Chapter 7: Pathogenesis

A microorganism is a pathogen if it is capable of causing disease; however, some organisms are highly pathogenic (i.e., they often cause disease), whereas others cause disease rarely. Opportunistic pathogens are those that rarely, if ever, cause disease in immunocompetent people but can cause serious infection in patients with reduced host defenses (immunocompromised) and, as discussed in Chapter 6, are frequent members of the body’s normal flora.

Virulence is a quantitative measure of pathogenicity and is measured by the number of organisms required to cause disease. The 50% lethal dose (LD₅₀) is the number of organisms needed to kill half of the hosts that are exposed to the pathogen, while the 50% infectious dose (ID₅₀) is the number needed to cause infection in half of the exposed hosts. Organisms with a lower LD₅₀ (or ID₅₀) are said to be more virulent than those with a higher LD₅₀ (or ID₅₀) because fewer organisms are needed to cause death or disease.

The infectious dose of an organism required to cause disease varies greatly among the pathogenic bacteria. For example, Shigella and Salmonella both cause diarrhea by infecting the gastrointestinal tract, but the infectious dose of Shigella is less than 100 organisms, whereas the infectious dose of Salmonella is on the order of 100,000 organisms. The infectious dose of bacteria depends primarily on their virulence factors (e.g., whether their pili allow them to adhere well to mucous membranes, whether they produce exotoxins or endotoxins, whether they possess a capsule to protect them from phagocytosis, and whether they can survive various nonspecific host defenses such as acid in the stomach).

There are two uses of the word parasite. Within the context of this chapter, the term refers to the parasitic relationship of the bacteria to the host cells (i.e., the presence of the bacteria is detrimental to the host cells). Bacteria that are human pathogens can be thought of, therefore, as parasites. Some bacterial pathogens are obligate intracellular parasites (e.g., Chlamydia and Rickettsia), because they can grow only within host cells. Many bacteria are facultative parasites because they can grow within cells, outside cells, or on bacteriologic media. The other use of the term parasite refers to the protozoa and the helminths, which are discussed in Part VI of this book.

People get infectious diseases when microorganisms overpower our host defenses (i.e., when the balance between the organism and the host shifts in favor of the organism). The organism or its products are then present in sufficient amount to induce various symptoms, such as fever and inflammation, which we interpret as those of an infectious disease.

From the organism’s perspective, the two critical determinants in overpowering the host are the number of organisms to which a person is exposed and the virulence of these organisms. Clearly, the greater the number of organisms, the greater is the likelihood of infection. It is important to realize, however, that a small number of highly virulent organisms can cause disease just as a large number of less virulent organisms can. The virulence of an organism is determined by its ability to produce various virulence factors, several of which were described previously.

The production of specific virulence factors also determines what disease the bacteria cause. For example, a strain of Escherichia coli that produces one type of exotoxin causes watery (nonbloody) diarrhea, whereas a different strain of E. coli that produces another type of exotoxin causes bloody diarrhea. This chapter describes several important examples of specific diseases related to the production of various virulence factors.

From the host’s perspective, the two main arms of our host defenses are innate immunity and acquired immunity, the latter of which includes both antibody-mediated and cell-mediated immunity. A reduction in the functioning of any component of our host defenses shifts the balance in favor of the organism and increases the chance that an infectious disease will occur. Some important causes of a reduction in our host defenses include genetic immunodeficiencies such as agammaglobulinemia and acquired immunodeficiencies such as acquired immunodeficiency syndrome (AIDS), drug-induced immunosuppression in patients with organ transplants, and cancer patients who are receiving chemotherapy. Patients with diabetes and autoimmune diseases also may have reduced host defenses. An overview of our host defenses is presented in Chapters 8 and 57.

In many instances, a person acquires an organism, but no infectious disease occurs because the host defenses were successful. Such asymptomatic infections are common and are typically recognized by detecting antibody against the organism in the patient’s serum.

The term infection has more than one meaning. One meaning is that an organism has infected the person (i.e., it has entered the body of that person). For example, a person can be infected with an organism of low pathogenicity and not develop symptoms of disease. Another meaning of the term infection is to describe an infectious disease, such as when a person says, “I have an infection.” In this instance, infection and disease are being used interchangeably, but it is important to realize that according to the first definition, the word infection does not have to be equated with disease. Usually, the meaning will be apparent from the context.

Bacteria cause disease by two major mechanisms: (1) toxin production and (2) invasion and inflammation. Toxins fall into two general categories: exotoxins and endotoxins. Exotoxins are proteins secreted by the cell, whereas endotoxins are lipopolysaccharides (LPS) that form an integral part of the cell wall of gram-negative bacteria. Endotoxins are not actively released from the cell and cause fever, shock, and other generalized symptoms. Both exotoxins and endotoxins by themselves can cause symptoms; the presence of the bacteria in the host is not required. Invasive bacteria, on the other hand, grow to large numbers locally and induce an inflammatory response consisting of erythema, edema, warmth, and pain. Invasion and inflammation are discussed later in the section entitled “Determinants of Bacterial Pathogenesis.”

Many, but not all, infections are communicable or contagious (i.e., they are spread from host to host). For example, tuberculosis is communicable (i.e., it is spread from person to person via airborne droplets produced by coughing), but botulism is not, because the exotoxin produced by the organism in the contaminated food affects only those eating that food.

An infection is epidemic if it occurs much more frequently than usual; it is pandemic if it has a worldwide distribution. An endemic infection is constantly present at a low level in a specific population. In addition to infections that result in overt symptoms, many are inapparent or subclinical and can be detected only by demonstrating a rise in antibody titer or by isolating the organism. Some infections result in a latent state, after which reactivation of the growth of the organism and recurrence of symptoms may occur. Certain other infections lead to a chronic carrier state, in which the organisms continuously grow with or without producing symptoms in the host. Chronic carriers (e.g., “Typhoid Mary”) are an important source of infection of others and hence are a public health hazard.

The determination of whether an organism recovered from a patient is actually the cause of the disease involves an awareness of two phenomena: normal flora and colonization. Members of the normal flora are permanent residents of the body and vary in type according to anatomic site (see Chapter 6). When an organism is obtained from a patient’s specimen, the question of whether it is a member of the normal flora is important in interpreting the finding. Colonization refers to the presence of a new organism that is neither a member of the normal flora nor the cause of symptoms. It can be a difficult clinical dilemma to distinguish between a pathogen and a colonizer, especially in specimens obtained from the respiratory tract, such as throat cultures and sputum cultures.

Most bacterial infections are acquired from an external source. However, some bacterial infections are caused by members of the normal flora and, as such, are not transmitted directly prior to the onset of infection.

A generalized sequence of the stages of infection is as follows:

1. Transmission from an external source into the portal of entry.

2. Evasion of primary host defenses such as skin or stomach acid.

3. Adherence to mucous membranes, usually by bacterial pili.

4. Colonization by growth of the bacteria at the site of adherence.

5. Disease symptoms caused by toxin production or invasion accompanied by inflammation.

6. Host responses, both nonspecific and specific (immunity), during steps 3, 4, and 5.

7. Progression or resolution of the disease.

1. Transmission

An understanding of the mode of transmission of bacteria and other infectious agents is extremely important from a public health perspective, because interrupting the chain of transmission is an excellent way to prevent infectious diseases. The mode of transmission of many infectious diseases is “human-to-human,” but infectious diseases are also transmitted from nonhuman sources such as soil, water, and animals. Fomites are inanimate objects, such as towels, that serve as a source of microorganisms that can cause infectious diseases. Table 7–1 describes some important examples of these modes of transmission.

Although some infections are caused by members of the normal flora, most are acquired by transmission from external sources. Pathogens exit the infected patient most frequently from the respiratory and gastrointestinal tracts; hence, transmission to the new host usually occurs via airborne respiratory droplets or fecal contamination of food and water. Organisms can also be transmitted by sexual contact, urine, skin contact, blood transfusions, contaminated needles, or biting insects. The transfer of blood, either by transfusion or by sharing needles during intravenous drug use, can transmit various bacterial and viral pathogens. The screening of donated blood for Treponema pallidum, human immunodeficiency virus (HIV), human T-cell lymphotropic virus, hepatitis B virus, hepatitis C virus, and West Nile virus has greatly reduced the risk of infection by these organisms.

The major bacterial diseases transmitted by ticks in the United States are Lyme disease, Rocky Mountain spotted fever, ehrlichiosis, relapsing fever, and tularemia. Of these five diseases, Lyme disease is by far the most common. Ticks of the genus Ixodes (deer tick) transmit three infectious diseases: Lyme disease, ehrlichiosis, and babesiosis, a protozoan disease. Dermacentor ticks (dog tick) transmit several diseases: Rocky Mountain spotted fever, tularemia, ehrlichiosis, anaplasmosis, and tick paralysis.

Bacteria, viruses, and other microbes can also be transmitted from mother to offspring, a process called vertical transmission. The three modes by which organisms are transmitted vertically are across the placenta, within the birth canal during birth, and via breast milk. Table 7–2 describes some medically important organisms that are transmitted vertically. (Horizontal transmission, by contrast, is person-to-person transmission that is not from mother to offspring.)

There are four important portals of entry: respiratory tract, gastrointestinal tract, genital tract, and skin (Table 7–3). Important microorganisms and diseases transmitted by water are described in Table 7–4.

The important bacterial diseases transmitted by foods are listed in Table 7–5, and those transmitted by insects are listed in Table 7–6. The specific mode of transmission of each organism is described in the subsequent section devoted to that organism.

Animals are also an important source of organisms that infect humans. They can be either the source (reservoir) or the mode of transmission (vector) of certain organisms. Diseases for which animals are the reservoirs are called zoonoses. The important zoonotic diseases caused by bacteria are listed in Table 7–7.

2. Adherence to Cell Surfaces

Certain bacteria have specialized structures (e.g., pili) or produce substances (e.g., capsules or glycocalyces) that allow them to adhere to the surface of human cells, thereby enhancing their ability to cause disease. These adherence mechanisms are essential for organisms that attach to mucous membranes; mutants that lack these mechanisms are often nonpathogenic. For example, the pili of Neisseria gonorrhoeae and E. coli mediate the attachment of the organisms to the urinary tract epithelium, and the glycocalyx of Staphylococcus epidermidis and certain viridans streptococci allows the organisms to adhere strongly to the endothelium of heart valves. The various molecules that mediate adherence to cell surfaces are called adhesins.

After the bacteria attach, they often form a protective matrix called a biofilm consisting of various polysaccharides and proteins. Biofilms form especially on foreign bodies such as prosthetic joints, prosthetic heart valves, and intravenous catheters, but they also form on native structures such as heart valves. Biofilms protect bacteria from both antibiotics and host immune defenses such as antibodies and neutrophils. They also retard wound healing, resulting in chronic wound infections, especially in diabetics. Biofilms play an important role in the persistence of Pseudomonas in the lungs of cystic fibrosis patients and in the formation of dental plaque, the precursor of dental caries.

The production of biofilms by bacteria such as Pseudomonas is controlled by the process of quorum sensing, which allows bacteria to coordinate the synthesis of particular proteins according to the density of the bacterial population. When the concentration of bacteria is low, these proteins are not expressed; but once the population reaches a critical high cell density, the individual members sense this and begin to synthesize these proteins, resulting in phenotypic changes that benefit the population as a whole. Examples of behaviors that are controlled by quorum sensing include biofilm formation, expression of virulence, and antibiotic resistance, all of which can contribute to pathogenesis.

Foreign bodies, such as artificial heart valves and artificial joints, predispose to infections. Bacteria can adhere to these surfaces, but phagocytes adhere poorly owing to the absence of selectins and other binding proteins on the artificial surface (see Chapter 8).

3. Invasion, Inflammation, & Intracellular Survival

One of the two main mechanisms by which bacteria cause disease is invasion of tissue followed by inflammation. (The inflammatory response is described in Chapter 8.) The other main mechanism, toxin production, and a third mechanism, immunopathogenesis, are described later in this chapter.

Several enzymes secreted by invasive bacteria play a role in pathogenesis. Among the most prominent are the following:

1. Collagenase and hyaluronidase, which degrade collagen and hyaluronic acid, respectively, thereby allowing the bacteria to spread through subcutaneous tissue; they are especially important in cellulitis caused by Streptococcus pyogenes.

2. Coagulase, which is produced by Staphylococcus aureus and accelerates the formation of a fibrin clot from its precursor, fibrinogen (this clot may protect the bacteria from phagocytosis by walling off the infected area and by coating the organisms with a layer of fibrin). Coagulase is also produced by Yersinia pestis, the cause of bubonic plague. See Chapter 20 for the role of coagulase in the pathogenesis of plague.

3. Immunoglobulin proteases. There are several examples of organisms that produce enzymes that degrade immunoglobulin (Ig) A and IgG. N. gonorrhoeae, Haemophilus influenzae, and Streptococcus pneumoniae produce IgA proteases, which inactivate this immunoglobulin at the mucosal surface. This leads to better adherence of these organisms to mucous membranes. S. pyogenes produces an enzyme that specifically cleaves IgG heavy chains, which reduces opsonization and complement activation, enhancing the virulence of this organism.

In addition to these enzymes, several virulence factors contribute to invasiveness by limiting the ability of the host defense mechanisms, especially phagocytosis, to operate effectively.

1. The most important of these antiphagocytic factors is the capsule external to the cell wall of several important pathogens such as S. pneumoniae and Neisseria meningitidis. The polysaccharide capsule prevents the phagocyte from adhering to the bacteria; anticapsular antibodies allow more effective phagocytosis to occur (a process called opsonization) (see Chapter 8). The vaccines against S. pneumoniae, H. influenzae, and N. meningitidis contain capsular polysaccharides that induce protective anticapsular antibodies.

2. A second group of antiphagocytic factors are the cell wall proteins of the gram-positive cocci, such as the M protein of the group A streptococci (S. pyogenes) and protein A of S. aureus. The M protein is antiphagocytic, and protein A binds to the Fc portion of IgG and prevents the activation of complement. These virulence factors are summarized in Table 7–8.

3. Leukocidins are pore-forming toxins that degrade the cell membrane of neutrophils and macrophages. The Panton-Valentine leukocidin produced by S. aureus is a good example.

Bacteria can cause two types of inflammation: pyogenic and granulomatous. In pyogenic (pus-producing) inflammation, neutrophils are the predominant cells. Some of the most important pyogenic bacteria are the gram-positive and gram-negative cocci listed in Table 7–8. In granulomatous inflammation, macrophages and helper T cells predominate. The most important organism in this category is Mycobacterium tuberculosis. No bacterial enzymes or toxins that induce granulomas have been identified. Rather, it appears that bacterial antigens stimulate the cell-mediated immune system, resulting in sensitized T-lymphocyte and macrophage activity. Phagocytosis by macrophages kills most of the bacteria, but some survive and grow within the macrophages in the granuloma.

Intracellular survival is an important attribute of certain bacteria that enhances their ability to cause disease. These bacteria are called “intracellular” pathogens and commonly cause granulomatous lesions. The best-known of these bacteria belong to the genera Mycobacterium, Legionella, Brucella, and Listeria. The best-known fungus is Histoplasma. These organisms can be cultured on microbiologic media in the laboratory and therefore are not obligate intracellular parasites, which distinguishes them from Chlamydia and Rickettsia. The intracellular location provides a protective niche from antibody and neutrophils that function extracellularly.

Intracellular bacteria use several different mechanisms to allow them to survive and grow inside cells. These include (1) inhibition of the fusion of the phagosome with the lysosome, which allows the organisms to avoid the degradative enzymes in the lysosome; (2) inhibition of acidification of the phagosome, which reduces the activity of the lysosomal degradative enzymes; and (3) escape from the phagosome into the cytoplasm, where there are no degradative enzymes. Members of the genera Mycobacterium and Legionella are known to use the first and second mechanisms, whereas Listeria species use the third.

The invasion of cells by bacteria is dependent on the interaction of specific bacterial surface proteins called invasins and specific cellular receptors belonging to the integrin family of transmembrane adhesion proteins. The movement of bacteria into the cell is a function of actin microfilaments. Once inside the cell, these bacteria typically reside within cell vacuoles such as phagosomes. Some remain there, others migrate into the cytoplasm, and some move from the cytoplasm into adjacent cells. Infection of the surrounding cells in this manner allows the bacteria to evade host defenses. For example, Listeria monocytogenes aggregates actin filaments on its surface and is propelled in a “sling-shot” fashion, called actin rockets, from one host cell to another.

The “Yops” (Yersinia outer-membrane proteins) produced by several Yersinia species are important examples of bacterial virulence factors that act primarily after invasion of human cells by the organism. The most important effects of the Yops are to inhibit phagocytosis by neutrophils and macrophages and to inhibit cytokine production (e.g., tumor necrosis factor [TNF] production) by macrophages. For example, one of the Yops of Y. pestis (Yop J) is a protease that cleaves signal transduction proteins required for the induction of TNF synthesis. This inhibits the activation of our host defenses and contributes to the ability of the organism to cause bubonic plague.

The genes that encode many virulence factors in bacteria are clustered in pathogenicity islands located on the bacterial chromosome or plasmids. For example, in many bacteria, the genes encoding adhesins, invasins, and exotoxins are adjacent to each other on these islands. Nonpathogenic variants of these bacteria do not have these pathogenicity islands. It appears that these large regions of the bacterial genome were transferred as a block via conjugation or transduction. Pathogenicity islands are found in many gram-negative rods, such as E. coli, Salmonella, Shigella, Pseudomonas, and Vibrio cholerae, and in gram-positive cocci, such as S. pneumoniae.

After bacteria have colonized and multiplied at the portal of entry, they may invade the bloodstream and spread to other parts of the body. Receptors for the bacteria on the surface of cells determine, in large part, the organs affected. For example, certain bacteria or viruses infect the brain because receptors for these microbes are located on the surface of brain neurons. The blood–brain barrier, which limits the ability of certain drugs to penetrate the brain, is not thought to be a determinant of microbial infection of the brain. The concept of a blood–brain barrier primarily refers to the inability of hydrophilic (charged, ionized) drugs to enter the lipid-rich brain parenchyma, whereas lipophilic (lipid-soluble) drugs enter well.

Two important diseases, diphtheria and pseudomembranous colitis, are characterized by inflammatory lesions called pseudomembranes. Pseudomembranes are thick, adherent, grayish or yellowish exudates on the mucosal surfaces of the throat in diphtheria and on the colon in pseudomembranous colitis. The term pseudo refers to the abnormal nature of these membranes in contrast to the normal anatomic membranes of the body, such as the tympanic membrane and the placental membranes.

4. Toxin Production

The second major mechanism by which bacteria cause disease is the production of toxins. A comparison of the main features of exotoxins and endotoxins is shown in Table 7–9.

Exotoxins

Exotoxins are produced by several gram-positive and gram-negative bacteria, in contrast to endotoxins, which are present only in gram-negative bacteria. The essential characteristic of exotoxins is that they are secreted by the bacteria, whereas endotoxin is a component of the cell wall. Exotoxins are polypeptides whose genes are frequently located on plasmids or lysogenic bacterial viruses (bacteriophages). Some important exotoxins encoded by bacteriophage DNA are diphtheria toxin, cholera toxin, and botulinum toxin.

Exotoxins are among the most toxic substances known. For example, the fatal dose of tetanus toxin for a human is estimated to be less than 1 μg. Because some purified exotoxins can reproduce all aspects of the disease, we can conclude that certain bacteria play no other role in pathogenesis than to synthesize the exotoxin. Exotoxin polypeptides are good antigens and induce the synthesis of protective antibodies called antitoxins, some of which are useful in the prevention or treatment of diseases such as botulism and tetanus. When treated with formaldehyde (or acid or heat), the exotoxin polypeptides are converted into toxoids, which are used in protective vaccines because they retain their antigenicity but have lost their toxicity.

Many exotoxins have an A–B subunit structure; the A (or active) subunit possesses the toxic activity, and the B (or binding) subunit is responsible for binding the exotoxin to specific receptors on the membrane of the human cell. The binding of the B subunit determines the specific site of the action of the exotoxin. For example, botulinum toxin acts at the neuromuscular junction because the B subunit binds to specific receptors on the surface of the motor neuron at the junction. Important exotoxins that have an A–B subunit structure include diphtheria toxin, tetanus toxin, botulinum toxin, cholera toxin, and the enterotoxin of E. coli (Figure 7–1).

The A subunit of several important exotoxins acts by catalyzing the addition of adenosine diphosphate ribose (ADP-ribose) to the target protein in the human cell (ADP-ribosylation). The modification of target proteins with ADP-ribose often inactivates it but can also hyperactivate it, either of which can cause the symptoms of disease. For example, diphtheria toxin and Pseudomonas exotoxin A ADP-ribosylate elongation factor-2 (EF-2), an essential factor required for eukaryotic protein synthesis. This modification inactivates EF-2, freezing the translocation complex, and results in the inhibition of protein synthesis.

On the other hand, cholera toxin and E. coli toxin ADP-ribosylate G_s protein, thereby activating it. This causes an increase in adenylate cyclase activity, a consequent increase in the amount of cyclic adenosine monophosphate (AMP), and the production of watery diarrhea. Pertussis toxin is an interesting variation on the theme. It ADP-ribosylates G_i protein and inactivates it. Inactivation of the inhibitory G proteins turns on adenylate cyclase, causing an increase in the amount of cyclic AMP, which plays a role in causing the symptoms of whooping cough.

Exotoxins are released from bacteria by specialized structures called secretion systems. Some secretion systems transport the exotoxins into the extracellular space, but others transport the exotoxins directly into the mammalian cell. Those that transport the exotoxins directly into the mammalian cell are especially effective because the exotoxin is not exposed to antibodies in the extracellular space.

Several classes of bacterial secretion systems (six and counting) have been identified, but the type III secretion system (also called an injectosome) is particularly important in virulence. This secretion system is mediated by a needlelike projection (sometimes called a “molecular syringe”) and by transport pumps in the bacterial cell membrane. The importance of the type III secretion system is illustrated by the finding that the strains of Pseudomonas aeruginosa that have this secretion system are significantly more virulent than those that do not. Other medically important gram-negative rods that utilize injectosomes include Shigella species, Salmonella species, E. coli, and Y. pestis.

The mechanisms of action of the important exotoxins produced by toxigenic bacteria are described in the following discussion and summarized in Tables 7–10, 7–11, and 7–12. The main location of symptoms of disease caused by bacterial exotoxins is described in Table 7–13.

Gram-Positive Bacteria

The exotoxins produced by gram-positive bacteria have several different mechanisms of action and produce different clinical effects. Some important exotoxins include diphtheria toxin, which inhibits protein synthesis by inactivating EF-2; tetanus toxin and botulinum toxin, which are neurotoxins that prevent the release of neurotransmitters; and toxic shock syndrome toxin (TSST), which acts as a superantigen causing the release of large amounts of cytokines from helper T cells and macrophages. The mechanisms of action and the clinical effects of exotoxins produced by gram-positive bacteria are described next.

1. Diphtheria toxin, produced by Corynebacterium diphtheriae, inhibits protein synthesis by ADP-ribosylation of EF-2 (see Figure 7–1).¹

   The resulting death of the affected cells leads to two prominent symptoms of diphtheria: pseudomembrane formation in the throat and myocarditis.

   The exotoxin activity depends on two functions mediated by different domains of the molecule. The toxin is synthesized as a single polypeptide that is nontoxic because the active site of the enzyme is masked. This molecule is cleaved and modified to yield two active polypeptides. Fragment A, derived from the amino-terminal end of the exotoxin, yields an enzyme that catalyzes the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD) to EF-2, inhibiting protein synthesis. Fragment B, derived from the carboxy-terminal end, binds to receptors on the outer membrane of eukaryotic cells and mediates transport of fragment A into the cells.

   As the bacteria synthesize and secrete the full-length exotoxin, the carboxy-terminal end binds to host cell membrane receptors. The toxin is transported across the cell membrane, triggering cleavage and modification that result in active fragment A, which then targets and inactivates EF-2. The specificity for this protein is due to a unique amino acid, a modified histidine called diphthamide, that is present only on EF-2. Since all eukaryotic cells carry out protein synthesis, there is no tissue or organ specificity. Prokaryotic and mitochondrial protein synthesis are not affected because a different, nonsusceptible elongation factor is involved. The enzyme activity is remarkably potent; a single molecule of fragment A will kill a cell within a few hours. Other organisms whose exotoxins act by ADP-ribosylation are E. coli, V. cholerae, and Bordetella pertussis.

   The tox gene, which codes for this exotoxin, is carried by a lysogenic bacteriophage called beta phage. As a result, only C. diphtheriae strains lysogenized by this phage cause diphtheria. (Nonlysogenized C. diphtheriae can be found in the throat of some healthy people.) This is an important example of lysogenic conversion, the process by which bacteria acquire new traits when lysogenized by a bacteriophage (see Chapter 4). Regulation of exotoxin synthesis is controlled by the interaction of iron in the medium with a tox gene repressor synthesized by the bacterium. As the concentration of iron increases, the iron-repressor complex inhibits the transcription of the tox gene.

2. Tetanus toxin, produced by Clostridium tetani, is a neurotoxin that prevents release of an inhibitory neurotransmitter involved in muscle relaxation. When the inhibitory neurons are nonfunctional, the excitatory neurons are unopposed, leading to muscle spasms and a spastic paralysis. Tetanus toxin (tetanospasmin) is composed of two polypeptide subunits encoded by plasmid DNA. The heavy chain of the polypeptide binds to gangliosides in the membrane of the neuron; the light chain is a protease that degrades the protein(s) responsible for the release of the inhibitory neurotransmitters (γ-aminobutyric acid [GABA] and glycine). The toxin released at the site of the peripheral wound may travel either by retrograde axonal transport or in the bloodstream to the anterior horn and interstitial neurons of the spinal cord. Inhibiting the release of the GABA and glycine leads to convulsive contractions of the voluntary muscles, best exemplified by spasm of the jaw and neck muscles (“lockjaw”).

3. Botulinum toxin, produced by Clostridium botulinum, is a neurotoxin that blocks the release of a different neurotransmitter, acetylcholine, at the synapse of the neuromuscular junction, producing a flaccid paralysis. Approximately 1 μg is lethal for humans; it is one of the most toxic compounds known. The toxin is composed of two polypeptide subunits held together by disulfide bonds. One of the subunits binds to a receptor on the neuron; the other subunit is a protease that degrades the protein(s) responsible for the release of acetylcholine. There are six serotypes of botulinum toxin (A–F), with toxins A, B, E, and F being the most important for human disease. Some serotypes are encoded on a plasmid, some on a temperate bacteriophage, and some on the bacterial chromosome.

4. Two exotoxins are produced by Clostridium difficile, both of which are involved in the pathogenesis of pseudomembranous colitis. Exotoxin A is an enterotoxin that causes watery diarrhea. Exotoxin B is a cytotoxin that damages the colonic mucosa and causes pseudomembranes to form. Exotoxins A and B are glucosyltransferases that modify target signal transduction proteins (Rho GTPases), which interferes with their signal transduction function. Glucosylation by exotoxin B causes disaggregation of actin filaments in the cytoskeleton, leading to apoptosis and cell death.

5. Multiple toxins are produced by Clostridium perfringens and other species of clostridia that cause gas gangrene. A total of seven lethal factors and five enzymes have been characterized, but no species of Clostridium makes all 12 products. The best characterized is the alpha toxin, which is a lecithinase that hydrolyzes lecithin in the cell membrane, resulting in destruction of the membrane and widespread cell death. The other four enzymes are collagenase, protease, hyaluronidase, and deoxyribonuclease (DNase). The seven lethal toxins are a heterogeneous group with hemolytic and necrotizing activity. Certain strains of C. perfringens produce an enterotoxin that causes watery diarrhea. This enterotoxin acts as a superantigen similar to the enterotoxin of S. aureus (described below).

6. Three exotoxins are produced by Bacillus anthracis, the agent of anthrax: edema factor, lethal factor, and protective antigen. The three exotoxins associate with each other, but each component has a distinct function. Edema factor is an adenylate cyclase that raises the cyclic AMP concentration within the cell, resulting in loss of chloride ions and water and consequent edema formation in the tissue (see Table 7–12). Lethal factor is a protease that cleaves a phosphokinase required for the signal transduction pathway that controls cell growth. Loss of the phosphokinase results in the failure of cell growth and consequent cell death. Protective antigen binds to a cell surface receptor and forms pores in the human cell membrane that allow edema factor and lethal factor to enter the cell. The name protective antigen is based on the finding that antibody against this protein protects against disease. The antibody blocks the binding of protective antigen, thereby preventing edema factor and lethal factor from entering the cell.

7. TSST is a superantigen produced primarily by certain strains of S. aureus but also by certain strains of S. pyogenes. TSST binds directly to class II major histocompatibility (MHC) proteins on the surface of antigen-presenting cells (macrophages) without intracellular processing. This complex interacts with the T-cell receptor of many helper T cells, resulting in activation of these T cells (see the discussion of superantigens in Chapter 58). This causes the release of large amounts of interleukins, especially interleukin-1, interleukin-2, and TNF. These cytokines produce many of the signs and symptoms of toxic shock.

8. Staphylococcal enterotoxin is also a superantigen, but because it is ingested, it acts locally on the lymphoid cells lining the small intestine. The enterotoxin is produced by S. aureus in the contaminated food and causes food poisoning, usually within 1–6 hours after ingestion. The main symptoms are vomiting and watery diarrhea. The prominent vomiting seen in food poisoning is caused by cytokines released from the lymphoid cells stimulating the enteric nervous system, which activates the vomiting center in the brain.

9. Exfoliatin is a protease produced by S. aureus that causes scalded skin syndrome. Exfoliatin cleaves desmoglein, a protein in the desmosomes of the skin, resulting in the detachment of the superficial layers of the skin. Exfoliatin is also called epidermolytic toxin.

10. Panton-Valentine (PV) leukocidin is a pore-forming exotoxin produced by methicillin-resistant strains of S. aureus (MRSA). It destroys white blood cells, skin, and subcutaneous tissue. The two subunits of the toxin assemble in the cell membrane to form a pore through which cell contents exit into the extracellular space.

11. Erythrogenic toxin, produced by S. pyogenes, causes the rash characteristic of scarlet fever. Its mechanism of action is similar to that of TSST (i.e., it acts as a superantigen). The DNA that codes for the toxin resides on a lysogenic bacteriophage. Nonlysogenic bacteria do not cause scarlet fever, although they can cause pharyngitis.

12. Exotoxin B is a protease produced by strains of S. pyogenes that cause necrotizing fasciitis. These strains are called “flesh-eating” streptococci.

Gram-Negative Bacteria

The exotoxins produced by gram-negative bacteria also have several different mechanisms of action and produce different clinical effects. Two very important exotoxins are the enterotoxins of E. coli and V. cholerae (cholera toxin), which induce an increase in the amount of cyclic AMP within the enterocyte, resulting in watery diarrhea (see Table 7–12). The mechanisms of action and the clinical effects of exotoxins produced by gram-negative bacteria are described next.

1. The heat-labile enterotoxin produced by E. coli causes watery, nonbloody diarrhea by stimulating adenylate cyclase activity in cells in the small intestine (Figure 7–2). The resulting increase in the concentration of cyclic AMP causes excretion of the chloride ion, inhibition of sodium ion absorption, and significant fluid and electrolyte loss into the lumen of the gut. The heat-labile toxin, which is inactivated at 65°C for 30 minutes, is an AB toxin. The B subunit confers specificity to the enterocytes in the small intestine by binding to a ganglioside receptor in the cell membrane. This enables the A subunit to enter the cell where it ADP-ribosylates its target G_s protein. This locks the G_s protein in the “on” position, which constitutively stimulates adenylate cyclase to synthesize cyclic AMP. This in turn activates cyclic AMP–dependent protein kinase, an enzyme that phosphorylates ion transporters in the cell membrane, resulting in the loss of water and ions from the cell. Most of the genes for the heat-labile toxin and for the heat-stable toxin (described next) are carried on plasmids.

   In addition to the labile toxin, there is a heat-stable toxin, which is a polypeptide that is not inactivated by boiling for 30 minutes. The heat-stable toxin affects cyclic guanosine monophosphate (GMP) rather than cyclic AMP. It stimulates guanylate cyclase and thus increases the concentration of cyclic GMP, which inhibits the reabsorption of sodium ions and causes diarrhea.

2. Shiga toxin is an exotoxin produced primarily by strains of E. coli with the O157:H7 serotype. These enterohemorrhagic strains cause bloody diarrhea and are the cause of outbreaks associated with eating undercooked meat, especially hamburger in fast-food restaurants. The toxin is named for a very similar toxin produced by Shigella dysenteriae. The toxin is a glycosidase that inactivates protein synthesis by removing adenine from a specific site on the 28S rRNA in the large subunit of the human ribosome.

   Shiga toxin is encoded by a temperate (lysogenic) bacteriophage. When E. coli Shiga toxin enters the bloodstream, it can cause hemolytic-uremic syndrome (HUS). Shiga toxin binds to receptors on the glomerulus of the kidney and on the endothelium of small blood vessels. Inhibition of protein synthesis results in death of vascular epithelial cells, leading to renal failure and microangiopathic hemolytic anemia. Certain antibiotics, such as ciprofloxacin, can increase the amounts of Shiga toxin produced by E. coli O157, which predisposes to HUS.

3. The AB enterotoxins produced by V. cholerae, the agent of cholera (see Chapter 18), and Bacillus cereus, a cause of diarrhea, act in a manner similar to that of the heat-labile toxin of E. coli (see Figure 7–2).

4. Pertussis toxin, produced by B. pertussis, the cause of whooping cough, is an exotoxin that catalyzes the transfer of ADP-ribose from NAD to an inhibitory G protein. Inactivation of this inhibitory regulator has two effects: one is to stimulate adenylate cyclase activity, leading to an increase in cyclic AMP concentration within the affected cells (see Table 7–12). This results in edema and other changes in the respiratory tract, leading to the cough of whooping cough. It also inhibits the signal transduction pathway used by chemokine receptors. This causes the marked lymphocytosis seen in patients with pertussis. The toxin inhibits signal transduction by all chemokine receptors, resulting in an inability of lymphocytes to migrate to and enter lymphoid tissue (spleen, lymph nodes). Because they do not enter tissue, there is an increase in their number in the blood (see the discussion of chemokines in Chapter 58).

Endotoxins

Endotoxins are integral parts of the cell walls of both gram-negative rods and cocci, in contrast to exotoxins, which are actively released from the cell (see Table 7–9). In addition, endotoxins are lipopolysaccharides, whereas exotoxins are polypeptides; the enzymes that produce the lipopolysaccharides are encoded by genes on the bacterial chromosome, rather than by plasmid or bacteriophage DNA, which usually encodes the exotoxins. The toxicity of endotoxins is low in comparison with that of exotoxins. All endotoxins produce the same generalized effects of fever and shock, although the endotoxins of some organisms are more effective than those of others (Figure 7–3). Endotoxins are weakly antigenic; they induce protective antibodies so poorly that multiple episodes of toxicity can occur. No toxoids have been produced from endotoxins, and endotoxins are not used as antigens in any available vaccine.

A major site of action of endotoxin is the macrophage. Endotoxins (LPS) are released from the surface of gram-negative bacteria in small pieces of outer membrane that bind to LPS-binding protein in the plasma. This complex binds to a receptor on the surface of macrophages called CD14, which activates toll-like receptor-4 (TLR-4). A signal cascade within the macrophage is then activated, resulting in the synthesis of cytokines such as interleukin-1 (IL-1), TNF, and nitric oxide (see later and Figure 7–3).

The findings of fever and hypotension are salient features of septic shock. Additional features include tachycardia, tachypnea, and leukocytosis (increased white blood cells, especially neutrophils, in the blood). Septic shock is one of the leading causes of death in intensive care units and has an estimated mortality rate of 30% to 50%. The endotoxins of gram-negative bacteria are the best-established causes of septic shock, but surface molecules of gram-positive bacteria (which do not have endotoxins) can also cause septic shock.

Two features of septic shock are interesting:

1. Septic shock is different from toxic shock. In septic shock, the bacteria are in the bloodstream, whereas in toxic shock, it is the toxin that is circulating in the blood. The clinical importance of this observation is that in septic shock, blood cultures are usually positive, whereas in toxic shock, they are usually negative.

2. Septic shock can cause the death of a patient even though antibiotics have killed the bacteria in the patient’s blood (i.e., the blood cultures have become negative). This occurs because septic shock is mediated by cytokines, such as TNF and IL-1, which continue to act even though the bacteria that induced the cytokines are no longer present.

The structure of the LPS is shown in Figure 2–6. The toxic portion of the molecule is lipid A, which contains several fatty acids. β-Hydroxymyristic acid is always one of the fatty acids and is found only in lipid A. The other fatty acids differ according to species. The polysaccharide core in the middle of the molecule protrudes from the surface of the bacteria and has the same chemical composition within members of a genus.

The somatic (O) antigen is a polysaccharide on the exterior that differs in each species and frequently differs between strains of a single species. It is an important antigen of some gram-negative bacteria and is composed of 3, 4, or 5 sugars repeated up to 25 times. Because the number of permutations of this array is very large, many antigenic types exist. For example, more than 1500 antigenic types have been identified for Salmonella based on different sugars in the O antigen. Some bacteria, especially N. meningitidis and N. gonorrhoeae, have lipooligosaccharide (LOS) containing very few repeating sugar subunits in the O antigen.

The biologic effects of endotoxin (Table 7–14) include the following:

1. Fever due to the release by macrophages of IL-1 (endogenous pyrogen) and IL-6, which act on the hypothalamic temperature-regulatory center.

2. Hypotension, shock, and impaired perfusion of essential organs due to nitric oxide–induced vasodilation, TNF-induced increased capillary permeability, bradykinin-induced vasodilation, and increased capillary permeability.

3. Disseminated intravascular coagulation (DIC) due to activation of the coagulation cascade, resulting in thrombosis, a petechial or purpuric rash, and tissue ischemia, leading to failure of vital organs. The coagulation cascade is activated when tissue factor is released from the surface of endothelial cells damaged by infection. Tissue factor interacts with circulating coagulation factors to cause widespread clotting within capillaries. A positive D-dimer test provides laboratory evidence for a diagnosis of DIC.

4. Activation of the alternative pathway of the complement cascade, resulting in inflammation and tissue damage. C5a is potent chemokine that attracts neutrophils to the site of infection.

5. Activation of macrophages, increasing their phagocytic ability, and activation of many clones of B lymphocytes, increasing antibody production. (Endotoxin is a polyclonal activator of B cells, but not T cells.)

The end result of the above five processes is called the systemic inflammatory response syndrome, or SIRS. The most common clinical signs of SIRS are fever, hypotension, tachycardia, tachypnea, and leukocytosis.

Damage to the vascular endothelium plays a major role in both the hypotension and DIC seen in septic shock. Damage to the endothelium allows the leakage of plasma and red cells into the tissue, resulting in the loss of blood volume and consequent hypotension. Damaged endothelium also serves as a site of platelet aggregation and activation that leads to the thousands of endovascular clots manifesting as DIC.

The evidence that endotoxin causes these effects comes from the following two findings: (1) purified LPS, free of the organism, reproduces the effects, and (2) antiserum against endotoxin can mitigate or block these effects.

Clinically, the presence of DIC in the patient can be assessed by the D-dimer laboratory test. D-Dimers are cleavage products of fibrin (fibrin split products) that are detected in the blood of patients with DIC.

Endotoxins do not cause these effects directly. Rather, they elicit the production of cytokines such as IL-1 and TNF from macrophages.² TNF is the central mediator because purified recombinant TNF reproduces the effects of endotoxin and antibody against TNF blocks the effects of the endotoxin. Endotoxin also induces macrophage migration inhibitory factor, which also plays a role in the induction of septic shock.

Note that TNF in small amounts has beneficial effects (e.g., causing an inflammatory response to the presence of a microbe), but in large amounts, it has detrimental effects (e.g., causing septic shock and DIC). It is interesting that the activation of platelets, which results in clot formation and the walling off of infections, is the same process that, when magnified, causes DIC and the necrosis of tumors. It is the ability of TNF to activate platelets that causes intravascular clotting and the consequent infarction and death of the tumor tissue. The symptoms of certain autoimmune diseases such as rheumatoid arthritis are also mediated by TNF; however, these symptoms are not induced by endotoxin but by other mechanisms, which are described in Chapter 66. Some of the important beneficial and harmful effects of TNF are listed in Table 7–15.

Endotoxins can cause fever in the patient if they are present in intravenous fluids. In the past, intravenous fluids were sterilized by autoclaving, which killed any organisms present but resulted in the release of endotoxins that were not heat inactivated. For this reason, these fluids are now sterilized by filtration, which physically removes the organism without releasing its endotoxin. The contamination of intravenous fluids by endotoxin is detected by a test based on the observation that nanogram amounts of endotoxin can clot extracts of the horseshoe crab, Limulus.

Endotoxin-like pathophysiologic effects can occur in gram-positive bacteremic infections (e.g., S. aureus and S. pyogenes infections) as well. Since endotoxin is absent in these organisms, a different cell wall component—namely, lipoteichoic acid—causes the release of TNF and IL-1 from macrophages.

Endotoxin-mediated septic shock is a leading cause of death, especially in hospitals. The results of attempts to treat septic shock with hemoperfusion to adsorb endotoxin or by administering antibodies specific to lipid A and TNF have been mixed. Treatment with activated protein C (drotrecogin-alfa, Xigris) was initially thought to be effective, but adverse effects, such as bleeding, and controversy regarding its effectiveness caused Xigris to be withdrawn from the market in 2011. Protein C is a normal human protein that functions as an anticoagulant by inhibiting thrombin formation. It also enhances fibrinolysis, which degrades clots once they are formed. These are the features of protein C that led it to be considered as a treatment for endotoxin-mediated septic shock.

5. Immunopathogenesis

In certain diseases, such as rheumatic fever and acute glomerulonephritis, it is not the organism itself that causes the symptoms of disease but the immune response to the presence of the organism. For example, in rheumatic fever, antibodies are formed against the M protein of S. pyogenes, which cross-react with joint, heart, and brain tissue. Inflammation occurs, resulting in the arthritis, carditis, and chorea that are the characteristic findings in this disease.

The fact that certain viruses can cause cancer is well established, but the observation that some bacterial infections are associated with cancers is just emerging. Several documented examples include (1) the association of Helicobacter pylori infection with gastric carcinoma and gastric mucosal-associated lymphoid tissue (MALT) lymphoma, and (2) the association of Campylobacter jejuni infection with MALT lymphoma of the small intestine (also known as alpha-chain disease). Support for the idea that these cancers are caused by bacteria comes from the observation that antibiotics can cause these cancers to regress if treated during an early stage.

S. aureus causes inflammatory, pyogenic diseases such as endocarditis, osteomyelitis, and septic arthritis, as well as nonpyogenic, exotoxin-mediated diseases such as toxic shock syndrome, scalded skin syndrome, and food poisoning. How do bacteria that belong to the same genus and species cause such widely divergent diseases? The answer is that individual bacteria produce different virulence factors that endow those bacteria with the capability to cause different diseases.

The different virulence factors are encoded on plasmids, on transposons, on the genome of temperate (lysogenic) phages, and on pathogenicity islands. These transferable genetic elements may or may not be present in any single bacterium, which accounts for the ability to cause different diseases. Table 7–16 describes the different virulence factors for three of the most important bacterial pathogens: S. aureus, S. pyogenes, and E. coli. Figure 7–4 describes the importance of pathogenicity islands in determining the types of diseases caused by E. coli.

A typical acute infectious disease has four main stages (Figure 7–5):

1. The incubation period, which is the time between the acquisition of the organism (or toxin) and the beginning of symptoms (this time varies from hours to days to weeks, depending on the organism).

2. The prodrome period, during which nonspecific symptoms such as fever, malaise, and loss of appetite occur.

3. The specific-disease period, during which the overt characteristic signs and symptoms of the disease occur.

4. The recovery period, also known as the convalescence period, during which the illness abates and the patient returns to the healthy state. IgG and IgA antibodies protect the recovered patient from reinfection by the same organism.

After the recovery period, some individuals become chronic carriers of the organisms and may shed them while remaining clinically well. Others may develop a latent infection, which can recur either in the same form as the primary infection or manifesting different signs and symptoms. Although many infections cause symptoms, many others are subclinical (i.e., the individual remains asymptomatic although infected with the organism). In subclinical infections and after the recovery period is over, the presence of antibodies is often used to determine that an infection has occurred.

Because people harbor microorganisms as members of the permanent normal flora and as transient passengers, this can be an interesting and sometimes confounding question. The answer depends on the situation. One type of situation relates to the problems of a disease for which no agent has been identified and a candidate organism has been isolated. This is the problem that Robert Koch faced in 1877 when he was among the first to try to determine the cause of an infectious disease, namely, anthrax in cattle and tuberculosis in humans. His approach led to the formulation of Koch’s postulates, which are criteria that he proposed must be satisfied to confirm the causal role of an organism. These criteria are as follows:

1. The organism must be isolated from every patient with the disease.

2. The organism must be isolated free from all other organisms and grown in pure culture in vitro.

3. The pure organism must cause the disease in a healthy, susceptible animal.

4. The organism must be recovered from the inoculated animal.

At the time, these principles provided a general but rigorous guideline for collecting evidence in support of identifying etiological agents of disease. We now recognize that there are infectious agents that do not fulfill all of Koch’s postulates, and proving disease causation rests on a constellation of observations. Exceptions to Koch’s postulates include the following:

1. Pathogens isolated from patients who are not manifesting symptoms (i.e., asymptomatic carriers) such as Salmonella typhi.

2. Pathogens that cannot be cultivated on existing media, such as T. pallidum (the causative agent of syphilis) or prions (infectious proteins that cause Creutzfeldt-Jakob disease).

3. Exposures to pathogens, for example, M. tuberculosis, that do not always result in disease in all hosts.

A second consideration for determining whether an isolated organism is actually responsible for a disease involves the diagnosis of a patient’s illness. In this instance, the signs and symptoms of the illness can often suggest a constellation of possible causative agents. The recovery of an agent in sufficient numbers from the appropriate specimen is usually sufficient for an etiologic diagnosis. This approach can be illustrated with two examples: (1) in a patient with a sore throat, the presence of a few β-hemolytic streptococci is insufficient for a microbiologic diagnosis, whereas the presence of many would be sufficient, and (2) in a patient with fever, α-hemolytic streptococci in the throat are considered part of the normal flora, whereas the same organisms in the blood are likely to be the cause of bacterial endocarditis.

In some infections, no organism is isolated from the patient, and the diagnosis is made by detecting a rise in antibody titer to an organism. For this purpose, the titer (amount) of antibody in the second or late serum sample should be at least four times the titer (amount) of antibody in the first or early serum sample.