increased affinity for its antigen because of somatic mutation of 19 genes. c. Mature B cells have surface 19M and IgD that serve as the B-cell receptor and bind antigen. B cells can also present antigen to Th cells via class II MHC. d. B cells respond to two types of antigens. (1) T-cell-independent antigens activate B cells in the absence of CD4+ Th cells.

These antigens are usually composed of polysaccharides or carbohydrates with repeating structures. (2) T-cell-dependent antigens (most all protein antigens) require B- and T-cell interaction. The T cell then drives B cells to switch antibody classes (class switching) via direct contact and by cytokine secretion. 2. T lymphocytes. Two major types of T cells exist and are classified based on the expression of the cell surface proteins CD4 or CDS. Cellular differentiation (CD) proteins are present on many cells of the body. They reflect the function of the cell and are thus "markers" for particular cells. For example, CD3 is found in the membrane of all mature T cells, whereas CD4 and CDS proteins are found on certain subsets of these mature T cells. a. Th cells are CD4 positive. There are two distinct subsets of Th cells with different functions (Th1 and Th2). The Th2 cell's function is to stimulate B lymphocytes to proliferate and differentiate into antibody-producing cells. The Th1 cell's function is to promote cytotoxic T-cell (CDS+ cells) responses and the delayed-type hypersensitivity response. (1) Activation and proliferation of Th cells depends on corecognition of specific antigenic peptides and MHC class II molecules on antigen-presenting cells such as macrophages, dendritic cells, or B cells (APCs). (2) Activated Th cells produce cytokines, differentiation factors, and inflammatory cytokines. b. Cytotoxic T cells are CD8 positive. They lyse cells such as virus-infected cells and tumor cells. Cytotoxic T cells act by recognizing foreign antigen and MHC class I molecules with their T-cell receptor. 3. Natural killer (NK) cells represent lO-15% oflymphocytes in the peripheral circulation. a. NK cells kill certain tumor cells (without damaging normal tissues) and defend against viral infection. Unlike T cells, NK cells recognize foreign antigen that does not have to be presented on MHC molecules. b. NK cells mediate antibody-dependent cellular toxicity (ADCC). This function allows NK cells to kill antibody-coated target cells. e. NK cells are activated by cytokines such as 1FN-y and 1L-2.

Note T-celi-independent antigens induce IgM antibody only and do not cause immunologic memory (anamnesis). Much of this antibody is secreted by CDS B celis.

Note There are two subsets of helper T celis. Th 1 releases IL-2 and IFN-y, whereas Th2 releases other interleukins (e.g., 4, 5, 6, 10). Th1 celis stimulate proliferation and cytoxic responses of cytotoxic T celis and macrophages, whereas Th2 celis stimulate B-celi maturation, differentiation, and immunoglobulin class switching.

In a Nutshell • Helper T celis

--7

• Cytotoxic T celis

CD4+

--7

CD8+

Note ADCC may be mediated by NK celis, eosinophils, or neutrophils, ali of which have Fc receptors

KAPLAN ' . I meIIlea

415

Immunology

THE IMMUNE RESPONSE Immune cells and the responses they generate can be categorized into innate and acquired. The innate responses are necessary for immediate reponse to a pathogen and for establishing local inflammation to recruit circulating phagocytes and, later, activated T cells and B cells. The T and B lymphocytes make the acquired response and are responsible for many of the classic characteristics of immunity, including memory, antigen specificity, and tolerance to self. The characteristics of antigen presentation and recognition by T and B cells are discussed below. A. Innate versus acquired immunity. The immune system is composed of cells that defend against foreign invaders by both nonspecific mechanisms (termed innate or natural immunity), and antigen-specific mechanisms (known as acquired immunity). The nonspecific response is typically observed first because no prior exposure to antigen is necessary. This response may not be sufficient to clear the foreign antigen, and an antigen-specific mechanism produced by Band T cells may be required.

In a Nutshell

1. Innate immunity (also known as natural immunity) is present at birth in all individuals.

This response does not increase upon repeated exposure to a given antigen.

Innate Immunity (Also Called Natural Immunity)

a. The first line of defense is intact skin and mucous membranes.

• Present at birth

b. Innate immunity allows elimination of foreign substance (i.e., bacteria or virus) without previous exposure.

• Composed of skin, mucous membranes, secretions such as saliva and tears, phagocytic cells, and NK cells Acquired Immunity • Developed in response to a specific immunogen exposure • Composed of antibodies (lgG, IgA, etc.) and lymphocytes (B and T cells)

c. Innate immunity is enhanced by either natural antibodies or natural cytotoxic cells,

including macrophages, neutrophils, eosinophils, and NK cells. 2. Acquired immunity is established late in fetal life, and development continues during childhood. Continued exposure to foreign antigen will stimulate acquired immunity. a. Responses are specific to individual antigens because of antigen-specific recognition by surface antibody on B cells and by T-cell receptors (TCRs) on T cells. b. Individual lymphocytes recognize their appropriate antigens (discussed below) and are activated to proliferate into effector cells and memory cells with the same antigen specificity (clonal expansion). c. May have anamnestic response, in which subsequent response to previously recog-

nized antigen is more avid and often magnified (this is a result of the production of memory T and B cells and somatic mutation of Ig molecules). d. Antigen is eliminated due to specific antigen recognition and effector functions (discussed below). 3. Self-tolerance describes the absence of immune responses to one's own tissue antigens. It is necessary to prevent autoimmune responses. Mechanisms of self-tolerance include the following: a. Thymic deletion, the elimination of clones of developing T cells with receptors against self-antigens, occurs during cell maturation in the thymus. b. Anergy of B lymphocytes bearing receptors for self-antigens is maintained by the lack of costimulatory signals when naive cells encounter self-antigens. B. Antigens and antibodies. An antigen is any substance that can be specifically bound by an antibody or T-cell receptor, whereas an immunogen is an antigen that induces an immune response. Because antibodies and TCRs recognize different types of antigens, and antibody production requires helper Th-cell activation and assistance, immunogens typically possess both antibody and T-cell-reactive regions.

416

meClical

Basic Immunology

1. An epitope or antigenic determinant is a specific site on the antigen that is recognized by

a B- or T-cell receptor, i.e., the part of the antigen to which an antibody binds. a. Most antigens express many epitopes. b. B-cell epitopes are short peptides of 10-25 amino acids that usually occur at the hydrophilic surface, whereas T-cell epitopes may be embedded within the protein. 2. Antibodies are able to bind to epitopes on a wide variety of molecules, including proteins, carbohydrates, nucleic acids, and small organic molecules. TCRs are only able to recognize peptides bound to major histocompatiblity (MHC) proteins on the surface of a cell (discussed below). 3. Many macromolecules can be antigenic. a. Proteins (glycoproteins, lipoproteins, or nucleoproteins) are the most common form of antigen and are usually very good immunogens. (1) Their antigenicity is based on amino acid composition, three-dimensional con-

formation, and/or biochemical properties (such as charge, etc.). (2) Peptides derived from processed proteins and bound to cell-surface MHC proteins (either class I molecules for CD8 T cells or class II molecules for CD4 cells) are the only type of antigen that T-cell receptors can recognize. b. Large, repetitive polysaccharides can activate B cells with little or no helper T aid (but cannot be recognized alone by TCRs) and so are considered T-cell-independent antigens. c. Nucleic acids can be recognized by antibodies but are poor immunogens because they cannot act as T-cell antigens unless they are bound to a specific protein carrier. Nucleic acids can stimulate antigen-presenting cells to make cytokines that enhance T-cell responses. d. Lipids are usually not immunogenic. When lipids are coupled to protein antigens, they tend to induce T-cell-mediated delayed hypersenstivity rather than antibody production. e. Haptens are small molecules that can act as an antibody epitope but will not induce immune responses because they are not recognized as T-cell antigens. When combined with a carrier protein, the hapten-carrier complex can produce hapten-specific and carrier-specific antibodies because the Th cell can recognize carrier peptides. Drugs that become metabolized in the body are common sources of haptens. Many allergens (e.g., penicillin) are haptens. C. Major histocompatibility complex (MHC). The MHC is a collection of highly polymorphic

Note Thymus-independent antigens do not induce memory cell production.

Note Carrier Effect Poorly immunogenic or nonimmunogenic molecules acquire immunogenicity when they are chemically linked to proteins that serve as carriers and impart the diversity and T-cell reactivity needed.

genes encoding the proteins that regulate immune responses. These genes include, most notably, the class I and class II cell surface proteins and the class III genes that encode complement proteins. In humans, the MHC genes are termed human leukocyte antigens (HLAs) and are found on the short arm of chromosome 6. The HLA proteins are glycoproteins present on cell surfaces that enable T cells to recognize and bind antigenic peptides, i.e., they function in immune recognition. 1. HLA class I antigen (Figure VI-1-5). Class I proteins are membrane glycoproteins on the

surface of all nucleated cells and platelets. They bind endogenous peptides processed from protein synthesized in the cell's cytosol and are necessary for antigen recognition by CD8+ cytotoxic T lymphocytes (CTLs). In humans, the three types of class I genes are referred to as HLA-A, HLA-B, and HLA-C antigens.

KAPLA~.

meulCa

I

417

Immunology

Figure VI-1-5. HLA class I antigen.

In a Nutshell Class I MHC

a. The class I proteins share common structures, including three globular domains (with a molecular weight of 45 kD) in noncovalent association with ~2 microglobulin (not an HLA-coded protein). (1) The carboxy terminus lies within the cytoplasm, and the hydrophobic surface

traverses the membrane.

• Ali nucleated cells

(2) The outer two globular domains form the peptide-binding groove.

• Human class I genes = HLA-A, HLA-8, and HLA-C

(3) Each allele of each HLA class I protein has a different range of peptides that can bind to it.

• Cytotoxic T cells (CDS) recognize class I MHC on infected celis (viruses, intracellular bacteria, parasites, tumor antigens) Class II MHC • Class II is found only on celis that interact with helper T cells = antigenpresenting cells (8 celis, macrophages, dendritic cells) • Human class II genes = HLA-DR, HLA-DQ, and HLA-DP • Helper T cells (CD4) bind to class II MHC on antigenpresenting cells

418

iiieClical

b. Proteins synthesized in the cell cytosol are routinely degraded by proteases; peptides from these proteins are transported, through a peptide transporter known as the TAP complex, into the endoplasmic reticulum, where they have an opportunity to bind to freshly synthesized HLA class I proteins. (1) Most commonly, peptides of normal intracellular proteins are bound to HLA

class I proteins and displayed on the cell surface. (2) Proteins made by intracellular viruses, bacteria, parasites, or neoantigens made by transformed tumor cells will also be displayed on HLA class I proteins. c. Cytotoxic T cells recognize viral, intracellular bacterial, parasitic, or tumor antigens in association with class I molecules. CDS on the surface of these cells recognizes a nonpolymorphic region of the class I MHC molecule.

2. HLA class II antigen (Figure VI-1-6). HLA class II proteins are expressed on a more restricted set of cells, including antigen-presenting cells (dendritic cells, Langerhans cells, activated macrophages), B cells, and thymic epithelial cells involved in T-cell maturation. These proteins bind exogenous peptide epitopes processed from endocytosed molecules and are necessary for antigen recognition by Th cells. Class II genes encode for cell surface glycoproteins with two polypeptide components called a and ~ (Figure VI-1-6). In humans, the class II genes include HLA-DR, HLA-DQ, and HLA-DP.

Basic Immunology

Peptide groove

I

Figure VI-1-S. HLA class II antigen.

a. The class II proteins share a common structure, with each ex and ~ chain containing two globular domains. (1) The outer domains of the ex and groove.

~

chains combine to form the peptide-binding

(2) The ex and ~ chains are strongly associated in the cell membrane but are not linked by covalent bonds. (3) Each allele of each HLA class II protein has a different range of peptides that can bind to it. b. Class II proteins are found on cells that interact directly with Th cells, including B cells, monocyte-macrophages, Langerhans cells, dendritic cells, and thymic epithelium. c. Proteins endocytosed, pinocytosed, or phagocytosed by the cell are routinely degraded

by proteases; peptides from these proteins are able to bind to freshly synthesized HLA class II proteins. (1) Most commonly, peptides of normal extracellular proteins are bound to HLA class II proteins and displayed on the cell surface. (2) Proteins made by extracellular bacteria or parasites or injected proteins (e.g., vaccines) will also be displayed on HLA class II proteins. d. CD4 cells recognize bacterial, parasite, or injected proteins in association with class II. The CD4 molecule binds a nonpolymorphic region of class II. 3. HLA class III antigen. Class III genes encode for complement components or regulators of serum complement component levels. C2 and factor B are encoded by class III MHC genes.

meClical

419

Immunology

4. HLA disease associations. Many diseases are associated with increased frequency of certain HLA antigens. Ankylosing spondylitis is typically associated with HLA-B27; other diseases are also associated with specific HLA antigens: rheumatoid arthritis (DR4), Sjogren syndrome (DR3), and insulin-dependent diabetes mellitus (DR3 and DR4). a. Most of the HLA-associated diseases are of unknown etiology and have associated immunologic abnormalities. b. Tissue typing for organ transplantation involves matching the HLA class I and class II antigens. D. Antibodies. Antibodies act as antigen-specific receptors on B cells, and, when secreted by plasma cells, mediate humoral responses. Antibodies comprise approximately 20% of plasma proteins. They are produced by B cells in response to the introduction of foreign substances (i.e., antigens) into the body. Antibodies are bifunctional: They bind epitopes on antigens, thereby directly attacking the antigen, and they stimulate other biologic phenomena such as activating complement and binding Fe receptors on other lymphoid cells (opsonization, ADCC). 1. Structure. Antibodies consist of four polypeptide chains-two identical light chains and

two identical heavy chains-all bound together by disulfide bonds (Figure VI -1-7). Heavy and light chains have hypervariable regions at the amino terminus (responsible for antigen-binding specificity) and constant regions at the carboxy terminus. There are intrachain disulfide links that divide each chain into subunits of 110 amino acids; thus light chains have two domains referred to as the variable and constant domains, whereas heavy chains have four or five domains-one variable domain and three or four constant ones.

Hypervariable regions

iI

I I

",.Ught chain

It. t.

Disulfide bonds

" . Heavy chain

"'*" ~

....

........ ..... --:::~~, ~

Hypervariable regions .. - - Papain site

~

T pFc'

Fc

....

, , ' ..... Hinge region ........ Pepsin site

J

Figure VI-1-7. Schematic structure of immunoglobulin molecule.

a. Light chains. The molecular weight of each light chain is approximately 23 kD and is composed of 220 amino acids. There are two subtypes of light chains: kappa and lambda. (1) Kappa chains differ from lambda chains on the basis of structural differences in the constant region. The genes coding for each are not on the same chromosome (2 and 22, respectively).

420

meClical

Basic Immunology

(2) Each antibody molecule has either two kappa or two lambda chains. (3) A specific immunoglobulin always has identical kappa or lambda chains. (4) The ratio of chain subtypes is constant within a species. b. Heavy chains. The molecular weight of each heavy chain is 50-75 kD. Each is composed of 450-580 amino acids. (1) There are five different isotypes corresponding to the individual heavy chain gene utilized. Structural differences in the constant regions of the various types of heavy chains account for their different biologic properties. (a) 19G has gamma (y) heavy chains. (b) 19A has alpha (ex) heavy chains. (c) 19M has mu (f.l) heavy chains. (d) 19E has epsilon (£) heavy chains. (e) 19D has delta (8) heavy chains. (2) Heavy chains are further divided into subclasses based on the number of interchain disulfide bridges. For example, 19G 1 and 19G4 have two disulfide bonds, 19G2 has four disulfide bonds, and 19G3 has 15 disulfide bonds. (3) Genes coding for heavy chains are on the same chromosome (14). c. Disulfide bonds link together the different components of the immunoglobulins by

binding between cysteine residues. The bonds confer the three-dimensional structure of immunoglobulins. (1) 1nterchain bonds. There is usually one interchain disulfide bond between the

cysteine residue of the heavy chain and the penultimate or ultimate carboxy terminal cysteine residue of the light chain. 1nterchain disulfide bonds between heavy chains are variable in number; this helps to differentiate subclasses of immunoglobulins. (2) 1ntrachain disulfide bonds also confer structural properties by linking cysteine residues located on the same polypeptide chain. d. The hinge region is located between the second and third 220-amino-acid segments of the heavy chains. This region allows flexibility in the F(ab)2 portion of the molecule and consists of flexible glycine residues flanked by proline-rich inflexible segments. 2. Immunoglobulin fragments generated by enzymatic digestion allow the functional regions of 19 to be characterized. a. Papain digestion of an immunoglobulin yields three fragments, two Fab fragments Cab" stands for "antigen-binding") and one Fc fragment ("c" stands for "crystalline"; the form this region takes in low ionic strength buffers). (1) The Fab fragment contains the amino terminal half of the heavy chain and the

entire light chain. Its molecular weight is 50 kD. (2) The Fc fragment contains the two carboxy terminal halves of the heavy chain held together by disulfide bonds. This region is responsible for binding to Fc receptors on phagocytic cells and for activating complement components. b. Pepsin digestion of an immunoglobulin yields one F(ab')2 fragment and two pFc fragments.

Mnemonic The "e" in Fe could stand for: • Crystallizable • Carboxy terminus of the molecule • Complement-binding region (CHR) • Cell tropism (e.g., FcyR on phagocytes, Fc£R on basophils, FcyR on syncytiotrophoblasts)

meClical

421

Immunology

(1) The F(ab')2 fragment is composed of the two light chains, each of which is disul-

fide bonded to the amino terminal portion of a heavy chain (see Figure VI-l-7). The F(ab')2 fragment maintains bivalent antigen-binding ability and is 100 kD in size. (2) The pFc' fragment is composed of the two free carboxy terminal heavy chains.

Note that the pepsin cleavage site is below the disulfide bonds, thus generating the two free heavy chain fragments that individually have no biologic functions. 3. Antigen-binding sites are found in the variable region located in the 1l0-amino-acid segment of the amino terminal end of the heavy and light chains. a. The variability of amino acid sequence confers antigen-binding specificity to individual immunoglobulin molecules. This site has both framework and hypervariable or complimentarity determining subregions. Framework subregions are relatively conserved amino acid sequences that preserve the three-dimensional structure of the variable region and stabilize the hypervariable regions. Hypervariable regions are folded to form the antigen-binding site (also called the paratope). b. The affinity (or binding strength) of an antibody is defined as the concentration of antigen at which half of the antibodies are in a complex with antigen and half are free. The relative affinity of an antibody to antigen is measured in moles per liter and is referred to as the dissociation constant, Kd • Higher numbers mean greater affinity. c. Different structurally related antigens may be cross-reactive and bound by the same antigen-combining site on different antibodies. This is important in diseases such as rheumatic fever, where streptococcal antigens are structurally related and cross-reactive with heart tissue (cardiac myosin). d. Biologic effects of antigen binding result primarily from cross-linking of immunoglobulin molecules resulting from multivalent antigen binding. 4. Idiotypes, allotypes, and isotypes a. Idiotype is the term used to describe the area of the variable region responsible for antigen specificity. These are the unique epitopes that are found in the paratope (see above) of the antibody. b. Isotype is the term used to describe the subclasses of immunoglobulins ("G-A-M -E-D") that are distinguished by unique constant regions encoded by the heavy chain gene. The individual isotypes have unique effector mechanisms, such as the ability to bind complement or mediate hypersensitivity responses (described below).

Note Monoclonal Antibody Plasma cell + myeloma cell = hybridoma. The hybridoma secretes antibody identical to the one produced by the plasma cell. The myeloma cell gives the hybridoma its immortality.

c. Allotype is the protein product of an allele that may be detected as an antigen by another member of the same species. It involves different alleles at a specific site in the constant region of the heavy chain. S. Immune serum contains a heterogeneous population of antibodies with varying affinity for antigens; the average affinity increases as the period of time after immunization lengthens due to somatic mutations. 6. Monoclonal antibodies are antibodies that are specific for a single epitope determinant. They are produced in vitro by hybridomas, created from the fusion of plasma cells to myeloma cells that are grown in tissue culture. E. Immunoglobulin genetics. The multiple genes encoding immunoglobulin molecules undergo a structured rearrangement process leading to the expression of functional protein. It is through this rearrangement that the diversity of immunoglobulin molecules is acquired. This section reviews the recombination events that are required for immunoglobulin synthesis.

422

meClical

Basic Immunology

1. Genes coding for light and heavy chain variable regions

a. Kappa light chain is encoded by a single locus on chromosome 2. It has a constant domain and a variable domain. Lambda light chain is encoded by a gene complex on chromosome 22. (1) The variable domain is encoded by two separate gene segments. The variable segment (VK or VA) codes the first 95 amino acids, and the joining segment OK or Til,) codes the final 12 amino acids. (2) There are an estimated 350 variable genes and six joining gene segments encoded

in the human K chain gene. b. Heavy chains (VDJ complex) are encoded by a single region on chromosome 14. (1) The constant region (CH ) sequences for each of the heavy chain isotypes are in

tandem on the chromosome. There is a single copy of each gene. (2) There are four joining segments OH)' several hundred variable segments (VH)' and a dozen diversity segments (DH ). (3) The heavy chain is assembled via VDJ recombination. 2. Antibody diversity is generated by random combinations between individual heavy and light chains, as well as by random combinations of variable, joining, and diversity genes. These intrachain recombinational events occur in the following manner (Figure VI-1-8) for light chains. a. The DNA loops around, bringing a variable gene allele close to a joining gene segment. b. The introns interact in a specific manner (Figure VI-1-9), as detailed in sections c through e below.

G~rm line DNA

_fill[][][F!=f= V~ Vvx2 V vxn

j

~ Gene

I

J1 J2 J3 J4 J5

C x

V-J joining

In a Nutshell Antibody diversity is due to: • Recombination of immunoglobulin genes for:

rearrangement

- Variable region

Plasma cell DNA Vx1

J1 J2 J3 J4 J5

!

- Joining region Cx

Transcription

Plasma cell RNA

Nucleoplasm Cytoplasm

- Diversity region (in H chains only) • Recombination of heavy chains with light chains • Addition of nucleotides to DNA during genetic recombination by terminal deoxynucleotidyl transferase enzyme

Figure VI-1-8.lntrachain recombinational events in light chain synthesis.

• Somatic mutations

meClical

423

Immunology

c. The recognition signals adjacent to the V and J segments hold the two strands together so that the recombinase enzyme products of the RAG-l and RAG-2 genes can affect gene fusion, creating a bifunctional codon (a DNA segment coding for both the V- and J-region polypeptides). The recognition signals (recombinant signal sequences [RSS]) are composed of a nine-nucleotide segment (nonamer) and a seven-nucleotide segment (heptamer) separated by 12 or 23 base pairs. d. The spacer of nucleotides in the intron region of the DNA allows the strand to loop around and bring the bases into proximity for optimal interaction. This is called the 1223 spacer because there are 12 nucleotides in one DNA segment and 23 in the other. e. The variable and joining gene segments are fused by the action of two recombinase enzymes. An endonuclease cleaves the DNA strand, splitting out the intron. The two gene segments are then united by DNA ligase.

C C Nonamer

Sp~'

12 bases (one turn)

A A A A A

T T T T T

C

G

A

T lIE

23 bases (two turns)

[

G

T Heptamer

G G

C A

G A

C T

C

G

Site of V/J recombinase action

G

/

A

c

T

INS

Figure VI-1-9. Recognition Signal interactions.

f. Once gene recombination has occurred, the DNA is transcribed into nuclear RNA and the intervening sequence between the bifunctional codon and the constant region gene is spliced out by an RNAse. g. The RNA then moves to the cytoplasm, where it can function as a messenger RNA molecule. h. A similar process is involved in synthesis of the heavy-chain mRNA with the exception that there are two recombinational events: A diversity gene combination with a joining gene precedes the joining of the variable gene with this bifunctional codon to form, in this case, a trifunctional codon.

424

meClical

Basic Immunology

3. General features of class (isotype) switching and gene recombination a. Mature B lymphocytes expressing IgM and IgD may switch antibody classes after contact with antigen to produce IgA, IgG, or IgE. b. The immunoglobulin produced has the same variable region of light and heavy chain as IgM and IgD on the cell surface, except for occasional mutational changes in the V region genes (somatic hypermutation). c. The constant region genes are maintained, allowing the synthesis of the different iso-

types all with the same antigen specificity. After VDJ recombination, a B cell may receive signals through cytokines or other cascades that a different isotype of immunoglobulin may be more appropriate. The cells will class switch to anyone of the downstream constant regions (y, ex, or £ chain genes). 4. Allotypes are antigens on immunoglobulin molecules encoded at one genetic locus and inherited as alleles. Not all members of the species have the same particular allotype in their genome. a. Km allotypes are a derivation of K light chains in humans from one of three alleles at a single locus, km. b. Gm allotypes are a derivation of the human IgG heavy-chain isotypes Gl, G2, G3, and G4. c. a2m allotypes are a derivation of the human a2 heavy-chain isotype from one of two alleles at a single locus, a2m (1 or 2).

d. Allelic exclusion is a term used to describe the phenotypic expression of a single allele in cells containing two different alleles for that genetic locus (one from each parent). The synthesis of a functional gene product from a light-chain gene suppresses recombination on all other light-chain genes; the same is true for heavy chains. Therefore, recombination events create an active H -chain or L-chain gene on only one of the two chromosomes. F. Properties of immunoglobulin subclasses. As previously noted, there are five immunoglobulin subclasses: IgG, IgA, IgM, IgE, and IgD (remember them by"G-A-M-ED"). They can be distinguished by the type of heavy chain they possess and by their distinct biologic functions. 1. IgG provides the major defense against bacteria and toxins. There are four subclasses,

varying by heavy-chain isotype (Gl, G2, G3, G4).

In a Nutshell

a. The relative amounts of serum IgG are IgGl, 60-70%; IgG2, 14-20%; IgG3, 4-8%; IgG4,2-6%.

Major Functions of IgG

b. IgG (molecular weight of 150 kD) comprises 85% of the total immunoglobulin, with serum concentration 800-1,700 mg/dL. It has a half-life of approximately 21 days, except IgG3, which has a half-life of 7 days.

• Opsonization

c. IgG is important in the secondary immune response to antigen and provides long-

lasting immunity.

• Passive immunity in fetus

• Complement activation • ADCC

d. IgG is the only class of immunoglobulin that crosses the human placenta into fetal circulation; it is responsible for protecting the newborn during the first 4-6 months of life. e. Three subclasses of IgG fix complement (IgG3 > IgG 1 > IgG2). f. The Fc region binds to Fcy receptors on polymorphonuclear leukocytes, monocytes, protein A of staphylococcal strains, and some B lymphocytes.

meClical

425

Immunology

2. IgM is important in the primary immune response to antigen it is usually the first antibody detected in the serum after exposure to a specific antigen.

In a Nutshell Major Features of IgM • First antibody synthesized • Found in B-cell membrane • Pentamer with J chain in serum • Very efficient activator of complement

a. Monomers serve as cell-surface receptors on mature, naive B lymphocytes. (1) The cell-surface form contains a hydrophobic segment of 25 amino acids in the

carboxy terminal of the heavy chains that serves to anchor the immunoglobulin to the membrane. (2) The secreted form contains an alternative hydrophilic chain terminus. b. Circulating IgM is a pentamer of five immunoglobulin molecules (10 heavy 11 chains and 10 light chains) and one disulfide-linked J chain, with a total molecular weight of approximately 900,000 daltons. The cysteine-rich J chain is linked by a disulfide bond to the penultimate cysteine residues of two 11 chains from separate monomers. c. The half-life of IgM in serum averages 5 days. The serum concentration, 50-190

mgldL, represents 10% of immunoglobulin in plasma. d. IgM fixes complement very efficiently because each circulating molecule has 10 Fc sites. e. Agglutination of antigens via the pentameric structure of IgM allows the cross-linking of antigens. f. Isohemagglutinins, rheumatoid factors, and heterophile antibodies are all IgM. 3. IgA has an important barrier function on mucosal surfaces and functions in the secretory immune response. a. 19A represents 15% of the total serum immunoglobulin. The serum half-life of 19A is 6 days with a concentration of 140-420 mg/dL.

In a Nutshell

b. The serum form is usually monomeric, consisting of two heavy chains (aI, (2) and two light chains with a molecular weight of 170 kD.

Major Features of IgA

c. The secretory form (sIgA) is found in tears, colostrum, saliva, milk, and other secretions.

• Secretory immunity

(1) It is a dimer joined by a polypeptide J chain.

• In secretions found as a dimer with a J chain and secretory piece (slgA)

(2) sIgA contains a 70,000-dalton secretory component added to polymeric 19A by epithelial cells as it passes through to the luminal side. (3) The secretory component confers stability to the molecule, making it less susceptible to proteolysis in the gastrointestinal tract. (4) It is produced by plasma cells in the lamina propria of the gastrointestinal and respiratory tracts. d. Subclasses of 19A are based on two heavy chain isotypes.

In a Nutshell Major Features of IgO • Very low concentration in plasma • High levels in membrane of mature B cell • Functions in antigen recognition by B cell

426

iileilical

(1) 19A1, 90% of serum 19A, is susceptible to bacterial proteases.

(2) 19A2, 60% of secretory 19A, is resistant to proteases. 4. IgD functions as a cell surface antigen receptor in naive B lymphocytes. IgD is very susceptible to proteolysis. a. Its serum concentration is very low, ranging between 0.3-4.0 mgldL with a serum half-life of 3 days and a molecular weight of 170 kD. b. IgD functions primarily as a receptor for antigen on B cells and stimulates B-cell proliferation. There are no subclasses of IgD. It disappears after antigenic stimulation.

Basic Immunology

5. IgE, also called the "reaginic antibody:' is associated with allergic response and immediate hypersensitivity. a. IgE has a serum half-life of 2 days in plasma and a serum concentration of <0.06 mg/dL. b. The Fc region of 19E binds to the surface of basophils and mast cells. When antigen cross-links two IgE molecules, mast cells degranulate and release leukotrienes, histamine, eosinophil chemotactic factors, and heparin, which results in an immediate hypersensitivity reaction. The half-life ofIgE is increased when it is bound to cell surfaces. c. IgE offers some protection against metazoan parasites, mainly via the release of

ECF-A and the induced inflammatory response at the site of worm infection. Eosinophils degranulate in the area and release major basic protein (MBP). This protein has been shown to have a direct toxic effect upon schistosomes and, presumably, acts as a general metazoan poison.

Heavy chain Serum concentration (mglmL) Serum half-life (days) Placental transfer Complement activation Binding to mast cells/basophils

IgG2

IgG3

y1

Major Features of IgE • Very, very low concentrations in plasma • Homocytotropic for basophils and mast cells • Reaction with antigen when bound to mast cell or basophil causes release of histamine and production of leukotrienes • Immediate hypersensitivity (type I)

Table VI-I-I. Characteristics of immunoglobulin isotypes. IgGl

In a Nutshell

IgG4

IgAl

IgA2

y4

a1

a2

IgM

IgE

IgD

9

3

1

0.5

3

0.5

1.5

0.00005 0.03

21

20

7

21

6

6

10

2

+

+

+

+

+

+

+

In a Nutshell Isotype

Ig Function

IgG

•

---7

Opsonization

• ADDC 3

• Placental passage • Complement activation

+ IgA

---7

•

Mucosal (secretory) immunity

IgM

---7

•

Complement activation

IgE

---7

•

Basophil and mast cell sensitization

IgD

---7

•

Antigen receptor on B cells

+

G. T-cell receptors (TCR) act as antigen-specific receptors, allowing T cells to function in cellmediated responses. They do not recognize antigen alone, but only in the context of MHC molecules. 1. Their expression is interwoven with T-cell maturation in the thymus. In the thymus, T cells mature and undergo positive and negative selection. Negative selection deletes auto reactive cells. T cells develop immunologic specificity in a manner similar to that used by B cells; that is, they use genetic recombination among variable, joining, and diversity genes to achieve their immunologic repertoire. The gene pools are different than those for B-cell (and antibody) diversity, but the processes are very similar. 2. The T-cell receptor is composed of two distinct polypeptide chains, most commonly an alpha and a beta chain that are homologous to the light- and heavy-chain components of the Fab of immunoglobulin molecules. A small percentage (1-5%) of T cells have different polypeptides, gamma, and delta chains; very little is known about the function of this T-cell subset. a. Each chain has two domains marked by intrachain disulfide bonds. These domains, one variable and one constant, have a structure and function similar to that of the antigen-binding fragment of antibody molecules.

KAPLA~.

meulCa

I 427

Immunology

b. There are three complementarity-determining regions in the variable domain of each chain. c. At the carboxy end of the constant domain is a short connecting sequence that has a

cysteine residue involved in disulfide linking of the two chains. Following this is a transmembrane segment; each chain has a cytoplasmic tail of 5-12 amino acids. 3. T-cell receptor genetics a. The genes encoding the TCR are expressed only in cells of the T-cell lineage. Their expression is induced during residence in the thymus under the influence of various cytokines. b. The four loci (a, B, y, and 8) have a chromosomal organization that is similar to the multigenic organization of the immunoglobulin genes. Separate V-, D-, and J-gene segments rearrange during T-cell maturation to form functional genes encoding mRNAs for the individual chains. c. The alpha chain is encoded on chromosome 14; there are 50 variable gene segments

and 70 joining gene segments. The beta chain genes are found on chromosome 7; there are 57 variable gene segments, 13 joining segments, and 2 diversity gene segments. The alpha chain has a single constant domain, whereas the beta chain may be one of two distinct constant domains. d. The mechanisms by which TCR DNA is rearranged to form functional receptor genes follows the same pattern as that found in immunoglobulin genetics. (1) Heptamer/nonamer recognition signal sequences containing either 12 (one

turn) or 23 bases (two turns) occur flanking each V-, D-, and J-gene segment in the TCR germ line DNA. (2) A recombinase enzyme recognizes the recognition signals and catalyzes V-J and V-D-J joining.

Note The genetic rearrangements that occur in the generation of the T-cell repertoire are very similar to those seen in B-cell development.

(3) The constant region of each chain is encoded by a gene segment that has multiple exons corresponding to the functional domains in the protein. The first exon encodes the majority of the domain. A short exon encodes the connecting sequence; this is followed by two exons encoding the transmembrane region and the cytoplasmic tail. (4) There is no secreted form of the TCR, so differential processing of primary RNA transcripts does not occur. H. Types of immune responses. Immune responses usually involve some level of inflammation and are an acquired or adaptive response of the body. 1. Infection at a tissue site causes inflammation, which enhances antigen delivery by

Note Adjuvants are immunostimulants that increase the immune response by their inflammatory action. They also prolong contact with the immunogen by acting as a depot of antigen deposition.

428

meClical

follicular dendritic cells and macrophages in that tissue, to lymphocytes found in nearby secondary lymphoid organs and recruits circulating phagocytes and NK cells to attempt to resolve the infection. Activation and clonal expansion of antigen-specific lymphocytes in the secondary organ requires several days to generate a large number of effector lymphocytes and some memory lymphocytes. The effector lymphocytes then circulate through the blood and extravasate at the site of infection due to upregulation of adhesin molecules on endothelial cells and locally produced cytokines (1L-8). 2. Humoral immune responses are generated against most antigens and require the secretion of antibody by plasma cells (terminally differentiated B cells) in response to a specific immunogenic stimulus.

Basic Immunology

a. The primary immune response is the first response to antigenic exposure. During the initial lag phase, lasting the first 3-5 days, no free antibody can be measured. During this phase, Th cells and B cells are being activated, and activated B cells are differentiating into antibody-secreting plasma cells (the specifics of Th- and B-cell activation are discussed later). Thereafter, an exponential (log) phase-rise in detectable antibody in the circulation occurs. (1) IgM is primarily produced. After the first IgM response, class switching occurs, and the level of IgM usually declines. (2) Later in the response, higher-affinity IgG becomes detectable; in response to an oral or mucosal antigen, slgA becomes detectable in secretions as welL b. The secondary immune response is a memory (anamnestic) response to previously encountered antigen. Memory Band T cells are responsible for this phase. (1) In a secondary response, high-affinity IgG class antibody levels rise more rapidly; this requires less antigen to elicit a response. The ability to respond may persist for years due to the presence of long-lived memory B cells and T cells. This memory response explains the efficacy of booster injections of vaccines. (2) Similarly, a secondary response to an oral antigen rapidly generates higher levels of 19A, much of which is transcytosed across the mucosal epithelial cells onto the external mucosal surface. (3) Secondary responses to large extracellular particles, such as some helminths, pollens, or enzymes (e.g., those found in cat saliva), may produce unusually high levels of IgE. IgE may be important in the response to helminths, but IgE made in response to pollens and enzymes can lead to allergies.

In a Nutshell Acquired immunity can be divided into two broad categori es: • Humoral, or antibody, responses - Primary response is always IgM - Secondary response is a result of class switching and results in synthesis of IgG, IgA, and/or IgE (predominantly IgG) • Cellular responses involve thymus-derived lymphocytes and involve the activation of helper cells, cytotoxic cells, and macro phages.

3. Cellular immune response. Effector (activated) Th cells have multiple functions, including helping activate B cells for most humoral responses, helping activate CTLs, and acting as effector cells in cell-mediated responses such as the killing of virally infected cells and some tumor cells. a. Helper T-cell activation requires presentation of foreign peptides on RLA class II proteins on antigen-presenting cells. (1) Activation of naive Th cells in primary responses typically occurs in the T-dependent areas of lymph nodes, spleen or, sometimes, the Peyer patches of GALT. (2) Activation of memory Th cells in secondary responses can occur in the lymph node, spleen, mucosal tissues, or in the cutaneous lymphoid tissue.

In a Nutshell • THl cells: IL-2, IFN-y • TH2 cells: IL-4, 5, 6, 10

b. Th cells secrete cytokines that lead to the differentiation and proliferation oflymphocytes. (1) Activation and clonal expansion of B cells in response to most antigens requires

that B-cell-bound antibodies bind their cognate antigen, and that an effector Th cell binds to the B cell and secretes cytokines such as IL-4, -5, -6, and -10. These interleukins favor the development of humoral immune responses. The helper cell subset that produces these cytokines is called a Th2 cell. (2) Another subset of helper T cells, called the Thl cells, secrete IL-2 and IFN-y, which cause proliferation and differentiation of cytotoxic T cells and macrophages, respectively. c. T cells are the major cell type needed for cell-mediated responses.

meCtical

429

Immunology

(1) Some effector Th cells and the effector CTLs leave the lymph node via the efferent lymphatic (or the spleen via the red pulp) and circulate through the blood. They bind to adhesion proteins expressed on inflamed endothelial cells and extravasate into the tissues. (2) Effector CTLs will bind to any virally infected or tumor cell that displays the appropriate epitope associated with a HLA class I MHC protein. This binding allows the CTL to secrete perforins and granzymes and kill the target cell. (3) Effector CD4+ (helper) cells can secrete cytokines that activate innate cells, e.g., NK cells (to help kill virally infected or tumor cells), or macrophages and neutrophils. Excessive activation of macrophages causes the macrophages to release inflammatory cytokines that can cause delayed-type hypersensitivity. 1. Regulation. All immune responses must be regulated. Antigen presentation can affect the strength of the response, as can immune suppression. Anti-idiotypic antibodies may playa role in downregulating normal humoral responses. 1. Immunologic tolerance describes the specific depression of immune responses induced

Clinical Correlate Some allergies can be controlled by the ingestion of the allergen. This has proven to be of value in some patients with pollen allergies (they eat honey).

by previous antigen exposure. The degree of immune tolerance may be partial or complete. That is, one may elicit tolerance to one epitope but not to another on a specific molecule. Important factors in tolerance induction include: a. The form of antigen. For example, de aggregated antigen (ultracentrifuged soluble foreign gamma globulin) induces tolerance. Also, antigen coupled to isologous immunoglobulin, lymphocytes, or inert carriers can elicit immunologic tolerance. b. Route of exposure. Intravenous and oral administration of the tolerizing antigen is most effective in tolerance induction. In contrast, subcutaneous administration of antigen induces immune responses. c. The age of the recipient. Neonates are more prone to tolerance induction by mecha-

nisms that are not entirely understood but probably relate to immunologic immaturity and the paucity of immunocompetent cells.

Clinical Correlate Frequently administered low doses of antigen may be the reason that large organ transplants (e.g., liver) survive well in most recipients.

d. Dosage of antigen. High doses of antigen may overwhelm the immune system, leading to specific immune suppression. Frequently administered low doses of antigen cause unresponsiveness, particularly in T cells. Levels of antigen required for B-cell tolerance are much higher than those needed for T-cell tolerance. 2. Immunosuppression is described as the active immunologic unresponsiveness. This can occur via cellular, physical, or chemical means. The biologic effects of immunosupression are shown in Table VI-1-2. a. Cellular suppression in the immune response is evidenced via the Thl and Th2 subsets of Th cells. IFN -y, produced by Thl cells, suppresses the Th2 response. IL-lO, produced by Th2 cells, suppresses the Thl response.

Clinical Correlate A serious consequence of . . . Immunosuppression IS opportunistic infections. Recurrence of CMV infection in bone marrow transplant recipients is a major cause of morbidity.

430

iiie&ical

b. Physical immunosuppression (1) X-ray and ultraviolet radiation can suppress immune responses by the elimina-

tion of lymphocytes. B cells are more sensitive to radiation than are T cells. Bone marrow and lymphoid tissues are especially sensitive. (2) Surgical intervention can be used by the removal oflymphoid tissue such as the thymus, spleen, and lymph nodes. (3) Chemical agents can successfully suppress immune responses. Corticosteroids can cause peripheral blood lymphopenia, inhibit RNA and DNA synthesis, decrease macrophage responses, decrease monocyte chemotaxis, and decrease

Basic Immunology

IgG responses. Purine or pyrimidine analogs (e.g., azathioprine) inhibit IgG response. Folic acid antagonists interfere with DNA and protein synthesis in lymphocytes. Alkylating agents (e.g., cyclophosphamide) reduce the number of lymphocytes in the spleen. 3. Anti-idiotypic responses are other mechanisms that downregulate or control ongoing immune responses. Anti-idiotypic immune responses are antibody responses directed toward the paratope of another specific antibody. The antibody-combining site itself acts as an antigen and induces the formation of specific antibody. Such antibodies, directed to the combining sites (idiotypes) of other antibodies, are known as anti-idiotypic antibodies.

Table VI-1-2. Biologic effects of immunosuppression. Specific Immunosuppression Monoclonal antibodies to: T cells

Biologic Effects Specific cell lysis and inhibition of macromolecules (cytokines)

Cytokines (IL-I, IL-2, TNF-a, IFN-y) Cytokine receptors (IL-Ir, IL-2r, TNFr) Adhesion molecules (ICAM) Specific allergens

Low-dose tolerance induction

Nonspecific Immunosuppression

Biologic Effects

Anti-inflammatory drugs Corticosteroids (prednisone) Nonsteroidal anti-inflammatory drugs (NSAIDs) Aspirin (acetylsalicylic acid) Ibuprofen (phenylacetic acid) Indomethacin (indoleacetic acid)

Inhibitors of cytokines, nitric oxide synthase, cyclo-oxygenase, adhesion molecules

Inhibitors of cyclo-oxygenase, prostaglandin synthesis

DNA metabolism inhibitors Cyclophosphamide Chlorambucil Methotrexate Azathioprine

Alkylating agent for DNA Alkylating agent for DNA Folic acid analog Purine analog

Inhibitors of T-cell signaling Cyclosporine FK 506 (tacrolimus) Rapamycin (sirolimus)

Immunophilin/calcineurin signaling inhibitor Immunophilin/calcineurin signaling inhibitor IL-2 receptor signaling inhibitor

a. The network theory of immune regulation states that certain lymphocyte clones may recognize the idiotypes of other lymphocyte clones as antigens. b. Every lymphocyte is in dynamic equilibrium, exposed to stimulatory and suppressor signals.

KAPLAN'dme leaI

431

Immunology

c. It is now believed that this regulatory system is important after a given lymphocyte has been activated by antigen. The passive transfer of anti-idiotypic antibody against certain dominant idiotypes may suppress the total immune response. d. Idiotypes on Band T lymphocytes may be cross-reactive and mimic the action of an antigen, such that anti-idiotypic antibodies may suppress both humoral and cellmediated immunity.

CELLULAR INTERACTIONS IN IMMUNE RESPONSES A. Antigen processing. The mechanism whereby antigen is internalized and re-expressed on the antigen-presenting cell (APC) membrane associated with MHC class I or class II molecules is called antigen processing. There are two separate routes that dictate with which MHC molecule the peptide will associate. 1. The exogenous route of antigen processing occurs when a cell takes in foreign antigen by

In a Nutshell • Endogenous antigens synthesize within the cell, bind with class I MHC molecules, and are presented on the cell surface. • Exogenous antigens are internalized and digested, and fragments are complexed with class II MHC molecules to be presented on the cell surface.

phagocytosis or pinocytosis. The antigen is engulfed and is found in endosomes inside the cells. Proteolytic enzymes digest the antigen into small peptide fragments. The endosomes fuse with exocytic vesicles that bear class II molecules on the interior of their membranes, and the peptide epitopes then bind in the antigen-binding groove of the class II MHC. The endosome then travels to the cellular membrane and fuses with the membrane, thereby expressing the MHC peptide on the surface of the cell. 2. Many antigens are synthesized within the cell. Examples of such antigens include tumor antigens and viral proteins. These proteins are processed by the endogenous route for antigen processing. These proteins are synthesized in the cytoplasm of the cell, and some are broken down into peptide fragments. They are transported to the lumen of the endoplasmic reticulum, through the TAP peptide transporter complex, where they associate with class I MHC molecules for presentation to CD8 T cells. The MHC I-peptide complex is transported through the Golgi to the cell surface. B. The T-cell receptor (TCR) and its interaction with antigen

1. The TCR is antigen (epitope) specific. It binds to the epitope located in a groove in the

appropriate class I or II MHC molecule on the surface of the APe. This binding is noncovalent and is identical to the interaction of an epitope with the Fab portion of an antibody molecule. The major distinction is that the interaction of this membrane-bound receptor with the MHC-associated epitope induces a signal transduction cascade. There is a considerable amount of interplay with other cell membrane molecules in the activation of these immunocompetent lymphocytes. 2. The TCR is noncovalently linked to the CD3 molecule, and engagement of the TCR by antigen stimulates CD3 to transmit biochemical signals into the interior of the cell. These signals are required to activate the T cell. When peptides are presented in association with MHC molecules, the antigen-binding region of the TCR recognizes processed peptide in association with the MHC molecule. Additionally, the CD4 molecule binds to the MHC molecule. CD8 molecules bind to class I MHC, but CD4 molecules cannot. The need for CD4 to bind to MHC II and CD8 to bind to MHC I explains why helper cells (CD4+) respond to processed antigens associated with class II, whereas cytotoxic T cells (CD8+) respond to antigens associated with class I MHC molecules. e. The T-cell-APC interaction. We have already discussed the physical interaction of the TCR with the MHC class I or class II-peptide complex. However, for the T cell to become activated, other molecules on the cell surface of the T cell and APe must interact. These accessory molecules are collectively known as adhesins and further strengthen the interaction between the T lymphocyte and the antigen-presenting cell.

432

meClical --------------

- - -

Basic Immunology

1. The T cell possesses adhesion molecules known as CD2 (LFA-l) and costimulatory molecules known as CD2S.

In a Nutshell

2. CD2 binds to LFA-3 on the APe. CD2S binds with B7 on the APe. 3. These interactions provide a more stable association between the APC and the T cell and, in addition, provide costimulatory signals across the T-cell membrane. Most important in this regard is costimulation provided by B7.

CD28

CD3

67

D. Overview of T-cell activation. Once the TCR-MHC-peptide-CD4 or CDS interaction and adhesins have made contact with the T cell, the T cell must also be signalled by the APCderived cytokine, interleukin-l (IL-1). 1. IL-l is secreted by the APC and interacts with IL-l receptors that are found on the surface of the T lymphocyte.

APC

Figure VI-1-10. T-cell-APC interaction.

2. This costimulatory signal provides information required for the T cell to express increased numbers of receptors for a second cytokine, interleukin-2 (IL-2). 3. The T cell also begins to secrete IL-2 in large amounts. 1L-2 then interacts with the IL-2 receptors of the T cell and stimulates the T cell to proliferate and grow. E. Cell signaling events. Many different events must be initiated before the T cell is fully activated. 1. The TCR-CD3 complex transmits intracellular signals via protein G and phospholipase e. This pathway results in the conversion of phosphatidylinositol bisphosphate (PIP 2) to diacylglycerol (DAG) and inositol triphosphate (IP 3 ). 2. DAG activates a protein kinase that mediates protein phosphorylations in the interior of the cell. 3. Calcium levels increase in the cell due to the action of IP3' and calmodulin kinase is activated. 4. CD4 (or CDS) also transmits intracellular signals via a tyrosine kinase mechanism.

Clinical Correlate

5. In addition, the interaction of IL-l with the IL-l receptor transmits intracellular messages via an adenyl cyclase mechanism. Costimulation provided by IL-l results in activation of the c-jun gene, whereas activation via the TCR activates the c-fos gene. The c-jun-c-fos heterodimer is responsible for activating genes for the synthesis of IL-2 and the IL-2 receptor.

Bruton agammaglobulinemia is caused by a defective tyrosine kinase (btk) located on the X chromosome.

F. Requirements for activation of cytotoxic T cells (Tc). Although Th cells can be fully activated by the T-cell-APC interaction (Figure VI-I-I0), cytotoxic T cells cannot. This is primarily because these cells do not secrete enough IL-2 to support their own growth. For this reason, Tc require activated Thl cells to supply the IL-2 they need. Although the tumor, graft, or vir ally infected cell itself is able to supply the Tc with peptide associated with MHC class I molecules, antigen-presenting cells must be able to take up some of the soluble antigens and present this antigen to Th cells. The Th cells can then supply the IL-2, which is needed by the Tc before it will develop into a fully functional cytotoxic T cell.

G. Requirements for activation of B cells 1. When B cells become activated, they require the interaction of native (unprocessed) antigen with their antigen receptor (membrane immunoglobulin). However, for class switching to occur, activated T cells must supply necessary Th2 cytokines, including IL-4, IL-S, IL-6, and IL-I0. B cells also need some IL-2, which may be supplied by a Thl cell. 2. Some antigens, notably polysaccharides, can stimulate B cells without the need for T-cell help. These antigens are said to be "thymus independent:' The antibodies made in such responses are IgM only; also, no immunologic memory is induced. This is due to the fact that Th cells do not respond to polysaccharides because they cannot be presented on MHC II.

I KAPLAlf medlea 411

Immunology

In a Nutshell Summary of Interleukin Role in Cell Activation • IL-l produced by macro phages activates Th cells. • IL-2 produced by Th1 cells is a signal for proliferation. IFN-y is also important in cell activation (i.e., macrophage activation). • IL-4,5 and 6 produced by Th2 cells are signals for maturation and class switching.

3. Protein antigens are "thymus dependent:' Immunoglobulins made to thymus-dependent antigens will include all classes of immunoglobulin because class switching will occur in these responses due to the presence of Th2-derived cytokines. H. Cytokines. Cytokine is a term used to describe a collection of proteins that regulate immunologic and inflammatory response to injury. Cytokines specifically modulate responses to antigen in several ways, including the regulation of cell growth and the control of differentiation of immunologic cells. Cytokines also mediate intercellular signaling that controls activation or suppression of immune cells. Normally, resting cells do not secrete cytokines; cells must be stimulated to produce them. Cytokines have both autocrine effects (i.e., exerts a local effect on the same cell that produces the cytokine) and/or paracrine effects (exerts an effect over a distance upon other cells). Cytokines are typically very potent agents that can act on their target cells at very low concentrations. Also, a single cytokine can have multiple functions, and many cytokines have similar effects. Cytokines often influence the synthesis of other cytokines, leading to cascades with both positive and negative regulatory mechanisms. 1. Interleukin-I (IL-I) is produced by macrophages and other antigen-presenting cells. IL-l

attaches to a high-affinity receptor on the target cell. The expression of IL-l receptor is decreased by internalization after the binding of IL-l. IL-l receptors are present on almost all cell types that have been examined. IL-l has several functions, including: a. Stimulation of T cells to secrete interleukin-2 (IL-2) and express IL-2 receptors b. Pyrogenic effects (causes fever) through the action on cells in the hypothalamic regulatory regions of the brain c. Chemotactic activity for neutrophils and monocytes

d. Increased expression of ICAMs on vascular endothelial cells e. Activation of macrophages and NK cells 2. Tumor necrosis factor (TNF) is produced by the same cells as IL-1. TNF has antitumor effects: It causes necrosis of tumors. TNF is the principal mediator of the host response to gram-negative bacteria. There are two TNF polypeptides: TNF-a and TNF-~. TNF-a (cachectin) is produced by macrophages and other cells. TNF-~ (lymphotoxin) is produced by T cells.

Clinical Correlate Deficiency of the y chain of the IL-2 receptor causes SCiD. This chain is also part of the receptors for IL-4, 7, 9, 13, and 15.

Clinical Correlate

3. Interleukin-2 (IL-2) is produced by activated CD4+ T cells. IL-2 is both an autocrine and a paracrine growth factor. It acts on the same cells that produce it and on nearby CD4+ and CD8+ T cells. IL-2 is not an endocrine growth factor because it does not circulate in the blood and does not act at a site that is far removed from the site of synthesis. The highaffinity receptor, IL-2R, is composed of a complex of three separate polypeptides that is upregulated on antigen-activated T cells. IL-2 has several biologic functions: a. Stimulation of the proliferation of other T-cell subsets b. Stimulation of the proliferation of activated B cells c. Activation of NK cells and enhancement of their cytolytic activity

IL-4 action is intimately involved in the induction of atopic allergies: • Enchances class switching to IgE • Increases mast cell numbers

434

meclical

d. Stimulation of lymphokine secretion 4. Interleukin-3 (IL-3), produced by Th cells and NK cells, supports the growth and differentiation of hematopoietic stem cells. 5. Interleukin-4 (IL-4) is produced by Th2 cells and mast cells. Its biologic functions include:

Basic Immunology

a. Induction of cells to express class II MHC antigens

In a Nutshell

b. Stimulation of B-cell proliferation

Interleukins

c. Regulation of immunoglobulin expression and isotype switching to IgG and IgE

IL-l ~

d. Mitogenic activity for T cells and mast cells 6. Interleukin-5 (IL-5) is produced by activated Th2 cells and activated mast cells. Its biologic functions include: a. Stimulation of B-cell proliferation, particularly with IL-2 and IL-4 b. Stimulation of bone marrow precursors to synthesize eosinophils

IL-2

~

IL-3

~

IL-4

~

IL-5

~

IL-6

~

IL-7

~

IL-8

~

c. Activation of mature eosinophils to kill helminths

d. Stimulation of B cells to class switch to 19A 7. Interleukin-6 (IL-6) is produced by Th2 cells and macrophages. IL-6 is synthesized in response to IL-l and, to a lesser extent, to TNF. Its biologic functions include: a. Stimulation of B cells to produce immunoglobulins b. Stimulation of liver to produce acute-phase proteins during an acute inflammatory response c. Functioning as a growth factor for certain plasma cell tumors (plasmacytomas and

myelomas) and hybridomas d. May also have antiproliferative activities on certain carcinomas, leukemias/lymphomas, and fibroblasts 8. Interleukin-7 (IL-7) is produced in the bone marrow and is secreted by thymic stromal cells. IL-7 stimulates pre-B cells, thymocytes, and mature T cells. It is important in the recovery from cyclophosphamide toxicity by stimulating myeloid precursors and megakaryocytes. 9. Interleukin 8 (IL-8) is produced by macrophages. It is chemotactic for neutrophils and also induces their adherence to vascular endothelium and extravasation into tissues. 10. Interleukin 10 (IL-1 0) is produced by Th2 cells. The biologic properties ofIL-I0 include: a. Downregulation of the expression of class II MHC molecules on B cells b. Inhibition of the growth of Th cell subsets that synthesize interferon and IL-2 (Thl cells) c. Stimulation of the growth of Th cell subsets that synthesize IL-4 (Th2 cells)

11. Interleukin 12 (IL-12) is a lymphokine produced by B cells and macrophages. It activates NK cells and induces Th differentiation into Thl-type cells (T cells that secrete IL-2 and interferon). It acts synergistically with IL-2 to enhance CTL production. 12. Interferons (IFN) are proteins with antiviral and antiproliferative activity. Interferons stimulate phagocytosis and other functions of macrophages and inhibit cell growth and viral replication.

IL-10

~

IL-12

~

a. Type I interferons are induced by the presence of virus or double-stranded RNA. IFN-a is produced by leukocytes, whereas IFN-[3 is produced by fibroblasts. b. Type II interferon (IFN-y) is induced by antigen and is produced by helper T cells (Thl cells). IFN-ystimulates NK cells, phagocytic cells, and antibody production more than type I IFN. IFN -y increases MHC molecule expression. It inhibits the growth of Th2 cells.

IFNy ~

• Stimulates other cells to proliferate, activate, and chemotax • Stimulates IL-2 secretion • Pyrogenic (fever inducing) • Produced byactivated T cells • Stimulates T cells (helper, cytotoxic, and natural killer cells) • Stimulates B cells • Secreted byactivated T cells • Stimulates bone marrow stem cells • Secreted by activated helper cells and mast cells • Stimulates B cells • Increases IgG and IgE • Secreted by activated helper cells • Promotes B-cell proliferation • Increases IgA and increases synthesis of eosinophils • Stimulates production of acute-phase reactants • Stimulates B cells • Stimulates pre-B and pre-T cells • Stimulates chemotaxis and adhesion of neutrophils • Inhibits cytokine release from macrophages • Inhibits interferon synthesis by Th 1 cells • Activates natural killer cells • Induces Th ~ Th 1 • Increases CTL numbers • Stimulates macrophages • Stimulates NK cells • Inhibits Th2 cells

KAPLA~.

meulCa

I

435

Immunology

13. Colony-stimulating factors (CSFs) stimulate the growth and development of pluripotent stem cells of bone marrow. CSF can be used to stimulate production of blood cells and help boost the immune system in an immunosuppressed patient. There are several types of CSF with distinct biologic functions: a. IL-3 (multilineage-CSF) and GM-CSF (granulocyte-macrophage CSF) stimulate all stem cells to produce hematopoietic cells. b. M-CSF (monocyte-macrophage CSF) stimulates macrophage production. c. G-CSF (granulocyte-CSF) stimulates neutrophil production. d. Erythropoietin stimulates RBC production. 14. Transforming growth factor-~ (TGF-~) was initially identified as a tumor-produced factor that would permit normal cells to survive in soft agar, a characteristic of malignant or transformed cells. TGF-~ is produced by T lymphocytes and antigen-presenting cells. The functions of TGF-~ include: a. Acting as a chemoattractant for macrophages b. Inducing interleukin production by macrophages c. Inducing class switching to IgA d. Inhibiting the growth of marrow stem cells

COMPLEMENT AND INFLAMMATION In a Nutshell IFN"'Y (from TH 1 cells) and IL-IO (from TH2 cells) are mutually inhibitory. This allows the immune system to focus its energies where they will be most beneficial: toward cell-mediated (fH 1) or antibody-mediated (fH2) effector systems.

Inflammation is an integral part of all immune responses. One of the major mechanisms for initiating inflammation is activation of the complement cascade, which also produces powerful opsonins, chemoattractants, and anaphylatoxins and can directly mediate killing through cell lysis. A. Complement. Historically, complement was the term used to describe the activity in serum, which, when combined with specific antibody, would cause bacterial lysis. Complement is now known to be a system of proteins that interact to playa role in humoral immunity and inflammation. Complement is a complex group of proteins and glycoproteins found in blood and tissue fluids. Complement can directly opsonize foreign material for phagocytosis after antibody activation (C3 component) and can participate directly in killing some cells and microorganisms. Finally, peptide fragments from the complement proteins can regulate inflammation. 1. Complement activation (Figure VI-1-11). There are two major pathways of complement activation: the classical pathway and the alternative pathway. The end result of either pathway is the formation of a membrane attack complex (MAC), which is a lipid-soluble pore structure that causes osmotic lysis of cells.

436

mectical

Basic Immunology

Classical Pathway

Alternative Pathway

Antigen-antibody complex

Cell walls

r

C1 (q,r,s)l. Cls C4 +

C21. C4b2a

~

C3bBb /

/~3~

C4b2a3b

C3b + B

P (Properdin)

C3a C3bBb3b(P)

~/ C5,C6,C7

/~ C5a

C5b,6,7

yC8,C9 Membrane attack lysis Figure VI-1-11. The complement cascade.

a. The classical pathway is the main antibody-directed mechanism for complement activation. It is the most rapid and efficient pathway. (1) Complement recognizes antigen-antibody complexes. The antibody must be

IgG or IgM. Complement activation requires one IgM molecule or two IgG molecules. (2) Antigen-antibody complexes bind to CI, which then activates the cascade. b. The alternative pathway of complement activation occurs as a result of binding directly to the surface of an infectious organism. This pathway is slow and less efficient than the classical pathway. (1) The alternative pathway primarily recognizes bacteria and depends upon the

interaction of a small amount of preformed C3b with protective surfaces. (2) It is important to remember that the two pathways both lead to lysis by the terminal complement components (C8 and C9); however, they are initiated by different complement components.

Clinical Correlate The alternative pathway protects the body from pathogens in the absence of antibody. The bacterial surface itself activates the cascade.

2. Classical pathway. The reaction occurs in the following order: Clq, Clr, CIs, C4, C2, and C3. Each component of the classical pathway has unique biologic functions. a. CI is a complex consisting of one Clq subunit associated, in a calcium-dependent manner, to two Clr and two CIs molecules. Clq binds to antigen-antibody complexes and is heat labile. In turn, Clr binds CIs, forming a heat-labile tetramer. Finally, CIs cleaves C4 and C2, with the latter complex being heat stable. b. C4 is produced and secreted by macrophages. The CIs protein acts on the a chain of C4, releasing the C4a and C4b products. C4b then attaches to the cell membrane. c. C2 is a heat-labile protein that mediates vascular leakage. It is produced by macrophages. C2 is activated by CIs to produce C2b and C2a. C2a, in association with

iiI.llical

437

Immunology

In a Nutshell Biologically Important C Proteins • (2a, (4a = weak anaphylatoxins • Ga, (Sa = strong anaphylatoxins • (Sa = potent chemotaxin • (3b = potent opsonin

In a Nutshell Classical Pathway • Activated by IgM or IgG complexed with antigen • (1, Q, and (4 are unique to the classical pathway (4b2a = 0 convertase (4b2a3b = C5 convertase

Alternative Pathway Activated by exposure to microbial surfaces, endotoxins, aggregated Ig • Ob + B + D + P (properdin) ---> ObBb(P) = 0 convertase

C4b, is the classical pathway C3 convertase, also known as C4b2a. This component can cleave many molecules of C3; this step is, therefore, the site of amplification of the complement pathway. C4b2a binds to cell membranes covalently. d. C3 is a heat -stable glycoprotein with two polypeptide chains, alpha and beta. C3 is responsible for the distinction between self and non-self. "Self" surfaces limit C3 deposition, whereas "non-self" surfaces allow rapid C3 deposition. C3 is activated by the classical pathway, the alternative pathway, thrombin, plasmin, and tissue proteases. (1) C3a is an anaphylatoxin, causes mast cell degranulation, and mediates vascular leakage.

(2) C3b causes opsonization and immunologic adherence via specific cell-surface receptors for this component. C3b receptors are found on granulocytes, monocytes, lymphocytes, and erythrocytes. C3b combines with the classical pathway C3 convertase (C4b2a) to form the classical pathway CS convertase (C4b2a3b). 3. Alternative pathway. There are several key components of the alternative complement pathway. a. Factor B is a protein similar to C2. b. Factor D is an enzyme that cleaves factor B. (The active product is termed Bb.) c. Properdin is a group of proteins involved in stabilizing C3bBb of the alternative pathway. d. There are several initiators of the alternative pathway. (1) C3 spontaneously degrades at a low rate to form C3a and C3b. (2) Lipopolysaccharides, the cell wall components of gram-negative bacteria (3) Bacterial and plant polysaccharides (4) Cell-membrane constituents

• ObBb3b(P) = (S convertase

(5) Aggregated 19A, IgG, IgE, and IgM

Final common Pathway

(6) Cobra venom factor

(S convertase converts (S to (Sa and (sb (Sb combines with (6 and G; (sb67 complex inserts into plasma membrane (8 and (9 join the complex to form the final membrane attack complex

(7) Endotoxins that complex with factor B to form C3 convertase e. The alternative pathway starts by the binding of C3b to factor B. The binding of C3b makes Factor B susceptible to proteolysis by factor D, and a complex of C3b and Bb (the proteolytic product) is formed. Properdin then forms a stable complex with C3bBb. The alternative pathway then continues as does the classical pathway. 4. The membrane attack complex (MAC) is formed from the reactions among C5, C6, C7, C8, and C9. a. CS is a heat-stable protein that consists of two polypeptide chains, alpha and beta. The alpha chain is cleaved by C5 convertase, resulting in C5b and soluble C5a. C5a is an anaphylatoxin and chemotactic factor. It also causes mast cell degranulation and mediates vascular leakage. b. C6 and C7. C5b, C6, and C7 combine to form the CSb,6,7 complex. The C5b,6,7 complex is lipophilic and inserts itself into the hydrophobic plasma membrane. c. C8 and C9 are the terminal components in cell lysis. C8 combines with C5b,6,7 to form CSb,6,7,8 and becomes stably attached to the cell membrane. C9 then polymerizes in the membrane, leading to osmotic lysis of the cell.

438

meClical

Basic Immunology

5. The products of the complement cascades have several biologic roles, including viral neutralization, lysis of infected cells, and direct pathogen lysis. Complement activation can also mediate immune adherence (the focusing of antigen on macrophage or lymphocyte surfaces) and promote phagocytosis. Finally, biologic responses such as anaphylaxis, kinin activity, smooth muscle contraction, vasodilatation, and chemotaxis are mediated by individual complement components.

Clinical Correlate Deficiencies in complement components predispose patients to certain diseases: G deficiency

->

Increased susceptibility to pyogenic infections

Q deficiency

->

Increased incidence of connective tissue disorders

B. Inflammation is a pathologic state initially characterized by pain, redness, heat, and swelling. These features of inflammation are caused by increased vascular permeability, leading to an infiltration of leukocytes (primarily neutrophils and macrophages, although small numbers of eosinophils and basophils can also be found). Lymphocytes can also enter sites of inflammation, where the release of cytokines can enhance the inflammatory response. A variety of compounds or chemicals can elicit inflammatory responses, and a variety of chemotactic factors are responsible for the migration of cells to sites of inflammation.

(5-8 deficiency ->

infections (meningococcal, gonococcal)

1. Vasoactive and smooth muscle constrictors a. Histamine (1) Histamine is stored as granules in mast cells, basophils, and platelets, with higher concentrations found in the intestine, lung, and skin. (2) Histamine is released from mast cells when antigen contacts IgE bound on the mast cell. It may also be released by nonimmunologic mechanisms, e.g., trauma or cold. (3) Histamine interacts with target cell receptors HI, H2, and H3. HI causes contraction of smooth muscle, increases vascular permeability, and elevates intracellular cyclic GMP. H2 increases gastric acid secretion, respiratory mucus production, and intracellular cyclic AMP. H3 is found in the central nervous system and functions in the negative feedback inhibition of histamine release and synthesis. (4) Histamine can be isolated from inflammatory sites in early inflammation, but its concentration dwindles within 1 hour. b. Arachidonic acid products. Arachidonic acid is derived from cell membrane phospholipids after conversion from linoleic acid. It is degraded via two pathways: the cyclooxygenase pathway and the lip oxygenase pathway. (1) The cyclo-oxygenase pathway converts arachidonic acid to prostaglandin G2 (PGG 2 ). PGG 2 is converted to PGH2 , which is ultimately converted to thromboxane ~ (T~), other more stable prostaglandins (PGF2 cx, PGE2 , PGD 2 ), and prostacyclin (CPGI 2 ).

Recurrent

Neisseria

esterase inhibitor deficiency

(1

->

Hereditary angioneurotic edema

In a Nutshell The inflammatory response can be triggered by local tissue damage that results in enzyme activation or by mast cell degranulation caused by anaphylatoxin interaction with specific receptors (e.g., OaR, C5aR) on the cytoplasmic membrane. Allergen reacting with cell-bound IgE can also trigger degranulation.

(2) The lipoxygenase pathway produces the leukotrienes, including LTC 4 , LTD 4 , and LTE 4 , which are collectively known as the slow-reacting substance of anaphylaxis. When administered by aerosol, these leukotrienes are more potent constrictors of human airway smooth muscle than is histamine. LTB4 is chemotactic for neutrophils, eosinophils, and macrophages. c. Platelet-activating factor (PAF) is derived from cell-membrane lipid and is synthesized by basophils, neutrophils, monocytes, and epithelium. (1) PAF activates platelets by initiating the release of platelet granule constituents,

causing platelets to clump. (2) PAF stimulates the synthesis of prostaglandins and leukotrienes. (3) PAF also increases the adhesiveness of neutrophils for endothelial cells.

meClical

439

Immunology

d. Adenosine is an inflammatory agent derived from mast cells after ATP breakdown. It interacts with Al and A2 receptors on the cell membrane. The A2 receptor is associated with increased levels of intracellular cyclic AMP, an effect that is blocked by methylxanthine drugs.

Clinical Correlate 2. Chemotactic factors

Eosinophils are important cells of the innate immune system; they augment acquired immunity to metazoans. They contain basic proteins in their granules that are toxic to worms.

a. Eosinophil chemotactic factors (ECF). There are six major eosinophil chemotactic factors: histamine, ECF-A; eosinophil stimulation promoter; soluble immune complexes, C5a; and hydroxyeicosatetraenoic acid (HETE), a lipoxygenase pathway product. b. Neutrophil chemotactic factors. There are six neutrophil chemotactic factors. Their primary role is to attract neutrophils to sites of inflammation. (1) High-molecular-weight neutrophil chemotactic factor (HMW-NCF)

(2) LTB 4 , which is derived from arachidonic acid metabolism, via the lip oxygenase pathway (3) PAF (4) IL-l

(5) IL-8 (6) C5a Phospholipid

t

Phospholipase

Arachidonic Acid ~ 5-Lipoxygenase

Cyclo·oxygenase / ' Thromboxane A2 ~ Prostaglandin (PG) 12 ~ PGF . . PGE:

¥'

PGG 2

5-HPETE

... ...

...

GH P 2

LTA4

... PGD 2

12-Lipoxygenase

12-HPETE

t

-+ LTB4

~+:}

SRS-A

LTE4

12-HETE Eicosanoids are derived from arachidonic acid and mediate inflammation.

Figure VI-1-12. Eicosanoid formation pathways.

3. Enzyme mediators a. Neutral proteases are products of mast cells. (1) Tryptic protease has trypsin-like potency and is capable of cleaving C3 to form C3a. It also alters blood-clotting proteins. (2) Neutral protease has a specificity similar to that of chymotrypsin. One of its substrates may be angiotensinogen.

440

meClical

Basic Immunology

b. Acid hydrolases are found in the lysosomes of many cell types. They degrade membrane components such as chondroitin sulfate. 4. ProteoglY<:alls function in the storage and release of other vascular mediators. a. Heparin is an anticoagulant that modulates tryptase activity. Tryptase, the major protein of human lung mast cells, can activate C3 directly in the absence of heparin. It is stored in mast cell granules in association with histamine. b. Chondroitin sulfates are structural molecules that function as binding sites for other mediators within mast cells. 5. Toxic oxygen molecules are important compounds that function in killing microorganisms and in the activation of neutrophils, eosinophils, and mast cells. They include singlet oxygen superoxide anion, hydrogen peroxide, and hydroxyl radicals.

Prostaglandin B1 (PGB 1)

Increases vascular permeability Increases pain response Aids in synthesis of DNA, RNA, and collagen

Prostaglandin B2 (PGB 2)

Blocks release of interleukin 1 OL-1) and

Prostaglandin F

Decreases vascular permeability

LTC4, LTD 4, LTE4

Slow-reacting substance of

interleukin 2 (IL-2)

anaphylaxis A chemotactic factor Activates neutrophils

Figure VI-1-13. Actions of eicosanoids.

6. Kinins are polypeptides (e.g., fibrin-split peptides) formed from precursors in the plasma. a. Kinins are labile proteins with physiologic effects similar to histamine. b. Bradykinin, a major kinin, functions as a vasodilator by increasing capillary permeability and producing erythema and edema. It also causes smooth muscle contraction. c. Elements of the kinin system include: (1) Hageman factor (coagulation factor XII) (2) Coagulation factor XI (3) Prekallikrein (4) High-molecular-weight kininogen

Clinical Correlate Enzyme activation during the complement cascade or as a part of the inflammatory response can activate the clotting system (Hageman factor, etc.) and lead to disseminated intravascular coagulation (OIC).

IMMUNOLOGIC METHODS A. Qualitative and quantitative tests for antigens and antibodies 1. Immunodiffusion is a method historically used to detect antigen-antibody complexes,

visible as a precipitate in a medium (such as agar in a petri dish) at optimal pH and temperature. KAPlA~. I meulca

441

Immunology

a. Double immunodiffusion is the most common method whereby both antibody and antigen diffuse toward each other and are allowed to complex and precipitate. When antibody and antigen diffuse through a semisolid medium, they form stable immunocomplexes that can be visualized as a white precipitate. This method is commonly used as the Ouchterlony radial immunodiffusion technique (or test). (1) Double immunodiffusion can be adapted to semiquantitate antigen or antibody

levels. Antibody is placed in the central well with varying antigen concentrations in surrounding wells on an agar plate. Precipitation lines allow an estimation of antigen concentration. This technique is also useful for determining precipitating antiserum titer by reversing antibody/antigen location. (2) Double immunodiffusion can also be used to determine reactant purity or relatedness (Figure VI-1-14). Antibody is placed in the central well with various related antigens in peripheral wells. Identical antigens cause precipitate lines to meet and are continuous. Nonidentical antigens are distinguished by precipitate lines that cross. Partial identity between antigens is demonstrated by precipitate lines that do not cross but are not continuous, with the interpretation that the antigens share some common epitope( s).

A. Identity reaction

B. Nonidentity reaction

c. Partial identity reaction

Figure VI-1-14. Double immunodiffusion technique-schematic representation.

2. Electrophoresis is a method used to separate proteins in an electric field. a. Zone electrophoresis separates proteins by surface charge. This method can quantitate serum protein and immunoglobulins. The medium is inert and does not impede or stimulate the flow of molecules; cellulose acetate or agarose are the preferred media. This method detects abnormalities associated with paraproteinemias, such as multiple myeloma, Waldenstrom macroglobulinemia, and hypergammaglobulinemia. Abnormal proteins spike in these disorders (in the y-globulin region). One can detect cerebrospinal fluid (CSF) protein oligo clonal band abnormalities, such as those that occur in multiple sclerosis. (1) A decrease in aI-globulin may indicate aI-antitrypsin deficiency. (2) An increase in aI-globulin may indicate an increase in the acute-phase reactants of inflammation. (3) An increase in a 2-globulin may indicate nephrotic syndrome or hemolysis with secondary hemoglobin in the serum. b. Immunoelectrophoresis is related to the electrophoretic methods described previously with the exception that antibodies are moved within an electric field. This technique distinguishes polyclonal from monoclonal increases in y-globulin. It detects heavy and light chain paraproteins; it is specifically used to characterize light chains in the urine of patients with plasma cell dyscrasias and autoimmune diseases. It also detects reduced or total absence of immunoglobulins found in a variety of immune deficiency disorders.

442

meClical

Basic Immunology

c. Counterimmunoelectrophoresis (one dimensional double electroimmunodiffusion) causes the rapid diffusion of antigen and antibody in a gel medium toward each other, leading to a semiquantitative measurement of antigen in biologic fluids. This method is useful in the analysis of CSF and in the serum diagnosis of several diseases, including cryptococcosis, meningococcal meningitis, Haemophilus inJluenzae meningitis, and staphylococcal endocarditis.

3. Radioimmunoassay (RIA) is an extremely sensitive method that can be used to quantitate any substance that is immunogenic or haptenic and can be labeled with a radioactive isotope. A known amount of labeled antigen and an unknown amount of unlabeled antigen are mixed with a known amount of specific antibody. The antigens compete for binding sites on the antibody. The antigen-antibody complexes are then separated from unreacted antigen by physiochemical or immunologic means, and the radioactivity of the precipitate is determined. Because this is a competition reaction for sites on the antibody molecule, the more radioactive (hot) the complex is, the less unlabeled (cold) antigen is in the sample. If the complex is cold, this means that there was a greater amount of unlabeled (cold) antigen than radioactive (hot) antigen. Conversely, a very hot precipitate would indicate low levels of the unlabeled antigen in the unknown sample. The concentration of an unknown sample can be determined by referring to a standard curve constructed from data obtained by allowing known amounts of unlabeled antigen to be added to the reaction mixture. 4. Agglutination, performed in tubes or microtiter plates, is a method whereby antibodies are mixed with antigen-coated particles. Antigen-antibody complexes form a visible clump, providing a visual endpoint. IgM is 750 times more efficient than IgG in agglutination due to the pentameric structure of IgM, so that cross-linking is more extensive. Latex fixation is a type of agglutination reaction where latex particles are used as passive carriers for adsorbed antigen. This test is used in the detection of rheumatoid factor (RF), which is a pentameric IgM directed against IgG. IgG is passively absorbed to the latex particles, and determinants on IgG then react with IgM RH.

Note RIA has wide application in the medical laboratory in detection of biologic substances: • Hormone levels: FSH, T4, insulin, and LH • Serum proteins: IgE, tumor markers • Metabolites: cyclic AMP, folic acid, vitamin B'2 • Drugs: digoxin, cocaine, morphine • Microbial agents: hepatitis B antigen or antibody, hepatitis A antigen or antibody

a. Direct agglutination can quantitate antibody titers as determined by the agglutination of a serially diluted unknown quantity of antibody with fixed quantity of insoluble antigen (RBC, bacteria, fungi, etc.). After several hours of incubation, particles are examined for evidence of clumping. It is termed "direct" because antibody directly agglutinates the target particle antigen. b. Indirect (passive) agglutination is a method whereby soluble antigen is coupled to RBCs or inert particles. There are many antigens that will spontaneously bind to RBCs, such as E. coli antigens, Yersinia species antigens, viruses, antibiotics, and DNA. It is termed "indirect" because the antigen must first be physically linked to a particle such as a bead or RBC, thereby allowing visualization of the agglutination reaction. c. Hemagglutination inhibition is when the presumed antigen to be identified is used to prevent agglutination between antigen-coated RBCs and specific antibody. This method is used to determine relative antigen concentrations in serum and other biologic fluids. It is performed using a known amount of specific antibody incubated with a serially diluted, unknown quantity of soluble antigen. Antigen-coated RBCs are then added to bind unbound antibody, and the agglutination reaction is allowed to occur. The amount of inhibition of agglutination reflects the amount of antigen present in the sample.

d. The Coombs test (antiglobulin test) measures amounts of antibodies too low to effectively agglutinate by themselves. This test is used in blood typing, in the evaluation of hemolytic disease of the newborn, and in autoimmune hemolytic anemias. The direct Coombs test detects serum proteins adherent to RBCs taken from a patient. The indirect Coombs test detects incomplete antibodies in serum. Serum is

meClical

443

Immunology

incubated with RBCs. The antibody-coated cells are then agglutinated by a Coombs antiglobulin serum. 5. Immunofluorescence is a method used to detect antibodies or antigens in tissue or fluids. This procedure is important in the diagnosis of systemic lupus erythematosus (SLE) and in several dermatologic diseases. Patients with SLE produce anti-DNA antibodies and other autoantibodies directed against several intracellular proteins. Immunofluorescence can be performed by direct and by indirect methods. a. Direct immunofluorescence is a technique used to identify antibody bound to tissue. To identify antibody (e.g., IgG) in tissue, fluorescein-labeled antihuman immunoglobulin conjugate is overlaid directly on the tissue. Sections are then examined with a fluorescence microscope. If antibodies are directly present in the tissue, they will be bound by the fluorescent-tagged antibody. b. Indirect immunofluorescence is a serologic test for antibodies in the serum or plasma (as in patients with SLE). Normal tissue is used as the substrate and is overlaid with dilutions of serum. If antibody is present to normal tissue components, it will bind to the substrate. Finally, fluorescein-labeled antihuman immunoglobulin is overlaid on the tissue and is then examined with a fluorescence microscope. c. There are a number of cutaneous diseases that use immunofluorescence in the diagnosis. (1) SLE exhibits immunoglobulin deposits at the epidermal basement membrane

zone in approximately half of all patients. (2) Pemphigus is diagnosed by cell-surface deposits of IgG on keratinocytes. (3) Bullous pemphigoid is diagnosed by a linear band of IgG or C3 at the epidermal basement membrane zone. (4) Henoch-Schonlein vasculitis is possibly caused by autoantibodies to 19A. 6. Complement assays are used to determine complement levels primarily in serum. a. Hemolytic assay (also known as CHso) is the antibody-mediated hemolysis of RBCs by complement. Known amounts of RBCs are incubated with antibodies to RBCs. Because a specific amount of complement produces hemolysis, the quantity of serum producing hemolysis reflects the amount of complement in the test serum. b. Immunoassays can be performed using antibodies to individual complement components followed by immunochemical determinations (such as with electrophoresis, as described previously). 7. Complement fixation is a method whereby the consumption of complement is used to measure levels of antibodies and antigen. Antigen and antibody are reacted in the presence of complement. The antigen-antibody complexes fix some but not all complement. Unfixed complement is measured by adding RBCs sensitized with a hemolysin antibody. The erythrocytes mayor may not be lysed by the remaining unfixed complement. A reciprocal relationship exists between the degree of lysis and the amount of antigen present. This method has been applied to the quantitation of HBsAg, antiplatelet antibodies, immunoglobulins, and anti-DNA. 8. Monoclonal antibodies refers to the production of antibodies with a single, identical specificity. This procedure is typically performed with immunized animals, although human B cells can be used as a source for making monoclonal antibodies. The method involves the hybridization of plasma cells with a myeloma tumor cell. The plasma cell pro-

444

KAPLA~. I meulCa

Basic Immunology

vides the myeloma cell with the genetic information to produce specific antibody, and the myeloma cell provides immortality to the plasma cell, which can then live indefinitely in tissue culture. 9. Enzyme-linked immunosorbent assay (ELISA) (Figure VI-I-IS) is one of the most sensitive types of immunoassays for the detection of specific antibodies and/or antigen. a. In one version of this procedure, the antigen is attached to a solid surface (such as a plastic microtiter plate) followed by an incubation step with serum antibodies. The specific antibody, if present, binds to antigen. Unbound antibody is washed away, and the surface is then incubated with enzyme attached to class-specific antiimmunoglobulin antibody (for example, an antihuman IgG antibody generated in a rabbit). The enzyme causes a color change of the substrate. Color change, as measured by spectrophotometry, quantifies the amount of antibody present. b. A similar procedure can be used to quantitate antigens in a specimen. In this instance, antigen-specific antibody is added to the solid surface first and acts as an antigen trap or sponge.

Antigen on glass bead

Substrate (colorless)

Antibody in serum

Changed substrate (colored)

•

i

Assay for amount of ----. changed substrate (colorimeter)

Anti-immunoglobin conjugated to enzyme

Figure VI-1-1S. Enzyme-linked immunosorbent assay (ELISA).

10. Western blot a. The Western blot is a modified ELISA procedure with two major changes. First, the antigens to be assayed are separated by an immunoelectrophoretic step, usually carried out in polyacrylamide gels. Because antigen-antibody interactions do not occur in this medium, it is necessary to transfer the separated proteins to nitrocellulose, a medium in which antigens and antibodies readily react. This is accomplished by a second electrophoretic step, where the antigens are moved from the polyacrylamide film to one of nitrocellulose. This is commonly referred to as the "blot;' likening the procedure to the blotting action of an ink blotter or a sponge that absorbs liquids. The nitrocellulose containing the separated antigens is then flooded with antiserum. KAPLA~. I 445 meulca

Immunology

Clinical Correlate ELISA tests for AIDS often yield fast-positive results, so any positive result is confirmed by another, more specific test, the Western blot. ELISA is the screening test for HIV; Western blot is the confirmatory test

The excess proteins are washed away, and the ftlm is flooded with a tagged antibody against the antiserum used. The second change from the standard ELISA occurs here. Enzymes are not usually used as the tag; more commonly, radioactive isotopes are used, and the position of the antibodies is determined by autoradiography. b. This procedure is employed in confirming the diagnosis of HIV after a positive ELISA test. An antigen "soup" prepared from a lysate of HIV-infected lymphocytes is separated into individual protein bands by gel electrophoresis. The antigens are then blotted to nitrocellulose, and the patient's serum is added. The excess human serum is washed away, and then an isotopically labeled antihuman gamma globulin is added to the plate. The presence of reactions is determined by autoradiographic techniques. If the patient has antibodies that are reactive with one or more of the HIV antigens, the test is positive; if the antibodies reacted with normal lymphocyte antigens (also present in the lysate), but not with HIV antigens, then the conclusion is made that the original ELISA screening test gave a false-positive reaction. 11. Polymerase chain reaction (PCR) is a technology for identifying the presence of DNA in a test sample. PCR is diagnostically useful to confirm the presence of DNA from specific pathogens in blood, CSF, and tissue specimens. It is performed by purifying DNA from the sample, which is then incubated with synthetic "primer" DNA complementary to the pathogen DNA. Precursors of DNA synthesis, nucleotides, and polymerase are then added, causing the replication of DNA fragments. The sample is then electrophoresed in agar gels to identify its size and specificity. PCR requires sophisticated expertise and lab instrumentation. B. Tests for cellular immune function 1. Delayed-type hypersensitivity (DTH) skin tests measure individual immunocompetence and may indicate previous exposure to certain microorganisms or chemicals. There are two methods for performing DTH tests. a. Patch testing is used by dermatologists and allergists to determine specific chemical substances responsible for contact dermatitis. b. Intradermal injection of antigen. This procedure tests previous contact with certain infectious agents, e.g., mumps tuberculosis. Interpretation of DTH is made within 48 or 72 hours of the application or injection of antigen. Induration (an area of hardness) arises around the injection site within 24-48 hours postinjection. Induration results from mononuclear cell inftltration and edema. The response indicates the degree of cellular immunity. In contrast, erythema or the wheal and flare response at the injection site disappearing within 12-18 hours indicates immediate-type hypersensitivity. It must be remembered that DTH responses are of little or no value in infants and in immunosuppressed patients because their T-cell responses are compromised. Also, highly sensitized or previously immunized individuals may have local and systemic adverse reactions, so caution should be observed.

Clinical Correlate The CD4 count must be less than 200/I-LL before the diagnosis of AIDS can be made. These patients are severely immunodepressed.

2. In vitro lymphocyte assays a. Basic lymphocyte purification and enumeration. T and B lymphocytes are separated from peripheral blood most easily by density gradient centrifugation. Quantity alone may be important (as in following AIDS patients), but function should also be assayed. Leukocytes are counted by microscopy or flow cytometry using antibodies to cell surface antigens. Monoclonal antibodies to class-specific and subclass-specific T antigens are now used in the detection of specific T-cell subsets (i.e., CD4 and CD8 T-cell subsets). b. CD4/CD8 ratio. In immunocompetent individuals, this ratio is typically 2:1; in patients with AIDS, the ratio is drastically reduced. Other conditions causing a

446 - - - -

meClical ~--------~-----------

Basic Immunology

decreased ratio include: CMV infection, burns, graft versus host disease, SLE with renal disease, measles, herpes infections, and myelodysplastic syndromes. Conditions causing an increase in this ratio include rheumatoid arthritis, SLE without renal disease, primary biliary cirrhosis, atopic dermatitis, psoriasis and Sezary syndrome. c. T-cell functional assays. In general, T-lymphocyte function can be examined in

several ways. For example, normal functioning T cells will proliferate in tissue culture in response to mitogens such as concanavalin A, phytohemagglutinin, and pokeweed mitogen. Antigen-specific responses can also be measured in culture by stimulation with specific purified antigens (such as tetanus toxoid) or allogeneic lymphocytes (a mixed lymphocyte reaction). The degree of stimulation is determined by measuring the uptake of tritiated thymidine (radioactivity) in the responding cells. d. B-Iymphocyte assays. B cells express many cell-specific molecules on their surfaces that can be detected by monoclonal antibodies. As with T cells, B cells are activated by mitogens such as staphylococcal protein A, pokeweed mitogen, and lipopolysaccharide (LPS). Functional B-cell assays are most commonly measured by the secretion of immunoglobulin and are examined by methods such as ELISA or RIA. (1) Surface immunoglobulins (sIg) on B cells can also be detected. Fluorescein-

tagged anti-immunoglobulin antiserum is mixed with B cells, unbound immunoglobulin is removed, and surface immunoglobulin is then detected by counting in a fluorescence microscope or by flow cytometry. (2) Cytoplasmic immunoglobulins are found in certain lymphoid cancers such as Waldenstrom macroglobulinemias and chronic lymphocytic leukemia. Intracellular immunoglobulin is determined by direct immunofluorescence with fluorescin-tagged antiheavy chain or antilight chain sera on preparations of purified lymphocytes. 3. Phagocyte function assays. The phagocyte function index measures the relative ability of blood phagocytes (neutrophils and macrophages) to ingest a fixed number oflatex particles in a specific period of time. a. Intracellular killing ability can be measured by several procedures. (1) Nitroblue tetrazolium (NBT) dye reduction test is a method whereby blue formazan is generated by NADPH oxidase during the ingestion of latex particles or microorganisms. The dye is visualized microscopically. (2) Chemiluminescence

Clinical Correlate NBT reduction is absent in phagocytic cells from patients with chronic granulomatus disease.

(3) Direct counts of surviving organisms (4) Lysosomal enzyme release (degranulation). The enzymes released are primarily~­ glucuronidase and acid phosphatase. b. Specific neutrophil function tests (1) Chemotaxis is the ability to migrate toward a source of chemical stimulus (such

as cytokines). A Boyden chamber (two compartments separated by a porous micropore filter) is commonly used to detect chemotactic movement. Cells are placed on one side of the filter, and a chemotactic agent is placed on the other side of the fIlter. If chemotactic activity is present, the cells will crawl through the pores of the membrane toward the chemotactic agent. (2) The phagocytic ability of neutrophils can be determined by measuring radioactivity after ingestion of radioactive particles or via staining and direct enumeration of ingested microbes.

meCtical

447

Clinical Immunology This chapter discusses immunopathology, transplantation, and tumor immunology. Immunopathology includes tV\lO broad areas: immunodeficiency states and autoimmune or hyperimmune reactions. Note that although the pathogenesis of autoimmunity is discussed in this chapter, most of the individual autoimmune disorders are reviewed in Organ Systems Books 1 and 2 (Volumes III and IV) in the context of the primary system involved. Summaries of autoimmune disorders, syndromes, and diseases are presented in table format throughout the chapter.

IMMUNODEFICIENCIES In immunodeficiency, there is a failure to produce cellular immunity, humoral immunity, or both. Several genes have been identified that cause congenital immune deficiency. Acquired immune deficiency may be caused by human immunodeficiency virus 1 and 2 (HIV 1, HIV 2) or may be iatrogenic when patients are treated with steroids, radiation, or chemotherapy. Some diseases (particularly lymphomas) cause immunodeficiency because they involve the cells and organs of the immune response. In view of the complex nature of the immune response, it is not surprising that a wide array of deficiencies exist, heralded primarily by recurrent infection, chronic infection, unusual (opportunistic) infecting agents, and a poor response to treatment. Occasionally, other manifestations, such as hepatosplenomegaly and diarrhea, are seen. When an immunodeficiency syndrome is suspected, the workup of the patient includes an evaluation of the native and acquired immune capabilities. These assays include an analysis of phagocytic functions (chemotaxis, phagocytosis, and killing), complement levels, antibody production, and cell-mediated immunity.

Clinical Correlate Most major opportunistic pathogens are normal flora agents like Staphylococcus, Candida, etc. Environmental agents include water bacteria such as Pseudomonas and Legionella and fungi such as Aspergillus and Rhizopus.

A. Primary immunodeficiency diseases represent defects in selected members of the immune system. With few exceptions, the primary biologic error for these defects is not known. Immunodeficiency diseases occur more frequently in children than in adults (3:2), with a male/female ratio of 5: 1. 1. Antibody deficiency disorders are congenital or acquired, partial or total, and are typically recognized because of recurrent opportunistic bacterial infections or treatment failure. a. X-linked (Bruton) agammaglobulinemia (1) Etiology. Usually arises in males after 6 months of age because this is the time when maternally derived immunoglobulins have totally disappeared. It arises from a failure of B-cell maturation, resulting in the absence of immunoglobulins, plasma cells, and germinal centers of lymphoid tissue. B-cell maturation is arrested because of a deficiency of a tyrosine kinase enzyme. The patient's blood contains pre-B cells (lymphocytes with intracytoplasmic mu chain), but B cells with surface IgM are absent. Antibody deficiency (IgM, IgG, and IgA) below 95% confidence limits for appropriate age- and race-matched controls is a hallmark of this syndrome. Cell-mediated immune responses are generally intact.

meClical

449

Immunology

(2) Clinical features. The clinical symptoms include repeated infections with extracellular pyogenic organisms and also increased enteroviral infections. pneumococcal, other streptococcal, and Haemophilus infections are most common; staphylococcal, Pseudomonas, and meningococcal infections are less common. Infections include pneumonia, sinusitis, meningitis, furunculosis, otitis, septicemia, conjunctivitis, echovirus, and encephalitis. Chronic complications result from the development of bronchiectasis or persistent enterovirus infection. There is also an increased incidence of autoimmune diseases. (3) Therapy. Replacement therapy with gammaglobulin is the usual treatment.

Table VI -2-1. Immunodeficiency syndromes. Immunodeficiency Syndrome

Specific Defects

Immune Abnormalities

Ataxia telangiectasia Bare lymphocyte syndrome

Kinase gene MHC class II

Bruton X-linked agammaglobulinemia Common variable hypogammaglobulinemia DiGeorge syndrome Selective IgA deficiency Transient hypogammaglobulinemia Wiskott-Aldrich syndrome

Tyrosine kinase

T cells, antibody Decreased T cells and APC function, decreased humoral response Arrested B-cell development, no antibodies Decreased antibodies

X-linked hyper-IgM syndrome

Unknown Thymus development Unknown; Unknown Th cell defects Glycosylated proteins CD40 ligand

Decreased T cells IgA decreased Transient decreases of IgG, IgA Decreased antibodies to glycosylated antigens Increased IgM, no isotype switching

Severe Combined Immune Deficiencies ADA deficiency PNP deficiency X-linked

Adenosine deaminase Purine nucleotide phosphorylase Multiple cytokine receptors

Absence of Band T cells Absence of Band T cells Absence of T cells

b. Common variable (B lymphocyte) hypogammaglobulinemia (1) Etiology. It is a congenital or acquired syndrome with equal sex distribution. The defect may appear at any age (usually between the ages of 15 and 30) and is in the abnormal terminal differentiation of B cells. Bacterial infections predominate but are less severe than in the X-linked form. Immunoglobulin-bearing B lym phocytes are normal in number, but serum antibody deficiency is profound because B cells proliferate but fail to differentiate into plasma cells. Lack of Th-cell function and excessive suppressor T-cell function have been demonstrated in some patients. (2) Clinical features. Tonsils and lymph nodes are normal sized or enlarged, and splenomegaly is common. Clinical presentations include alopecia areata, thymoma, lymphoreticular malignancy, hemolytic anemia, gastric atrophy, and

450

meClical

Clinical Immunology

achlorhydria. There is also a high incidence of pyogenic infections, autoimmune diseases, and malabsorption. (3) Treatment is with replacement immune gamma globulin. c. Selective IgA deficiency is the most common immunodeficiency state (greater than 1/1,000 population), particularly in individuals of European descent.

(1) Etiology. The disease is marked by reduced levels of serum and secretory IgA. (2) Clinical features. The majority of patients appear healthy but may have increased incidence of respiratory infections. IgA deficiency patients are predisposed to certain diseases, including atopy, autoimmune disorders (such as SLE, pernicious anemia, and rheumatoid arthritis), and certain malignancies. Milk allergies are common as a result of antibodies to secretory IgA. Patients with IgA deficiency may have anaphylactic reactions to blood transfusion due to preformed antibodies to IgA; they should not be treated with pooled gamma globulin preparations. d. Transient hypogammaglobulinemia of infancy (1) Etiology. Results when the onset of immunoglobulin synthesis, particularly IgG synthesis, is delayed beyond the norm. The cause is unknown but may be associated with a temporary deficiency of Th cells. Most infants go through a transient period of hypogammaglobulinemia between the fifth and sixth months of life. Many normal children experience recurrent respiratory infections. A few infants will have a developmental delay in IgG synthetic ability. (2) Clinical features. Infants with this disorder suffer recurrent pyogenic infections of, for example, the skin, meninges, or respiratory tract, usually caused by various gram-positive microorganisms. The situation usually cures itself within 16-30 months of age. (3) Treatment involves antibiotics, gamma globulin, or both. 2. Cellular immunodeficiency disorders a. Thymic hypoplasia or aplasia (DiGeorge syndrome) is a condition caused by the dysmorphogenesis of the third and fourth pharyngeal pouches, leading to hypoplasia or aplasia of the thymus and parathyroid glands and congenital heart disease. Immunoglobulin concentrations are usually normal but may be elevated. T cells are absent. Fetal thymic transplantation is recommended in complete disease associated with neonatal tetany (due to lack of parathyroids) and congenital defects of the heart, great vessels, and face (low-set ears). Patients are prone to viral and fungal infections. b. Cellular immunodeficiency with immunoglobulins (Nezelof syndrome) presents clinically with lymphopenia, abnormal thymus, and reduced lymphoid tissue, but with no cardiac or endocrine abnormalities. Serum immunoglubulins are normal or elevated. The patient usually presents with recurrent or chronic pulmonary infections, oral or cutaneous candidiasis, chronic diarrhea, gram-negative sepsis, or severe varicella. This syndrome can be distinguished from pediatric AIDS patients in a number of ways: patients with Nezelof syndrome usually have normal CD4:CD8 ratios, the peripheral lymphoid tissue is hypoplastic, and the thymus is small. 3. Severe combined immunodeficiency (SCID) is a group of disorders with defects in humoral and cell-mediated immunity. The incidence is 1:100,000 births. The thymus gland lacks lymphoid cells, as do peripheral lymphoid organs such as the spleen, Peyer patches, etc. Patients usually succumb to infection within the first year. There are two modes of inheritance.

Clinical Correlate Bruton disease is approximately 100 times less frequent than selective IgA deficiency, but clinical disease is almost a certainty with Bruton disease.

In a Nutshell Bruton Agammaglobulinemia • X-linked (males) • Pre-B cells are normal, but B cells are absent • Low circulating antibody levels • Normal cell-mediated immunity • Recurrent bacterial infections Common Variable Hypogammaglobulinemia • Equal sex distribution • B cells normal but do not differentiate to plasma cells • Low circulating antibody levels Selective IgA Deficiency • Low levels of IgA • Present with respiratory infections; milk allergies common Transient Hypogammaglobulinemia of Infancy • Occurs between the time maternal antibodies disappear and onset of meaningfullgG production by the infant (5-6 months) • Not considered a pathologic condition but will mimic Bruton in early life

meclical

451

Immunology

Clinical Correlate Children with congenital infections often suffer a wasting syndrome and have general immunodeficiency. The agents include: • Toxoplasma • Rubella

• Cytomegalovirus • Herpes simplex, HIV • Syphilis (Treponema) These are called the ToRCHeS agents.

Clinical Correlate Children with Down syndrome (chromosome 21 trisomy) have a lOO-fold higher mortality from respiratory infections than normal children. They have thymic hypoplasia and phagocytic defects but normal B-cell functions.

a. Autosomal recessive (Swiss-type agammaglobulinemia) is the congenital absence of both cellular and humoral immune function due to severe T- and B-celllymphopenia. Opportunistic infections lead to death. This syndrome may be associated with lymphopenia and hypoplasia of the thymus gland. There is an absence of T cells and an inability to mount a humoral immune response. (1) About 50% of the autosomal recessive form of the disease (about 20% of the

total) is caused by a deficiency in the enzyme adenosine deaminase (ADA). ADA is widely distributed in mammalian tissues but is particularly abundant in lymphocytes. Its deficiency leads to the accumulation of metabolites (e.g., deoxyadenine triphosphate) that are toxic to lymphocytes because they block DNA synthesis. (2) Another form of scm is caused by deficiency of purine nucleotide phosphorylase, an enzyme involved in purine catabolism; this defect leads to accumulation of metabolites toxic to lymphocytes. (3) Advances in genetic engineering have made gene therapy available for treatment of the ADA deficiency patients. In this approach, lymphocytes or stem cells removed from the affected individuals are transfected with a retroviral vector containing a functional ADA gene obtained from a normal donor. These engineered cells are then reinjected into the patient. The advantage of using stem cells as opposed to mature lymphocytes is that the former are capable of self-renewal; hence, repeated infusion of engineered cells is not required. b. X-linked recessive is the most common SCID (sometimes abbreviated XSCID). The disease clinically resembles the autosomal recessive form, but no severe leukopenia occurs. Most patients have normal numbers of B cells with few or no circulating T cells. This disorder is caused by a lack of the gamma chain of the IL-2 receptor, a molecule also necessary to form receptors for IL-4, -7, -9, -13, and -15. There have been successful treatments with transplantation of bone marrow or fetal liver cells from an HLA genotypically matched donor, confirming the hypothesis that the stromal microenvironments of the thymus and bone marrow of these patients are capable of supporting T- and B-cell differentiation of normal stem cells. 4. Partial combined immunodeficiency disorders a. Wiskott-Aldrich syndrome is an X-linked genetic disorder presenting clinically as a triad of thrombocytopenic purpura, eczema, and recurrent opportunistic infections with organisms having polysaccharide capsules. There is a 12% incidence of fatal malignancy; patients usually die before the second decade of life. The disease is characterized by alterations in immunoglobulin serum concentrations, most notably decreased IgM. T-cell deficits become apparent as the patient matures. Bone marrow transplantation has been used with some therapeutic success. b. Ataxia-telangiectasia is a complex autosomal recessive disorder including cerebellar ataxia, chronic sino pulmonary infection, oculocutaneous telangiectasias, variable humoral and cellular immunodeficiency, and a high incidence of malignancy. Patients usually have low IgA and/or IgE levels, decreased humoral immune response to new antigens, and decreased cellular immunity. The thymus is hypoplastic and is similar in appearance to an embryonic thymus. Ataxia-telangiectasia patients are exquisitely sensitive to radiation-induced chromosomal damage as a result of defective DNA repair mechanisms. c. Hyperimmunoglobulinemia E with recurrent infection syndrome (Job syndrome), an autosomal dominant disorder, presents clinically with high serum IgE concentrations and blood and sputum eosinophilia. Patients have recurrent severe staphylo-

452

meClical

Clinical Immunology

coccal abscesses and pruritic dermatitis. Often there are abnormally low anamnestic antibody responses to booster immunizations and poor antibody and cell-mediated responses to new antigens. In addition, there is abnormal neutrophil and monocyte chemotaxis. d. Immunodeficiency with spindle cell thymoma is typically a disease of adults. Patients present with recurrent infections, benign thymoma, hypogammaglobulinemia, and cell-mediated immune deficiency. There is also a deficiency of circulating B cells and pre-B cells in the bone marrow. Hematologic disorders may be one or several of the following: thrombocytopenia, eosinopenia or eosinophilia, agranulocytosis pancytopenia, or progressive lymphopenia. e. Chronic mucocutaneous candidiasis is a disease where skin and mucous membranes have recurrent candidal infection; invasive systemic infection is rare. The central defect is an absence of a T-cell response to candidal antigens. There may be endocrinopathies, possibly due to autoantibody formation, as well as iron deficiency. Immunologic responses vary among patients. Some patients may have normal immune components, whereas some may have IgA deficiency or deficient cellmediated immunity.

In a Nutshell Hyper-lgE Syndrome • IgE = >3,000 IUjmL • ~ Antibody responses

· i

Respiratory infections, especially s. aureus

• Eosinophilia • Dermatitis • Growth retardation

B. Acquired immunodeficiency. Acquired immunodeficiency syndrome is the defining infec-

tious disease of our generation. 1. Causative agent. AIDS is caused by the human immunodeficiency virus (HIV; see Figure VI -2-1 for an illustration of the HIV replication cycle). a. HIV was first suspected as the etiologic agent of AIDS in 1981. In some communities in central African countries, the rate of infectivity is as high as 30-40% among the adult population. Two types of HIV, termed HIV-l and HIV-2, are responsible for most infections worldwide. b. HIV is a C-type retrovirus belonging to the Lentivirus family. Its genome encodes nine proteins, the most important of which are gag (encoding viral core proteins), pol (reverse transcriptase), and env (envelope proteins). c. The RNA core is surrounded by a lipid envelope that is derived from the host plasma membrane. The viral membrane contains the transmembrane protein gp160. This protein is commonly detected in Western blot analysis as two fragments-gp41, which promotes the fusion of viral envelope with the host cell membrane, and gp120, an adhesion molecule that binds to CD4 on Th cells. d. The core proteins include reverse transcriptase and two nonglycosylated proteins designated p18 and p24. 2. Risk groups for HIV infection include homosexual males, intravenous drug users, children born to infected mothers, sexual partners of people in high-risk groups, and recipients of infected blood products and secretions. The latter risk is now minimal, given the excellent methods of screening blood products for potential contamination.

meClical

453

Immunology

Adhesion

Il-=--'%--llr---

RNA genome

gag

HIV fusion and entry

A.

Viral assembly and budding

B.

D. CD4 T-cell activation induces HIV replication and assembly

c. Figure VI-2-1. HIV replication cycle.

454

meClical

Clinical Immunology

3. Mechanism of transmission. HIV is primarily spread by contact with infectious bodily fluids such as semen and blood. Sexual intercourse, either homosexual or heterosexual, is the principle route of transmission, although vertical transmission of virus from mother to newborn can also occur. The sharing of needles by intravenous drug users continues to be an important source of infection. 4. Mechanism of infection. AIDS is characterized by a profound loss of CD4+ T cells. The virus binds to the CD4 molecule on the T cell via gp120. The chemokine receptor CXCR4 is required as a coreceptor on the target cell. Some HIV also infects macrophages and monocytes and requires another chemokine coreceptor, termed CCRS. Once the virus enters into the cell, the RNA is reverse transcribed and integrated within the host DNA. Exposure of the T cell to activating stimuli such as cytokines or antigens results in activation of the virus by stimulating transcription of virally encoded genes. The long terminal repeat (LTR) sequences of the viral genome contain binding sites for a mammalian transcription factor. Once activated, the virus replicates within the cell. Extensive viral budding can lead to death of the T cell. In contrast to the T cell, the macrophage is more resistant to death from viral infection and appears to serve as an important viral reservoir. This cytolysis leads to profound immune suppression. A number of opportunistic infections, as noted below, overwhelm the patient's weakened immune response. 5. Cellular consequences of HIV infection a. T cells. A central consequence of infection with HIV is a loss of CD4+ Th cells. However, in addition to a loss of CD4+ T cells, there is also a decrease in the response of T cells to antigen, impaired production of cytokines and such as IL-2 and IFN-y, decreased intracellular signaling. The hypermetabolic wasting syndrome seen in HIVinfected individuals may be caused by increased levels of cytokines such as TNF-a (cachectin).

Note The loss of CD4+ cells leads to defects in cell-mediated immunity, with an increased incidence of infection due to fungi, viruses, and mycobacteria.

Clinical Correlate HIV patients can present with a wide range of problems across organ systems. If you encounter an HIV (or high-risk group) patient on the exam, you should know that there is a high incidence of the following disorders in HIV patients: • Oral thrush (Candida)

b. B cells. Although patients with early HIV infection appear to have polyclonal activation of B cells with circulating immune complexes in their plasma, they steadily lose the ability to mount an effective antibody response to new antigens.

• Esophagitis (Candida, CMV,

c. Macrophages. HIV can enter the macrophage through binding of gp120 to CD4 and a second membrane receptor, CCRS (a chemokine receptor). Both circulating monocytes and macrophages serve as a reservoir for the virus. Although more resistant to death from viral infection, infected macrophages have decreased IL-l secretion, as well as an impaired ability to present antigen to T cells.

- Bacterial (Sa/monella,

6. Natural history of HIV infection. The Centers for Disease Control (CDC) has proposed three clinical subgroups for AIDS-infected individuals: a. Category A. This group of individuals is characterized by an acute infection. After the initial infection, the patient may develop a syndrome resembling infectious mononucleosis, characterized by rash, sore throat, fever, or even aseptic meningitis. The illness typically resolves after 1-2 weeks, upon which seroconversion to HIV proteins arise. This group of individuals may also be characterized by asymptomatic infection. The disease becomes clinically latent and can remain so for 7-10 years. c. Category B. This group is characterized by persistent generalized lymphadenopathy together with fever, rash, and fatigue. AIDS-related complex (ARC) represents a nonspecific cluster of signs and symptoms of AIDS that is not accompanied by a decrease in CD4+ cells. A diagnosis of early ARC is made if the individual has one or two of the following symptoms: fatigue, fever, weight loss, persistent skin rash, oral hairy leukoplakia, herpes simplex, and oral thrush. Advanced ARC is determined if the individual has two or more of these symptoms.

HSV) • Diarrhea Shigella, Yersinia, Campy/obader, Mycobaderium aviumintracellu/are, C diffici/e) - Parasitic (Cryptosporidium, Isospora, Giardia, Entamoeba) - Viral (CMV colitis) - Fungal (Candida) • Intestinal neoplasms: lymphoma, Kaposi sarcoma • Pneumonias (especially Pneumocystis carinit) (Continued)

meClical

455

Immunology

Clinical Correlate (conrd) • Tuberculosis and atypical mycobacterial infection • Fungal respiratory disorders: histoplasmosis, coccidioidomycosis • Pulmonary neoplasms: Kaposi sarcoma, lymphoma • Hematologic problems: anemia, leukopenia, thrombocytopenia • Neurologic disorders - Cryptococcal meningitis - Toxoplasmosis - Progressive multifocal leukoencephalopathy - CMV encephalopathy - CMV retinitis - AIDS dementia - CNS lymphoma • Sexually transmitted diseases • Skin - Shingles - Kaposi sarcoma - Seborrheic dermatitis - Herpes simplex • Disseminated infections - Cytomegalovirus - M. avium-intracellulare

- Histoplasmosis • Papillomavirus-induced tumors - Carcinoma of the cervix, penis - Anal condylomas

d. Category C. This group is characterized by generalized disease, including neurologic opportunistic infections and secondary neoplasms. Ultimately, the disease progresses to the breakdown of the immune system; full-blown AIDS is characterized by the development of secondary tumors and numerous opportunistic infections. (1) Among the tumors common to AIDS is Kaposi sarcoma, which can be found in individuals even before breakdown of the immune system, caused by HSVS. (2) Non-Hodgkin lymphomas are increasingly common tumors that are found in severely immunocompromised patients. (3) HIV-infected individuals are also susceptible to opportunistic infection by protozoa (e.g., Cryptosporidia and Toxoplasma gondii) , fungi (e.g., Cryptococcosis, Candidiasis, and Pneumocystis carinii), bacterial (e.g., Mycobacterium aviumintracellulare and M. tuberculosis), and viruses (e.g., Cytomegalovirus [CMV], herpes simplex, and varicella-zoster). (4) Central nervous system (CNS) involvement is a common feature of AIDS (60%). It can result either from direct infection of the CNS by the virus or from opportunistic infections (e.g., toxoplasmosis). 7. Diagnosis a. Seroconversion against HIV proteins usually occurs within 2 months after infection or, in rare cases, later in the illness. Serum antibodies against surface envelope proteins gp41, gp120, and/or gp160 and at least one gag protein and one pol protein can be detected by ELISA and by Western blot. The latter assay is typically used to confirm seroconversion first identified by ELISA. b. Peripheral CD4 counts can additionally be used as a marker of disease progression and as a predictor of the risks for opportunistic infection. c. Polymerase chain reaction (PCR) can identify HIV RNA from blood, CSF, and tissues of infected individuals even before seroconversion. Additionally, PCR can differentiate HIV-l from HIV-2 infection. S. Prognosis and treatment a. Only a small fraction of patients (5%) develop overt clinical symptoms within the first 2 years of HIV infection. However, upon clinical expression of disease and opportunistic infections, the median survival is approximately 2 years. Treatment is directed at reducing the viral load and at treating the symptoms of opportunistic infection. The principal therapy of choice is nucleoside analogs that prevent reverse transcriptase activity of HIV and newly defined protease inhibitors. The most studied dideoxynucleoside analog is azidothymidine (AZT) , which is typically used in combination therapy with other analogs and with protease inhibitors, including indinavir, saquinavir, and ritonavir. Other commonly used nucleoside analogs are dideoxycytidine (ddC) , dideoxyinosine (ddI), didehydrodeoxythymidine (d4T), dideoxythiacytidine (3TC), and nevirapine. b. Treatment of opportunistic infection can take many directions depending on the presentation of individual patients. For example, trimethoprim-sulfamethoxazole (TMP-SMX) is now effective treatment for P. carinii pneumonia, as well as other opportunistic infections such as Listeria, Nocardia, and Toxoplasma. C. Phagocytic cell disorders are characterized by a heightened incidence and severity of

infections caused by bacteria, particularly pyogenic organisms such as staphylococci and streptococci. They may be classified into syndromes defective in the production and/or

456

mellical

Clinical Immunology

maturation of cells (production defects) or syndromes where cells are present but functionallyaberrent (functional defects). 1. Cyclic neutropenia is defined as a periodic neutropenia, usually occurring every 21 days, with corresponding intermittent bone marrow maturation arrest at the promyelocyte stage. Splenectomy may improve clinical symptoms, and GM-CSF will stimulate neutrophil production and reduce symptoms and infections. Patients are typically sick for 1 week with fever, malaise, ulceration of skin and mucous membranes, abdominal pain, sore throat, lymphadenitis, and arthritis. 2. Hereditary neutropenia is usually fatal by 1 year of age. It is characterized by the absence of peripheral blood neutrophils from birth and by the arrested maturation of bone marrow precursors. 3. Acquired neutropenia has several known causes, including: a. Hyposplenism (as seen in sickle cell disease) b. Neoplasms, particulary myelocytic leukemia and metastatic carcinoma c. Overwhelming infection from tuberculosis, typhoid fever, measles, infectious mononucleosis, and leishmaniasis d. Drug toxicity from many cancer chemotherapeutic agents, antibiotics, and diuretics e. Irradiation f. Hemodialysis and cardiopulmonary bypass patients 4. Opsonic defects are functional defects of phagocytic cells commonly caused by the lack of serum factors, primarily complement products. Opsonization refers to the clearance of organisms (or other target cells) via antibody or complement attaching to the target organism and by the engulfment of this complex after attachment to complement or Fc receptors. In newborns, opsonic defects can arise due to C3 deficiency or from IgM deficiency. Other complement "deficiencies" are primarily from a lack of receptors for C3b that normally reside on phagocytic cells. 5. Chemotactic defects refer to the impaired ability of cells to respond or move toward a chemical stimulus. As described previously, cells of the lymphoid system can normally migrate toward a variety of stimuli, such as cytokines and mediators of inflammation. Defects in chemotaxis can be due to serum inhibitors, possibly associated with alcoholic liver disease, recurrent staphylococcal infections, Hodgkin disease, or agammaglobulinemia. In addition, drugs (steroids, colchicine) or C5 defects can affect normal chemotaxis. Several syndromes are associated with impaired cellular chemotactic responses, including lazy leukocyte syndrome and leukocyte adhesion deficiency. a. Lazy leukocyte syndrome refers to inadequate chemotactic responses and reduced random mobility of leukocytes. Once again, bacteria are the major threat to these patients. b. Leukocyte adhesion deficiency is an impairment of leukocyte interaction caused by defective synthesis of the ~-chain component of the CD18 integrin (adhesion) molecule. The loss of the chain results in failure of diapedesis of neutrophils and impaired cell interactions among leukocytes. 6. Functional deficits of phagocytic cells included deficiencies in which the phagocytes are unable to kill ingested pathogens. a. Chediak-Higashi syndrome is an autosomal recessive disease in which neutrophils have decreased chemotactic response. Gigantic lysosomal granules form; the basic cell defect involves microtubule dysfunction. This disease may present as peripheral

meCtical

457

Immunology

neuropathy, and patients are susceptible to viral and bacterial enteric infections. Partial albinism is also present. b. Chronic granulomatous disease is characterized by the inability of phagocytes to displaya typical respiratory burst during phagocytosis. The defect is in the NADPH oxidase system. Infection with catalase-positive microorganisms such as Staphylococcus au reus, Salmonella, Nocardia, Aspergillus, and Serratia are common. In the laboratory, the disease is marked by the failure of granulocytes to reduce nitroblue tetrazolium (NBT) dye to formazan. D. Complement deficiencies are caused by defects (either genetic or functional) in individual complement components.

In a Nutshell

1. C2 deficiency is the most common complement component deficiency. Collagen vascular disease is commonly observed in C2-deficient patients. Encapsulated bacterial septicemia and CNS infections are also found in these patients.

Immune Deficiency

Predominating Opportunist

Bcell

Pyogenic bacteria

Tcell

Viruses and fungi; also mycobacteria

3. C5 deficiency clinically presents with infection by gram-negative rods and Neisseria species. C5 dysfunction is known as Leiner syndrome (eczema, diarrhea, recurrent gramnegative sepsis).

C3

Pyogenic bacteria

4. Late complement component deficiencies (C5-C8) can result in disseminated Neisseria meningitidis or N. gonorrhoeae infections.

(5-(8

Neisseria

5. CI inhibitor deficiency results in hereditary angiodema (HAE).

Phagocytes

Pyogenic bacteria

Note These reactions are called "immediate" because symptoms of the allergy will be seen in a patient 10-20 minutes after exposure to the allergen. There must have been a previous antigenic encounter to induce the sensitivity. Always think of type I (atopic, IgE-mediated) hypersensitivity when the symptoms (rash, wheezing, cramps) occur rapidly after antigen injection/ingestion.

2. C3 deficiency causes recurrent infections with encapsulated bacteria. It may also be associated with cirrhosis, immune complex disease, and SLE.

HYPERSENSITIVITIES Hyperimmune reactions cause disease syndromes when the normal protective functions of immunity become imbalanced. Excessive immunoglobulin E (lgE) can lead to allergy; antigen-antibody (Ag-Ab) complexes can provoke arteritis, arthritis, and glomerulonephritis; and cytotoxic antibodies can destroy red blood cells (RBCs), white blood cells (WBCs), platelets, or, less frequently, other cells in the body. Inappropriate or undesirable cell-mediated immunity can lead to graft rejection or demyelinating disease. There are four distinct types of hypersensitivity reactions characterized by the time course required for the induction of the response, the immune cells and soluble factors involved, and the types of antigen involved. A. Type I hypersensitivity (or immediate, atopic, or anaphylactic hypersensitivity) requires an initial exposure to antigen in order to sensitize the person. Re-exposure to the same antigen causes cross-linking of IgE receptors on the surface of basophils and mast cells (via FCE receptors). The mast cells then release a variety of pharmacologic mediators. The systemic reactions occur within minutes of secondary exposure to the allergen. Smooth muscle contraction leads to constriction of bronchi and bronchioles, and vasodilation and increased vascular permeability result in peripheral edema. The significant cutaneous response is a wheal and flare reaction ("hives" or urticaria). The typical clinical syndromes associated with type I hypersensitivity include asthma, atopic dermatitis, eczema and allergic rhinitis (hay fever). 1. The important preformed mediators (and their primary biologic functions) released from basophils and mast cells upon the cross-linking of IgE are: a. Histamine (smooth muscle contraction and vasodilatation) b. Heparin (anticoagulant)

458

meClical

Clinical Immunology

Bridge to Respiratory System IgE-Fc receptor on mast cell surface

I

=

====
c·Ircu Iatlng · Ig E

Asthma is discussed in detail in the Respiratory Pathology chapter of Organ Systems Book 1 (Volume III).

IgE binds to mast cell at Fc end -Allergen

Allergen binds to - - and bridges two IgE antibodies

Figure VI-2-2. IgE-mediated cell degranulation-part I.

Anaphylatoxin receptor

Antigen

\

_ _ _ Acetylcholine receptor

ea

. . . . . --.~ Granules

Leukotrienes (SRS-A) Neutrophil chemotactic factor Eosinophil chemotactic factor (ECF) Platelet-activating factor (PAF) Histamines

Figure VI-2-3. IgE-mediated cell degranulation-part II.

c.

Eosinophil chemotactic factor of anaphylaxis (ECF-A) (chemotaxis)

d. Platelet-activating factor (PAF; microthrombi and platelet degranulation with release of vasoactive compounds) e. Tryptase (activates C3; inhibited by heparin) 2. In addition, several biologically active compounds are newly synthesized during mast cell degranulation and the ensuing inflammatory response. These include compounds

meClical

459

Immunology

formed from the arachidonic acid breakdown product of cell membrane phospholipids (via phospholipase A2, which is activated during Ca2+ influx that accompanies IgE cross-linking by allergen): a. Leukotrienes (LT) are formed from arachidonic acid via the lip oxygenase pathway. (1) LTB4 (chemotactic for PMNs; increases vascular permeability in synergy with

PGE 2 ) (2) LTC4 , D 4 , and E4 are also known as the slow-reacting substances of anaphylaxis; they have the same activities as histamine and are responsible for the late manifestations of these allergies; they also cause mucus secretion into the airways. b. Prostaglandins (PG) are formed via the cyclo-oxygenase pathway from arachidonic acid. (1) PGD2 (smooth muscle contraction, vasodilatation)

(2) PGE2 (same as PGD 2 ; synergizes with LTB 4 ) (3) Thromboxane Az (bronchial constriction, platelet aggregation) c. The anaphylatoxins C3a and C5a are generated via tryptase action on the native complement proteins; they cause mast cell degranulation by reacting with C3a and CSa receptors on the cell membranes of mast cells or basophils (anaphylactoid reactions). d. Bradykinin is generated from kininogen by the action of kallikrein, activated Hageman factor (factor XIIa), or trypsin; it stimulates vasodilatation and increases vascular permeability. 3. Common allergens that induce type I or immediate responses include: a. Foods (milk, eggs, fruits, and shellfish) b.Pollen c. Drugs (e.g., penicillin) d. Insect venom e. Animal dander f. House dust, mite fecal pellets

Note

Anaphylactoid reactions do not require presensitization. They will occur shortly after a first-time encounter with a material that can activate the alternative complement pathway and cause mast cell degranulation or activate arachidonic acid metabolism.

4. Anaphylaxis refers to an immediate hypersensitivity response that is inducible in a normal host of any given species upon appropriate antigenic exposure (called sensitization). Anaphylaxis of type I hypersensitivity occurs rapidly and may be life threatening. Victims may die of asphyxiation due to respiratory tract edema and bronchial constriction. Atopy refers to an immediate hypersensitivity response that occurs only in genetically predisposed hosts upon sensitization to specific allergens. This condition differs from anaphylaxis in that it cannot be induced in normal hosts. As in anaphylactic reactions, the response in atopic reactions is characterized by smooth muscle contraction and increased capillary permeability with edema.

5. Treatment of type I hypersensitivity a. Avoidance. The most direct way to manage allergic disease is through environmental control. b. Hyposensitization is a form of immunotherapy aimed at stimulating the production of IgG blocking antibody, which binds the offending allergen and prevents its combining with IgE on the mast cell.

460

meClical

Clinical Immunology

c. Desensitization may be produced by a series of closely spaced, small injections of allergen. This "uses up" the IgE in the body without causing significant symptoms. Once the desensitizing injections are discontinued, the hypersensitivity will return when sufficient allergen-specific IgE is generated to sensitize the basophils and mast cells. This treatment has been used to desensitize patients to particular antibiotics that are required for therapy of their infection. d. Drug treatment involves administration of agents designed to: (1) Block mediator binding to target tissue (e.g., antihistamines) (2) Reduce inflammation (e.g., corticosteroids). In addition, corticosteroids such as prednisone increase beta-receptor numbers in tissues and inhibit phospholipases, thus blocking mediator synthesis and secretion. These compounds also interfere with cell adherence to the vascular endothelium and diapedesis. (3) Stimulate adenyl cyclase, the enzyme responsible for conversion of adenosine triphosphate (ATP) to cAMP and, thereby, block the release of mediators (e.g., epinephrine)

Bridge to Respiratory System These drugs are reviewed in greater detail in the Respiratory Pharmacology chapter of Organ Systems Book 1 (Volume III).

(4) Block the release of mediators by stabilizing mast cell membranes and inhibiting Ca2+ influx (e.g., cromolyn sodium) (5) Inhibit phosphodiesterase, an enzyme that converts cAMP to AMP, thus preserving levels of cAMP essential for blockage of mediator release (e.g., theophylline) B. Type II hypersensitivity, or antibody-mediated cytotoxicity, may be associated with autoimmune diseases. IgG or IgM antibody reacts with membrane-associated antigen on the surface of cells, causing the activation of the complement cascade and, ultimately, cell destruction. Alternatively, natural killer (NK) cell-mediated lysis (antibody-dependent cellular cytotoxicity; ADCC) of an antigen-bearing target cell may occur. The response may be transient or chronic.

Step 1

*

Step 3

Step 2 IgE binding to FceR

* ••

•

T-cell activation

**

•

Cross-linking of surface IgE by allergen

•

Allergen·

Trypase, chymase, serine protease

... Degranulation; histamine release

1

Tissue destruction

Figure VI-2-4. Immediate hypersensitivity reactions.

meClical

461

Immunology

1. Complement-mediated cytotoxicity results when IgM or IgG reacts with antigen, resulting in activation of the complement system and phagocytosis or lysis of cells where complement has been fIxed.

Clinical Correlate There are several autoimmune diseases that may be considered type II hypersensitivities even though the antibody is not "toxic." For example, in Graves disease, the antibody actually causes hyperthyroidism. Also anti-B l2 antibodies cause pemicious anemia.

2. ADCC results when cells with Fc receptors (especially NK cells, but also monocytes and neutrophils) bind to target cells coated with IgG, causing phagocytosis or lysis of target cells. 3. Examples of type II reactions a. Certain drug allergies b. Blood transfusion reactions (red cell lysis) c. Hemolytic disease of the newborn d. Goodpasture syndrome with antiglomerular basement membrane antibody formation 4. Therapy for cytotoxic reactions includes treatment of the underlying cause of the reaction as well as the subsequent manifestations of such a reaction. Examples of therapeutic measures include: a. Suppression of the immune response by means of corticosteroids, with or without cytotoxic immunosuppressive drugs (e.g., cyclophosphamide) b. Removal of offending antibodies via exchange transfusion (in the case ofRh hemolytic disease of the newborn) or plasmapheresis c. Withdrawal of the offending allergen in the case of drug-induced syndromes C. Type III hypersensitivity, or immune-complex-mediated hypersensitivity, is caused by anti-

bodies formed to foreign antigens (typically, horse antibodies to snake venom or diphtheria toxin) or by autoantigens such as DNA. It occurs only with complement-fIxing antibodies (i.e., IgG and IgM). Immune complexes of IgG or IgM with horse gamma globulin activate complement, resulting in the generation of C3b, which promotes neutrophil adherence to blood vessel walls. Immune complexes also generate the anaphylatoxins C3a and C5a, which lead to inflammation and tissue destruction. The hallmark signs of sickness become apparent at 7-14 days and include urticaria, angioedema, fever, chills, and malaise. 1. Serum sickness is the hallmark syndrome of type III hypersensitivity. It results from immune complex deposition in small vessels. Serum sickness may improve quickly or persist for months depending on the type of antigen and the levels of antibody. Drug hypersensitivity reactions to penicillin, streptomycin, sulfonamides, and phenylbutazone may also cause serum sickness.

2. The Arthus response is the cutaneous reaction of type III responses. The Arthus response is highly localized (in and around blood vessels), appears within an hour, and resolves within 12 hours unless there has been severe tissue necrosis. The Arthus response is easily demonstrated in animal models. The repeated immunization of the animal results in high levels of antibody (usually IgG). The antigen is then injected intradermally or subcutaneously and complexes locally with antibody. The complement cascade is activated, and the reaction site becomes edematous and hemorrhagic. The intravascular clumping of platelets may lead to tissue necrosis. 3. Immune complexes are the central elements in type III pathologic events. Tissue deposition is most likely triggered by increased vascular permeability caused by vasoactive substances described previously. In autoimmune disease, immune complexes are formed by autoantibodies (such as anti-DNA in SLE) with its antigen. Immune complexes may then bind preferentially to certain tissues. In SLE, anti-DNA immune complexes bind

462

iiieClical

Clinical Immunology

Lysis by:

Phagocytosis

~ Platelet C/)

Q)

C1-C9

'5 o

.0

Ettl

Complement mediated

()

<;:::

.(3 Q)

Cl..

(j)

Tissue cells Cell-expressing antigen

Antibody-dependent cell-mediated cytotoxicity (ADCC)

Figure VI-2-5. Type II hypersensitivity.

to kidney glomeruli, causing nephritis. In rheumatoid arthritis, rheumatoid factor immune complexes may be found in joint spaces. 4. Clinical features include urticaria, lymphadenopathy, edema, and fever. Occasionally, arthritis, vasculitis, and glomerulonephritis are seen. The hallmark of immune complex glomerulonephritis is its granular ("lumpy bumpy") appearance as detected by immunofluorescense using tagged antibody specific for immunoglobulin or complement. Poststreptococcal glomerulonephritis is characterized by proteinuria, hematuria, and red blood cell casts in urine. Serum sickness occurs 1-2 weeks after administration of antiserum, which acts as an antigen. Formation of circulating immune complexes is followed by deposition in blood vessels, joints, kidney, and heart. This results in an inflammatory reaction with necrosis, edema, vasodilatation, microthrombi, and ischemia. 5. Pathology

a. There is acute necrotizing vasculitis with fibrinoid necrosis and neutrophilic exudation in arterial walls. b. Glomeruli in the kidney swell, and there is proliferation of endothelial and mesangial cells with a neutrophilic inflammatory exudate due to activation of the complement cascade.

Clinical Correlate Horse antiserum will almost invariably cause serum sickness. It is much less frequent if human antiserum is used.

Clinical Correlate The major complement proteins that contribute to the glomerular lesions are Db, which opsonizes, and (Sa, which is chemotactic for PMNs.

meClical

463

Immunology

~~ .....

.•

~~....

G4a

1) Appearance of antigen

11

.

) ( G3a G5a

2)

Vasodilation; influx of granulocytes into tissue

Figure VI-2-6. Type III hypersensitivity.

c. Immunofluorescence shows lumpy deposits along the basement membrane of blood vessels and glomeruli. d. Endocarditis with sterile vegetations, edema, and congestion of valves may also be seen. 6. Therapy for immune-complex-mediated reactions a. Aspirin, antihistamines, and steroids to reduce inflammation b. Suppression of immune response by means of corticosteroids and cytotoxic immunosupppressive drugs (e.g., cyclophosphamide) c. Removal of immune complexes via plasmapheresis D. Type IV hypersensitivity, or delayed-type hypersensitivity (DTH). Unlike the other types of hypersensitivity responses, which are mediated by antibody, DTH is mediated by T cells that have been sensitized to a particular antigen. An example of DTH is seen in tuberculin

464

metlical

Clinical Immunology

skin testing (TB tests), which measures previous T-cell exposure to the TB organism. Contact hypersensitivity, such as the response to poison ivy, is another characteristic type IV response. l. Delayed hypersensitivity reactions are initiated by sensitized Th1 (TDTh ) cells. The reac-

tions manifest as inflammation at the site of antigen exposure, which usually peaks 48-72 hours after exposure. a. Pathogenic mechanisms. Tissue damage results from the activation of macrophages via IFN-y and leads to the release of cytokines and reactive oxygen metabolites (see below). b. Induction of delayed hypersensitivity requires that the T cells recognize immunogenic epitopes as well as determinants in major histocompatibility complex (MHC) gene products (MHC restriction). Cytokine products of helper T cells aid in regulating the response. (1) Helper T cells secrete IL-2, IFN-y, and lymphotoxin and help inflammatory

reactions.

Clinical Correlate Delayed hypersensitivity responses are very important in the body's control of various chronic pathogens, such as mycobacteria and most fungi. Cytotoxic T cells are important in antiviral and antitumor responses.

(2) IL-2 from the antigen-presenting cell also enhances the development of delayed hypersensitivity. c. Expression of delayed hypersensitivity

(1) On contact with epitope, activated TDTh cells secrete lymphokines.

(2) These products activate macrophages and recruit the immigration of more cells into the area. (3) The macrophages secrete a variety of biologically active compounds, including interleukins 1 and 6, tumor necrosis factor alpha (TNF-a), reactive oxygen metabolites, proteases, and other lysosomal enzymes.

Chemokines

Macrophage influx

IFN-'Y TNF-cx

Tissue destruction

TNF-~

L

-+ Increased adhesion molecules

IL-3 GM-CSF

Monocyte proliferation

Figure VI-2-7. Type IV hypersensitivity.

2. Contact hypersensitivity is characterized clinically by eczema on the skin at the site of contact with allergen. It is induced by compounds (such as poison ivy catechols) that cross the skin and become conjugated to normal proteins. This conjugate sensitizes the person. T cells are able to recognize the conjugate after presentation by the Langerhans cell. 3. Tuberculin-type hypersensitivity detects the previous exposure or sensitization of T cells to Mycobacterium tuberculosis. A protein extract (ppd) of the organism is injected under the skin. After 24 hours, the site of antigen exposure is infiltrated with lymphocytes and monocytes. At 48 hours, there is a more extensive infiltrate of mononuclear cells. The reaction is read by measuring the degree of induration; 12-14 mm is usually considered positive. 4. Granulomatous hypersensitivity is caused by the persistence of antigen (usually a microorganism) within macrophages. Microbial agents causing this type of reaction

meClical

465

Immunology

include M. tuberculosis and M. leprae. Characteristic target cells include epithelioid cells (which are derived from macrophages) and multinucleate giant cells. These reactions persist in the body for months or years.

FOREIGN INVADER Acute Inflammation Continued attraction for neutrophils

.

• ?

'<"A concept for granulomatous formation: P = Persistent foreign invader D = Destruction of foreign invader

Figure VI-2-8. Granulomatous hypersensitivity.

Clinical Correlate 5. Expression ofT-cell cytotoxicity may also be linked to delayed hypersensitivity.

Necrotizing granulomas are particularly characteristic of tuberculosis. In AIDS, even with TB, granulomas form poorly because of a lack of coordinated cytokine secretion.

a. Cytotoxic T lymphocytes (Tc) destroy only targets that they recognize antigenically. b. If recognition occurs, the Tc are activated to release cytotoxic proteins from intracytoplasmic granules, the most important of which are perforins and enzymes such as serine proteases and nucleases. (1) Perforin resembles the complement component C9.

(2) This molecule inserts into target cell membranes and polymerizes to form transmembrane channels that allow influx (and egress) of essential ions and lowmolecular-weight metabolites. c. After delivery of this lethal "hit;' the lymphocyte detaches from the target and searches out another "victim:'

d. Fragmentation of target-cell DNA suggests that another significant product of the Tc might be an endonuclease, or an endogenous endonuclease may be activated during the lytic process. e. Cell-mediated immunity and disease (1) TDTh cells recognize the pathogen's antigens and secrete IFN-'Y and other

lymphokines, thereby activating macrophages to cytotoxic activity and helping B-cell responses.

466

meclical

Clinical Immunology

(2) Tc recognize foreign antigens on most cells of the body and confer resistance by two mechanisms. (a) Secretion ofIFN-ythat induces the production of antiviral proteins in cells of the body

In a Nutshell Hypersensitivity Type I • Anaphylaxis

(b) Destruction of the cell via release of perforin with subsequent formation of transmembrane channels, Ca2+ ion influx, endonuclease activation, etc. (c) As a result, delayed hypersensitivity reactions provide resistance to chronic intracellular bacterial infections (e.g., tuberculosis), fungal, viral, and protozoan infections, and malignancies. (3) As another expression of cell-mediated immunity, delayed hypersensitivity also plays a role in the rejection of grafted tissues and organs. (Humoral immunity also may be involved in allograft rejection.) (4) Finally, delayed hypersensitivity reactions provide the basic mechanism of tissue injury in a variety of diseases, such as contact dermatitis and certain autoimmune diseases. 6. Hypersensitivity pneumonitis (also called extrinsic allergic alveolitis) refers to the lung parenchymal reaction to repeated inhalation of particulate allergens. a. Pathogenesis. Patients often have antibodies to the allergens, with deposition of antigenantibody complexes in target (lung) tissue during the early phase of the disease. Recent evidence suggests that, in addition to immune complexes, delayed hypersensitivity may also playa role in hypersensitivity pneumonitis, particularly in the chronic disease process. b. Symptoms include fever, chills, chest pain, cough, and dyspnea, which occur 4-8 hours after exposure. In severe, chronic cases, irreversible lung damage (fibrosis) may occur. 7. Therapy for delayed hypersensitivity reactions includes: a. Drugs (1) Aspirin

• Atopy Requires previous exposure that sensitizes mast cells and basophils • Mediated by IgE bound to mast cells or basophils that degranulate and release vasoactive mediators and chemotactic factors Examples: asthma, atopic dermatitis, eczema, and allergic rhinitis (hay fever) Type II • Cytotoxicity • Antibody reacts with cell-surface epitopes Complement-mediated lysis or phagocytosis • Examples: transfusion reactions, hemolytic anemia, and Goodpasture syndrome Type III • Immune-complex mediated • Activates complement Examples: serum sickness, Arthus reaction, rheumatoid arthritis, and lupus nephritis

(2) Nonsteroidal anti-inflammatory agents

Type IV

(3) Corticosteroids

• Delayed-type (DTH)

b. Allergen avoidance

AUTOIMMUNE DISEASES A defect in the central mechanism underlying self-recognition (autotolerance, self-tolerance) can result in autoimmune responses. These are immune responses to antigens present in the host's own tissue and can be mediated by humoral (circulating antibodies, immune complexes) or cellular (delayed hypersensitivity) mechanisms.

• Only type that is cell mediated (T cells) • Th 1 cells (T DTh) react with antigen in association with MHC class II --7 Iymphokines released Examples: tuberculin skin testing, contact dermatitis, transplant and graft rejection

A. Several theories have been proposed to explain autoimmune responses. They may represent an immunologic imbalance arising from the following: 1. An excess of helper T2 (Th2) cell activity can be produced by coupling of chemicals, drugs (e.g., hydralazine), or viruses to self-antigen. The modified antigens would then recruit a reactive Th2 cell and elicit autoantibody production. 2. Epstein-Barr virus or polydonal B-cell activation with materials such as bacterial lipopolysaccharide may result in autoantibody formation.

meclical

467

Immunology

Clinical Correlate Several autoimmune diseases seem to be linked to viral infections, particularly nervous system maladies. • Measles = multiple sclerosis, subacute sclerosing panencephalitis • Rubella = insulindependent diabetes • Influenza = Guillain-Barre

3. An inability to downregulate the immune response to self-antigen 4. Release of sequestered antigen (e.g., from lens of the eye, sperm) not ordinarily available for recognition by the immune system. Release might occur via such means as trauma or infection. 5. Molecular mimicry, in which a microbial antigen contains epitopes that are also found in self-antigens. The host responds to the infectious agent (e.g., M protein of Streptococcus pyogenes) and the resultant immune response damages autologous tissue (e.g., myosin of cardiac tissues). 6. Inappropriate expression of class I MHC molecules. Pancreatic cells of IDDM patients have high levels of these molecules compared with healthy controls, where the antigen is practically nonexistent. 7. Preferential activation ofThl cells B. Genetic predisposition 1. There is clearly a role for genetic factors in autoimmune disease. It is thought to be asso-

ciated with those major histocompatibility complex genes that code for the class II antigens, which are so important in presentation of antigens. Most autoimmune diseases appear to be associated with DRl, DR3, DR4, or DRS. Some examples include rheumatoid arthritis, DR4; insulin-dependent diabetes mellitus, DR3/DR4 heterozygotes; and SLE, DR3. Over 90% of patients with ankylosing spondylitis have the HLA-B27 antigen, compared with only 8% of the normal population. 2. Autoimmune diseases tend to occur at a higher frequency in women. For example, the incidence of SLE in women is four to six times that in men, and rheumatoid arthritis is three to four times more common in women. C. The frequency of autoimmune diseases increases with age. Most appear in the 20- to 40-year

age group. 0. General signs of autoimmune disease that may be of diagnostic importance include: 1. Elevated serum gamma globulin levels

2. Various autoantibodies 3. Depressed levels of serum complement 4. Immune complexes in serum 5. Lesions detected on biopsy (e.g., glomerular lesions resulting from deposition of immune complexes) E. Diagnostic tests are designed to detect antibodies specific to the particular antigen involved in the disease. Certain facts should be pointed out, however. 1. Autoantibodies are not unique to autoimmune disease; for example, antinuclear anti-

bodies may be found in tuberculosis, histoplasmosis, malignant lymphoma, and other neoplasms. 2. Patients with autoimmune disease may have more than one autoantibody and, in fact, may suffer from multiple autoimmune diseases. 3. Although SLE is associated with antinuclear antibodies and rheumatoid arthritis is associated with rheumatoid factor, both antibodies may be found in both diseases. Patients with SLE may have 10-15 different autoantibodies. F. Therapy for autoimmune disease involves several approaches.

468

meClical

Clinical Immunology

1. In certain organ-specific diseases, metabolic control may suffice. a. In thyrotoxicosis (Graves disease), antithyroid drugs such as propylthiouracil or methimazole may be prescribed. Surgical or radionuclide (1311) ablation of the gland is also effective.

Clinical Correlate Drugs are occasionally cited as precipitators of autoimmunity.

b. Vitamin Bl2 is given to patients with pernicious anemia. 2. Agents such as nonsteroidal anti-inflammatory drugs (e.g., aspirin, indomethacin), steroidal anti-inflammatory drugs (e.g., cortisone, which may also be immunosuppressive), and immunosuppressive cytotoxic drugs (e.g., cyclophosphamide, azathioprine) are useful in treating disease symptoms. 3. Anticholinesterase drugs and thymectomy are of value in myasthenia gravis. 4. Plasmapheresis, or plasma exchange therapy, appears to be of value in certain diseases (e.g., Guillain-Barre, systemic lupus erythematosus, and Goodpasture syndrome) by removing offending antibodies and immune complexes. 5. Splenectomy is of value in hemolytic diseases and idiopathic thrombocytopenic purpura. G. Systemic lupus erythematosus (SLE) is a collagen vascular or connective tissue disease with a chronic remitting and relapsing course and multisystem pathology.

• SLE may follow treatment with procainamide or hydralazine.

• D-penicillamine has been reported to induce myasthenia gravis, pemphigus, Goodpasture disease, and lupus. • L-dopa and a-methyl dopa are associated with hemolytic anemias.

1. Incidence is about 1 in 1,000 with a female/male ratio of 9:1. It is more common in African Americans.

Clinical Correlate

2. Pathogenesis is caused by a loss of self-tolerance, particularly to nuclear antigens. The disease is characterized by the presence of antibody against double-stranded DNA and a variety of other antigens. The disease is associated with certain HLA haplotypes (HLADR2, HLA-DR3), but environmental factors, such as viruses, drugs, and hormones, can lead to exacerbation of disease episodes. B-cell abnormalities are most prominent, with anti-self-antibodies waxing and waning.

A complication of splenectomy in the treatment of autoimmune diseases is the increased susceptibility to septicemia, particularly that caused by Pneumococcus. Patients can be protected by immunization with pneumovax, a polyvalent (23 serotypes) capsular vaccine.

3. Diagnostic tests a. Antinuclear antibody test (ANA) is an immunofluorescent test for antinuclear antibodies. It is very sensitive (positive in 99% of SLE patients) but not specific (positive in 10% of normal individuals); it is also elevated in other collagen vascular diseases such as rheumatoid arthritis. b. Extractable nuclear antigen tests. These tests have variable specificity and sensitivity. Anti-RNA-associated protein (anti-RNP) antibody is the most specific antibody for SLE, but it is present in only one third of lupus patients. c. Anti-double-stranded (native) DNA test is diagnostic if positive, and titers correlate with disease activity. Anti-Sm antibodies are also highly diagnostic. 4. Clinical diagnosis is established by the criteria of the American Rheumatism Association. There should be four of the following signs or symptoms during the period of observation: a. Facial erythema (malar "butterfly" rash) b. Discoid lupus

Clinical Correlate Anti-dsDNA antibody levels correlate with severity of nephritis.

c. Raynaud phenomena d. Alopecia e. Photosensitivity f. Oral or nasopharyngeal ulceration

meclical

469

Immunology

Clinical Correlate

g. Arthritis without deformity

Anti-Ro antibodies are associated with congenital heart block seen in infants bom to lupus mothers. These same infants may also have a transient annular erythematous skin rash. AntiRo antibodies are also found in high incidence in patients with Sjogren syndrome.

h. LE cells 1.

Chronic false-positive serologic test for syphilis for more than 6 months

j. Profuse proteinuria (>3.5 g1day)

k. Urinary cellular casts 1. Pleuritis or pericarditis

m. Psychosis or seizures n. One or more of the following: hemolytic anemia, leukopenia, or thrombocytopenia 5. Clinical features. SLE usually presents in the second or third decade. The onset may be insidious or acute. Most common presentations include fever of unknown origin, arthritis, rashes, and renal involvement. The course is highly variable with spontaneous exacerbations and remission. a. Treatment consists of corticosteroids and immunosuppressive drugs. b. Mortality is most often from renal failure or infections. 6. Pathology is predominantly due to deposition of DNA-anti-DNA complexes but may also result from direct autoantibody cytotoxicity in any organ. a. Blood vessels show acute necrotizing vasculitis in most affected tissues. Necrosis and fibrinoid deposits containing immunoglobulins, DNA, C3, and fibrinogen are seen, leading to a narrowed lumen. b. Joints are involved in 80-90% of cases. There is swelling and a mononuclear cell infiltrate of synovial membranes. Cartilaginous damage is rare.

Note Immune complexes seen in the lupus kidney glomerulus include: • IgG or IgM • Complement proteins

• DNA • Fibrinogen

470

meClical

c. Skin is involved in 80% of cases. The classical lesion is a maculopapular, erythematous rash over the bridge of the nose and malar eminences (butterfly rash). Microscopically, one sees liquefaction necrosis in the basal layer of the epidermis with immunoglobulin and complement deposition along the dermoepidermal junction. d. Kidney involvement can be demonstrated in 70% of patients by light microscopy and almost 100% of cases using the electron microscope or immunofluorescent techniques. e. The heart is involved in 50% of cases. Libman-Sacks endocarditis consists of bland verrucous vegetations of valves composed of fibrinoid material, necrotic debris, and inflammatory cells.

Clinical Immunology

Figure VI-2-9. Lupus nephritis (microscopic).

Figure VI-2-10. Lupus nephritis (immunofluorescence).

f. Serosal membranes are involved in 40% of cases. The pericardium and pleura are

involved with effusions, fibrinous exudates, or, eventually, fibrosis. g. Lymph nodes are involved in 60% of cases. They show enlargement, germinal center hyperplasia, and focal fibrinoid necrosis in vessels. h. In the eNS (30% of cases), there is vasculopathy, precipitating infarcts, and hemorrhages, primarily involving small vessels. 1.

In the liver (30% of cases), acute vasculitis with inflammatory infiltrates in the portal tracts is seen.

KAPLAN ' . I medlea

471

Immunology

In a Nutshell Systemic Lupus Erythematosus • Young women

j. The spleen (20% of cases) may show capsular fibrosis, onion skin lesions, and concentric perivascular fibrosis.

k. In the lungs (10% of cases), interstitial pneumonitis and fibrosing alveolitis are seen. 7. Other types of lupus

• Malar rash, joint pain, nephritis, endocarditis, pleuritis, pericarditis

a. Discoid lupus features skin lesions consisting of erythematous plaques with keratotic scaling, follicular plugging, and atrophic scarring. Multisystem involvement may occur after many years in 50% of patients.

• Positive ANA

b. Subacute cutaneous lupus features superficial nonscarring lesions associated with mild systemic disease and antinuclear antibodies. HLA-B8 and HLA-DR3 are present in these patients in greater frequency than in the normal population.

• Anti-dsDNA antibodies • Anti-Sm antibodies • Treated with steroids and immunosuppressive drugs SLE has long been a Boards' favorite. You should consider it every time a question presents a 15-45-year-old woman and then look for clues to seal the diagnosis (malar rash, anti-dsDNA, etc.). A clinical clue that is commonly given is kidney dysfunction (e.g., proteinuria, ankle edema, etc.). Kidney lesions often seen (60-70% of the time) include mesangial lupus glomerulonephritis, focal proliferative glomerulonephritis, diffuse proliferative glomerulonephritis, and membranous glomerulonephritis.

c. Drug-induced lupus. The development of antinuclear antibodies (but not anti-double-

stranded DNA antibodies) is seen after receiving certain drugs (e.g., isoniazid, procainamide, hydralazine). It is associated with individuals having HLA-DR4 antigen, which may cause them to be slow acetylators. H. Scleroderma is characterized by fibrosis throughout the body. It most often affects the skin but also involves the kidney, lungs, striated muscle, heart, and gastrointestinal tract. There is a 3:1 female/male incidence ratio. 1. Clinical features. Scleroderma usually presents in the third to fifth decades with edema

and thickening of the skin of the hands or Raynaud phenomena. Also, arthralgias, dysphagia, respiratory distress, cor pulmonale, intestinal obstruction, hypertension, and renal failure may occur. The disease is more severe in Mrican Americans. Patients experience progressive debilitation. Eighty percent of patients are alive at 2 years, but only 20% are alive at 10 years. Death is usually due to cardiac, renal, or pulmonary involvement. 2. Pathology a. Serologic abnormalities are varied and include antinuclear antibodies in 50% of cases. Diagnostic markers include anti-SCI-70 (nonhistone nuclear protein) and anticentromere antibody. The latter typically occurs in mild scleroderma (CREST). b. Damage to small blood vessels may be prominent, causing ischemic injury and scarring. As in SLE, damage may be mediated by immune complex deposition. c. Skin changes begin distally in the upper extremities and progress proximally, eventu-

ally involving all of the arms, chest, neck, and face. Lesions consist of increased dermal collagen, epidermal atrophy, and loss of skin appendages, which result in a limitation of movement, ulcerations, and a "drawn mask" appearance of the face. d. In the gastrointestinal tract, fibrosis and atrophy of the esophagus are prominent, with ulceration of the mucosa. Scleroderma occasionally involves the stomach and causes malabsorption in the small intestine. e. Striated muscle shows mixed inflammatory infiltrates and fibrosis. f. Joints show sclerosis of articular collagen and periarticular connective tissue. g. In the kidney, intimal proliferation and hyalinization of small arteries occur in two thirds of patients. h. The lungs show diffuse interstitial fibrosis. i. The heart shows perivascular interstitial fibrosis.

472

meClical

Clinical Immunology

3. The CREST syndrome (mild scleroderma) is a rather common collection of lesions characterized by calcinosis, Raynaud phenomena, esophageal dysfunction, sclerodactyly (scleroderma of the digits), and telangiectasia. 1. Sjogren syndrome is characterized by keratoconjunctivitis sicca (dry eyes), chronic arthritis, and xerostomia (dry mouth). It results from immunologic destruction of salivary and lacrimal gland duct epithelium. Sjogren syndrome may be a primary disorder (sicca complex) or associated with another autoimmune disorder, such as SLE. It is diagnosed by the presence of autoantibodies to the Ro/SSA and/or La/SSB auto antigen by ELISA.

Clinical Correlate Anticentromere antibodies are pathognomonic for CREST syndrome.

1. Pathogenesis. Sjogren syndrome results from a lymphocytic infiltrate (predominantly

helper T cells) and fibrosis of the ducts of lacrimal and salivary glands. It is associated with numerous autoantibodies. The primary form is associated with HLA-DR3. 2. Clinical features. Middle-aged women are most commonly affected. a. One third of patients have the primary form; two thirds are associated with another autoimmune disease such as SLE or rheumatoid arthritis. b. Patients present with blurred vision, a foreign body sensation in the eyes, cracks and fissures of the mouth, and dry mouth. The parotid gland is frequently enlarged. Vasculitis, Raynaud phenomenon, hyperviscosity syndrome, and peripheral neuropathy are also associated with Sjogren syndrome. c. The term "pseudolymphoma" is used to describe enlargement and a pleomorphic

infiltrate oflymph nodes. In this syndrome, there is a 40 times greater chance of developing B-celllymphomas. 3. Pathology. In Sjogren syndrome, there is a destruction of glandular structure by lymphocytic and plasma cell infiltrates. Eyes exhibit inflammation and ulceration of the corneal epithelium. The oral mucosa exhibits atrophy, inflammation, and ulceration. The process occasionally involves respiratory passages, the esophagus (forming webs), and the stomach (atrophic gastritis).

meclical

473

Immunology

Table VI-2-2. Summary of autoimmune disorders. Disease

Immune Reaction Direct Against

Nervous system: Guillain -Barre Multiple sclerosis Myasthenia gravis

Myelin Nervous tissue (myelin) Acetylcholine receptors

Musculoskeletal/Skin Bullous pemphigoid Pemphigus vulgaris PolymyositisDermatomyositis Rheumatoid arthritis

Hematologic Autoimmune hemolytic anemIas Idiopathic thrombocytopenic purpura (ITP) Pernicious anemia Systemic Systemic lupus erythematosus Scleroderma Other Sjogren syndrome

C C H

Skin basement membrane Squamous desmosomes Vessel walls of skin and muscle

H H

IgG

H

Endocrine Addison disease Adrenal microsomal proteins Graves disease TSH receptors Hashimoto thyroiditis Thyroglobulin Diabetes mellitus (type 1) ~ islet cellslinsulin Cardiovascular Rheumatic fever Respiratory/Renal Goodpasture syndrome

Humoral (H) or T-Cell Mediated (C)

H,C

H H

H,C H,C

Cardiac myosin

H

Alveolar and glomerular basement membranes

H

Red blood cells

H

Platelets

H

Intrinsic factor, and receptor

H

DNA, many others

H

Scl-70 centromere

H

Salivary and lacrimal gland duct epithelium

H

J. Mixed connective tissue disease is a disorder that exhibits the characteristics of SLE, scleroderma, and polymyositis. It is associated with high titers of anti-ribonucleoprotein antibodies, producing a speckled pattern with immunofluorescence. The female/male ratio is 12:1. The syndrome usually presents between the ages of 30 and 60. Paradoxically, there is little renal disease, and the course is rather benign.

474

meClical

Clinical Immunology

Table VI-2-3. Autoimmune diseases. Disease

Autoantigen

Pathology

Autoimmune hemolytic anemia

Rh blood group antigen on RBCs

RBC lysis, anemia

Autoimmune thrombocytopenia purpura Goodpasture syndrome

Platelet integrin

Abnormal platelet functions, bleeding

Type IV collagen of basement membrane IgG (rheumatoid factors)

Glomerulonephritis, pulmonary hemorrhage

Autoantibody-mediated diseases

Mixed essential cryoglobulinemia Pemphigus vulgaris Rheumatic fever

Systemic lupus erythematosus T-celI-mediated diseases Diabetes (IDDM) Multiple sclerosis, acute disseminated encephalomyelitis, experimental autoimmune encephalitis Rheumatoid arthritis

Vasculitis

Cahedrin (epidermis)

Skin blistering

Cross-reactive cardiac myosin and streptococcal M cell wall antigen DNA, snRNPs, histones, ribosomes, RoISSA, La/SSB

Myocarditis, arthritis, fibrotic heart valves Glomerulitis, arthritis, vasculitis, skin lesions

Pancreatic islet ~ cell

~-cell necrosis (lack of insulin production)

Myelin basic protein, proteolipid protein

CNS paralysis, T-cell infiltration in brain

Unknown agent in synovium

Joint inflammation, arthritis

Table VI-2-4 Disease

FemalelMale Ratio

HLA

Relative Risk

Ankylosing spondylitis

1:3

B27

88

Good pasture syndrome

1:1

DR2

16

Multiple sclerosis

10:1

DR2

10

Graves disease

4:1

DR3

4

Myasthenia gravis

1:1

DR3

3

Systemic lupus erythematosus

8:1

DR3

6

Diabetes (IDDM)

1:1

DR30rDR4

3

Rheumatoid arthritis

3:1

DR4

4

Hashimoto thyroiditis

5:1

DRS

3

iiieilical

475

Immunology

Clinical Correlate

AMYLOIDOSIS

The infectious amyloidopathies are caused by "slow-virus" (prion proteins) and are manifested as spongiform encephalopathies.

Amyloidosis is characterized by the deposition of amyloid protein in the extracellular space of various organs and tissues. Generally, immunologic mechanisms are thought to underlie the accumulation of amyloid, although the specific causes are diverse. A. Pathology 1. With ordinary stains, amyloid appears as an eosinophilic, amorphous substance in extra-

cellular spaces. 2. Under polarized light, amyloid displays "apple-green" birefringence when stained with Congo red. 3. Electron microscopy shows single fibrils 7.5-10 nm in diameter. X-ray crystallography reveals that the protein is arranged in a ~-pleated sheet. Two major types of amyloid fibrils are found. a. AL (amyloid light chains), which consist of immunoglobulin light chains or portions of light chains b. AA (amyloid-associated protein), made in the liver c. Other forms of amyloid derive from other body proteins folded into a ~-pleated sheet arrangement, e.g., amyloid associated with Alzheimer disease is derived from amyloid precursor protein (APP). B. Types of disease. Amyloidosis may be localized, affecting particular organs or tissues, or sys-

temic, affecting the entire body: 1. B-cell dyscrasia (e.g., multiple myeloma) is associated with systemic amyloid deposition

composed of immunoglobulin light chains (Bence-Jones protein). This is also known as primary amyloidosis. 2. Secondary (reactive) amyloidosis is characterized by systemic deposition of AA protein in association with prolonged chronic inflammatory states (e.g., rheumatoid arthritis, inflammatory bowel disease, tuberculosis). 3. Around two thirds of chronic hemodialysis patients develop amyloid deposits, consisting of ~-2 microglobulin, in and around joints. 4. Hereditary (familial) amyloidosis (Mediterranean fever) is inherited as an autosomal recessive and is systemic in nature. 5. Localized amyloidosis occurs in aging (senile cardiac amyloidosis, Alzheimer disease), in some endocrine disorders, and in some cases of B-cell or plasma cell dyscrasia. C. Clinical manifestations 1. Renal involvement causes proteinuria, cellular casts, and eventual renal failure.

2. Liver or spleen may be involved, with symptoms depending on the degree of infiltration. 3. Cardiac amyloidosis can produce arrythmias, cardiomyopathy, and heart failure. 4. The prognosis is poor in generalized amyloidosis (mean survival 1-3 years). Treatment is often unsatisfactory.

476

meClical

Clinical Immunology

TRANSPLANTATION AND TUMOR IMMUNOLOGY

Clinical Correlate

A. Transplantation immunology deals with immune response to grafted tissues. The grafts may be from one part of the body to another site (e.g., autografts, as in mandible reconstruction, where a portion of the iliac crest is used to fashion a new jaw), or they may be from another person (e.g., allograft or homograft, where an organ such as a kidney, heart, or lung is removed from a donor and placed in a recipient whose organ(s) are failing).

Larger organs such as liver and heart! lung transplants are less immunogenic than skin and kidney transplants.They may also survive better because of the vast amount of antigens they contain.

1. Autologous grafts are accepted as "self" and do not induce an immune response.

2. Allografts are composed of foreign tissues and the recipient will respond immunologically; the relative immunogenicity varieties between organs. 3. Some grafts may involve synthetic material, as occurs in heart valve replacement and artificial heart implantation surgery. Only rarely are tissues and/or organs transplanted across species barriers. These grafts, termed xenografts, are usually rejected very rapidly and are not intended to be permanent organ replacements. 4. Grafting of normal tissue between identical twins or from one animal in an inbred line to another animal in that same line succeeds as if there were no immune barriers. These are isografts, and the tissues bear a syngeneic relationship. However, transplantation between genetically nonidentical animals will result in an immune rejection phenomenon, which may or may not be prevented or aborted by the use of immunosuppressive drugs. Rejection is due to the recipient's recognition of, and immunologic response to, glycoprotein histocompatibility antigens in the membranes of cells in the grafted tissue. Certain antigens of the red cell system may also act as transplantation antigens. One cannot violate incompatibility in the red cell system and expect graft survival. 5. The donor-recipient workup. The compatibility of donor and recipient must be maximized. a. ABO blood group compatibility is first established. b. Class I and Class II HLA tissue typing is performed. c. Cross-matching is used to test for recipient preformed antibodies against the donors HLA class I and class II antigens. d. Other cellular assays such as the mixed lymphocyte reaction (MLR) may be used to determine if the donor cells stimulate blastogenesis in the recipient's lymphocytes. 6. Histocompatibility gene complex a. HLA is an acronym for human leukocyte antigens, so termed because lymphoid cell membranes are particularly rich in these substances. The genes that dictate the immunologic specificity of these antigens occur in allelic form; they are polymorphic, e.g., multiple genes may occur at a single locus but only one will be expressed per locus (per chromosome). In addition they are codominant in expression, which means that if an individual is heterozygous, both parental antigens will be present on the cells. b. Classification of HLA molecules (1) Class I antigens: The HLA-A, -B and -C antigens are found on all nucleated

human cells. Structurally, the class I antigens are composed of a glycoprotein in noncovalent association with a nonpolymorphic beta-2-microglobulin. Class I molecules present endogenous antigens and are also involved in MHC restriction of cell-mediated cytolysis. (2) Class II antigens: HLA-D antigens (D = DP, DQ, and DR) are found chiefly on the surfaces of antigen-presenting cells, including macrophages/monocytes, resting T lymphocytes (in low amounts), activated T lymphocytes, and

metlical

477

Immunology

B lymphocytes. The HLA-D region antigens have been shown to be involved in antigen presentation by macrophages to T lymphocytes as well as in efficient collaboration between Band T cells. 7. Transplant rejection a. In graft rejection, an allogenic peptide is expressed in the groove of an allogenic MHC I molecule. This combination stimulates the host in much the same way as a viral peptide. T-cell-mediated rejection results in acute rejection 10-14 days after a graft is placed. It is mediated by both delayed-type hypersensitivity and T-cell cytotoxicity and requires helper T cells and cytotoxic T cells. b. Antibody-mediated rejection may be caused either by preformed (hyperacute) or subsequently formed antibodies (late acute rejection vasculitis). Hyperacute rejection may be mediated either by ABO blood group antibodies (usually IgM) or anti-HLA antibodies (usually IgG) from a previous transplant, or, in the case of multiparous women, from immunization during pregnancy. 8. Methods to minimize rejection a. Matching for at least four of the HLA antigens reduces the incidence of rejection (HLA A, B, C, DR).

b. Immunosuppression of the graft recipient with monoclonal antibodies versus helper T cells, azathioprine, steroids, or cyclosporin A kills lymphocytes or reduces IL-2 secretion. c.

Clinical Correlate Hyperacute rejection is caused by preformed antibody against the graft donor, as may occur in multiparous women, or across an ABO barrier.

Antilymphocyte serum decreases the level of lymphocytes, thereby prolonging graft survival.

d. With the use of high doses of immunosuppressive drugs, one often gets into trouble with infection and/or malignancy. About 25% of the deaths that occur in kidney transplants are caused by sepsis. 9. Pathology of rejection a. Hyperacute rejection shows arteritis, thrombosis, and ischemic necrosis as a result of an Arthus-like reaction. The graft does not become vascularized and appears dusky and mottled within seconds to minutes. b. Acute rejection has two components. The humoral component causes vasculitis; the cellular component causes death of tissue cells of the graft with accompanying decrease in function. It occurs weeks after transplantation. c. Accelerated graph rejection takes days to appear and is due to the reactivation of sensitized T cells. d. Chronic rejection shows intimal fibrosis, causing parenchymal ischemia, atrophy, and fibrosis. Its time course is months to years. 10. Graft-versus-host disease (GVH) results from transplantation of immunologically active cells into an immunocompromised recipient, resulting in a "rejection" reaction of these graft cells against host tissues. It most often occurs after bone marrow transplantation. Newer technology is able to remove immunologically active donor cells and minimize GVH. Cyclosporin A has been used successfully to control these adverse transplant events. B. Tumor immunology 1. Tumor antigens. Tumor-associated antigens (TAA) are those antigens present on tumor

cells but that may also be found on some normal tissues. TSA refers to tumor-specific antigens. These antigens are found only on tumor cells and not on normal tissues.

478

meclical

Clinical Immunology

Tumor-specific transplantation antigens (TSTA) are a specialized form of TSA; they are the antigens to which the immune system must respond if it is to eliminate the growth of the tumor in vivo. a. Antigens of chemically induced tumors. When tumors are induced in experimental animals using a chemical carcinogen, the TSTA of each individual tumor will be unique to that tumor. Tumor antigens of chemically induced tumors are mutational events that occur as a result of the chemical carcinogen that is also a mutagen. The mutation is random and would not be found in another tumor, unless millions were compared. The tumor antigen is a mutated normal cellular protein. b. Antigens of virally induced tumors. The TSTA of tumors that arise as a result of oncogenic viruses are completely cross-reactive. The genetic information for the TSTA induced by viral agents comes from the virus. The virus enters the cell and integrates its genome into the host cell DNA. Proteins derived from this genetic material are also processed by the endogenous route and expressed on the surface of the cell in association with MHC class I molecules. When a different virus is used for induction of the tumor, then different proteins are made and the TSTA is also different. c. Oncofetal antigens. Other types of antigens that may be found on tumor cells are

called oncofetal antigens. These antigens are normally present on tissues during fetal development, but their synthesis is repressed before birth. When cells undergo malignant transformation, these genes are derepressed, and the proteins are once again synthesized. In human medicine, oncofetal antigens have been used as markers for the growth of some tumors. (1) Alpha fetoprotein (AFP) is a serum protein secreted by the fetal liver. It is increased in most patients who have hepatomas, but it is also increased in other types of cancer. Originally, it was thought that AFP levels might be useful in the diagnosis of these malignant diseases; however, the protein is also elevated in a variety of nonmalignant disorders of the liver. Although diagnosis of malignancy by AFP levels is not possible, AFP levels have been used to establish prognosis and monitor the success of therapy for tumors that secrete this protein. (2) Carcinoembryonic antigen (CEA) is another human oncofetal antigen that is elevated in patients with carcinoma of the colon or pancreas. CEA levels are present in normal individuals at low levels because it is normally present in the gut, liver, and pancreas. Elevated CEA levels occur with other cancers, including lung, breast, and prostate. As with AFP, CEA levels can also be elevated in some nonmalignant disorders. Fifteen percent of heavy smokers will have elevated CEA levels. Although not useful for diagnosis of disease, elevated CEA levels can contribute to diagnosis, provide information regarding prognosis, and be used to monitor the success of therapeutic intervention for carcinoma of the colon.

Note There are several known oncogenic viruses: • Human papilloma virus ~ cervical and penile cancer • Epstein-Barr virus ~ Burkitt lymphoma, nasopharyngeal cancer • Hepatitis Band C viruses ~ hepatocellular carcinoma • HTLV 1 ~ adult T-cell leukemia

Note

oncofeta I antigens are found in fetal tissues and in malignant tissues. However, their production is also seen in nonmalignant conditions; hence, they are not diagnostically useful. Their value is in prognosis/ monitoring.

(3) Leukemia antigens. Specific antigens are associated with certain leukemias, making blood tests for these antigens useful in diagnosis. (a) Terminal deoxynucleotidyl transferase (TdT) is an enzyme marker for lymphocytic cells of the hematopoietic system. It is found in patients with leukemia and lymphomas. (b) Common acute lymphoblastic leukemia antigen (CALLA), also known as CD 10 antigen, is found on the surface of tumor cells in 80% of patients with acute B-celllymphoblasti.c leukemia (ALL) and in some patients with chronic myelogenous leukemia in blast phase. It is also found on immature B cells.

meClical

479

Immunology

Table VI-2-S. Useful tumor markers.

Clinical Correlate Natural killer cells are responsible for elimination of precancerous cells from the body (immune surveillance). Their numbers are sometimes increased via in vitro manipulations so that they can be used therapeutically.

Antigen

Cancer

Alpha fetoprotein (AFP) Carcinoembryonic (CEA) Chorionic gonadotropin Immunoglobulin Prostate-specific antigen (PSA) CALLA

Liver, testes Colon, some lung Trophoblastic Myeloma, B lymphoma Prostate B-celileukemia

2. Immune surveillance refers to the role of the immune system in preventing cancer in immunologically normal individuals. It is believed that one of the roles of the immune system is to regularly eliminate cells of the body that may undergo malignant transformation. The fact that immunodeficient patients have an increased incidence of tumors has been used to support this theory. Current thinking suggests that this function may be carried out by natural killer cells. 3. Mechanisms for rejection of tumors a. Antibody and complement. In many experimental and human situations, antibodies specific to tumor cells can be found in the serum of the tumor-bearing host. However, for the most part, experimental evaluation has shown that antibody is not very effective at eradicating tumor cells in vivo. b. Cytotoxic T cells (Tc). CD8+ cytotoxic T cells are very potent killers of tumor cells. These cells recognize foreign peptides on tumor cells and release a variety of molecules that can kill the tumor target. One of these molecules is perforin, which polymerizes in a manner similar to the membrane attack complex of the complement system and punches holes in the membrane of the tumor. Not only does this provide a mechanism whereby the target might lyse, but enzymes released by the Tc cell and that are toxic to the tumor cell can enter through these holes. These enzymes (e.g., proteases and nucleases) are called granzymes. TNF-~ and IFN-y are also produced by the Tc cell. These cytokines interact with their respective receptors on the membrane of the tumor cell and are able to promote killing of the tumor. c. Helper T cells. Although tumor antigens are expressed in conjunction with class I

MHC, there is evidence that class II MHC expression also occurs. This may be due to engulfment of dying tumor cells by macrophages and expression of peptides from these molecules via the exogenous antigen processing route. Helper T cells of the Thl type are very active in augmenting nonspecific mechanisms of tumor rejection. Lymphotoxin (also called TNF-~) produced by the Thl cell can kill tumor cells directly. Additionally, IFN-y released by the Thl cell activates natural killer (NK) cells to exhibit enhanced cytotoxicity toward tumor targets. Interferon also activates macrophages to release molecules that are cytotoxic to tumor cells (e.g., reactive oxygen and nitrogen intermediates). d. Natural killer cells. NK cells are cytotoxic to tumor cells; they secrete a variety of molecules that attack the tumor target. Natural killer cell cytotoxic factor (NKCF) is active in this regard. Additionally, these cells secrete granzymes and perforin. NK cells also release IFN -y and TNF. NK cells have the ability to recognize malignant cells and kill them, but they are not cytotoxic for normal cells. Receptors for MHC and other molecules are responsible for this interaction.

480

mellical

Clinical Immunology

e. Activated macrophages. Normally, macrophages are not cytotoxic in their resting state. However, after they come in contact with IFN-y they become activated and are highly cytotoxic to tumor cells. Like NK cells, activated macrophages kill tumor cells but do not kill normal cells. They release cytotoxic factors, including TNF-a; reactive oxygen intermediates (ROI), such as H 2 0 2 and superoxide anion; and the reactive nitrogen intermediate (RNI), nitric oxide. All of these molecules are toxic to tumor cells. f. Antibody-dependent cell-mediated cytotoxicity (ADCC) is a mechanism that helps cytotoxic cells come in better contact with tumor targets. Because NK cells and macrophages have Fc receptors, antibodies can associate with these cells via the Fc portion of the antibody molecule. If the antibody is directed at a tumor cell's surface antigen, then the antibody can help the NK cell or the macrophage come in close contact with the tumor cell. 4. Factors that favor tumor growth. Even though the immune system has a wide variety of weapons to inhibit the growth of tumors, the fact remains that the large majority of cancer patients will die if they are not treated by surgery and/or chemotherapy. Tumor immunologists have discovered a variety of mechanisms that tumors use to subvert the immune system. a. Tumor cell heterogeneity. Individual tumor cells isolated from a single tumor are very heterogenous. As the tumor grows and differentiates, the cells mutate in many ways, including antigenic make-up. This property allows the tumor cell to escape from Tc cells directed at specific antigens.

In a Nutshell Mechanisms of Tumor Rejection • Antibodies • Complement • Cytotoxic cells - T cells: TDTh, Tc - Macrophages - NK cells - ADCC cells

b. Rapid growth. Tumor cells may grow more rapidly than normal cells. The tumor cell may simply be able to outstrip the ability of the immune system to respond. Many tumors secrete growth-promoting factors (e.g., epidermal growth factor) that promote their own proliferation. c. Release of immunosuppressive factors. Many tumors release factors that are immunosuppressive. Included among these are alpha fetoprotein and the cytokine TGF-I3. d. Shedding of antigens. Tumors cells have been shown to shed their antigens to a much greater degree than normal tissues. This large amount of soluble antigen can be immunosuppressive via the induction of immunologic anergy or through blocking cytotoxic T-cell function. e. Serum-blocking factors. Factors have been found in the serum of tumor patients that are able to block the cytotoxic activity of the Tc cell. These factors are present in !he serum when a large tumor is present and are lost from the serum soon after surgical removal of the tumor. Tumor antigen, antitumor antibody, and antigen-antibody complexes can all function as serum-blocking factors. However, antigen-antibody complexes are by far the most efficient in their inhibition of the cytotoxic T-cell response. 5. Immunotherapy in cancer. The treatment of tumors by immunotherapy alone is not likely to be successful because of the various factors that were discussed previously. For this reason, methods to diminish the bulk of the tumor burden, including surgery, chemotherapy, radiation, or a combination of these modalities, should be instituted before immunotherapy. a. Monoclonal antibodies. Because of the ability of tumor cells to modulate their antigens, treatment with monoclonal antibodies alone is not successful. However, recent investigations are directed at adding very toxic molecules to these antibodies in the hope of targeting the toxin to the surface of the tumor cell. Such molecules are called immunotoxins and are the product of coupling toxins such as ricin or radioisotopes (e.g., 131I) to tumor-specific antibodies.

In a Nutshell Factors that Favor Tumor Growth • Rapid growth rate • Antigenic heterogeneity • Synthesis of immunosuppressants • Secretion of tumor antigens • Induction of non protective, competing antibodies

Note Ricin is a protein-synthesisinhibiting toxin much like diphtheria toxin.

meClical

481

Immunology

b. Tumor-infiltrating lymphocytes. Lymphocytes that infiltrate solid tumors are called tumor-infiltrating lymphocytes (TIL). These lymphocytes can be isolated from solid tumors, cultured in vitro in the presence ofIL-2, and reinfused into the tumor patient. Such approaches have been shown to be effective in helping to eradicate metastatic tumor foci. c. Lymphocyte-activated killer (LAK) cells. LAK cell therapy involves isolating peripheral blood natural killer cells from the tumor patient and culturing them in vitro in the presence of high concentrations of IL-2. These LAK cells are then infused back into the patient. This method of treatment has shown some promise in the treatment of renal carcinomas. d. Macrophage and NK cell activators. A variety of microbial products are able to activate macrophages and natural killer cells in vivo. The most commonly used of these products is the vaccine used for immunization against Mycobacterium tuberculosis, which is called Bacille Calmette Guerin (lBCG). The active fraction of BCG, called muramyl dipeptide, has also been used as a cell activator, as have certain cytokines (e.g.,IFN-y). e. Bifunctional monoclonal antibodies. Although most antibodies have two identical antigen-combining sites, it is possible to create antibodies in the laboratory that have two different antigen-combining sites. An antibody containing one antigen-combining site for tumor antigen and another for the CD3 antigen will focus T cells around the tumor cell. In addition, anti-CD3 can trigger T-cell activation without the need for engagement of the TCR with antigenic peptide in the groove of MHC molecules.

482

meClical