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 1.
Table1. Main Features of Exotoxins and Endotoxins
Exotoxins
Exotoxins are produced by several gram-positive and gram-negative bacteria, in contrast to endotoxins, which are present only in gram-negative bacteria as a component of the cell wall. The essential characteristic of exotoxins is that they are secreted polypeptides whose genes are frequently located on plasmids or lysogenic 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, a fatal dose of tetanus toxin for a human is estimated to be less than 1 μg. Because some purified exotoxins can repro duce 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 lose 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, the B subunit of botulinum toxin binds to specific receptors on the surface of the motor neuron at the neuromuscular 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 1).
Fig1. Mode of action of diphtheria toxin. The toxin binds to the cell surface via its binding subunit, and the active subunit enters the cell. The active subunit is an enzyme that catalyzes the addition of ADP-ribose (ADP-R) to elongation factor-2 (EF-2). This inactivates EF-2, and protein synthesis is inhibited.
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). Target protein modification 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 and death of the cell.
On the other hand, cholera toxin and E. coli toxin ADP-ribosylate Gs 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 Gi 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 host cell.
Several classes of bacterial secretion systems (at least nine!) have been identified. 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 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 similar systems 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 2, 3, and 4. The main location of symptoms of disease caused by bacterial exotoxins is described in Table 5.
Table2. Important Bacterial Exotoxins
Table3. Important Mechanisms of Action of Bacterial Exotoxins
Table4. Exotoxins That Increase Intracellular Cyclic AMP
Table5. Main Location of Symptoms of Disease Caused by Bacterial Exotoxins
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 macro phages. 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 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 poly peptides. 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 ADP-ribosylating enzyme is due to a unique amino acid, a modified histidine called diphthamide, that is present only on eukaryotic 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. As a result, only C. diphtheriae strains lysogenized by this phage cause diphtheria. This is an important example of lysogenic con version, the process by which bacteria acquire new traits when lysogenized by a bacteriophage. 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 com plex 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 neu rotransmitters (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 blood stream to the internuncial 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 Clostridioides 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 trans duction proteins (Rho GTPases), which interferes with their signal transduction function. Glucosylation by exotoxin B inter feres with actin in the cytoskeleton, which leads to apoptosis and cell death.
(5) Multiple toxins and tissue-degrading enzymes are produced by Clostridium perfringens and other species of clostridia that cause gas gangrene, although no single species of Clostridium makes all of the toxins. The best characterized is the alpha toxin (a lecithinase) that hydrolyzes lecithin in the cell membrane, destroying the membrane and killing the cell. Other enzymes produced are collagenase, protease, hyaluronidase, and deoxyribonuclease (DNase). Several other clostridial toxins with hemolytic and necrotizing activity have also been described. 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 adenyl ate 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 4). Lethal fac tor is a protease that cleaves a phosphokinase (MAP kinase) required for the signal transduction pathway that controls cell growth. Loss of the phosphokinase leads to cell death. Protec tive 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 by blocking protective antigen from binding, which prevents entry of edema and lethal factor.
(7) TSST is a superantigen produced primarily by certain strains of S. aureus and S. pyogenes. TSST binds directly to class II major histocompatibility (MHC) proteins on the surface of antigen-presenting cells, which engage the T-cell receptor of many helper T cells. This results in nonspecific T cell activation, causing the release of large amounts of interleukins (ILs), especially IL-1, IL-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 to 6 hours after ingestion. The main symptoms are vomiting and watery diarrhea. The vomiting seen in food poisoning is caused by serotonin (5-hydroxytryptamine) produced by mast cells stimulating the enteric nervous system, which activates the vomiting center in the brain.
(9) Exfoliatin (epidermolytic toxin) 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.
(10) Panton-Valentine (PV) leukocidin is a pore-forming exotoxin produced by methicillin-resistant Staphylococcus 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, is a superantigen that causes the characteristic rash of scarlet fever. It is encoded by a lysogenic bacteriophage.
(12) Exotoxin B is a protease produced by strains of S. pyogenes that cause necrotizing fasciitis, and are often called “flesh-eating” streptococci. Exotoxin B cleaves E-cadherin in the skin, immunoglobulins, and complement proteins.
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 4).
(1) The heat-labile enterotoxin produced by E. coli causes watery, nonbloody diarrhea by stimulating adenylate cyclase activity in cells in the small intestine and works exactly like Chol era toxin (Figure 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. Heat-labile toxin, which is inactivated at 65°C for 30 minutes, is an A-B toxin. The B subunit confers specificity to the enterocytes in the small intestine by binding to a ganglioside receptor in the cell membrane. This allows subunit A to enter the cell where it ADP-ribosylates its target Gs protein, locking it in the “on” position. This stimulates adenylate cyclase to synthesize cyclic AMP, which in turn, activates cyclic AMP–dependent protein kinase. This enzyme 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 (not inactivated by heat). This 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 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 enterotoxins produced by V. cholerae, the agent of cholera, and Bacillus cereus, a cause of diarrhea, act in a manner similar to that of the heat-labile toxin of E. coli (see Figure 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 4). 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, causing the marked lymphocytosis seen in patients with pertussis. The toxin inhibits signal transduction by all chemokine receptors, which inhibits lymphocyte migration and their ability to enter lymphoid tissue (spleen, lymph nodes). Because they do not enter tissue, there is an increase in their number in the blood.
Fig2. Mode of action of Escherichia coli and Vibrio cholerae enterotoxins. The enterotoxin (e.g., cholera toxin) binds to the surface of the enterocyte via its binding subunit. The active subunit then enters the enterocyte. The active subunit is an enzyme that catalyzes the addition of ADP-ribose (ADP-R) to the GS regulatory protein. This activates adenylate cyclase to overproduce cyclic adenosine monophosphate (AMP). As a consequence, cyclic AMP–dependent protein kinase activity increases, and water and electrolytes leave the enterocyte, causing watery diarrhea.
Endotoxins
Endotoxins are an integral part of the cell wall of gram-negative bacteria, in contrast to exotoxins, which are actively released from the cell (see Table1). In addition, endotoxins are LPS, whereas exotoxins are polypeptides. The enzymes that produce the LPS are encoded by genes on the bacterial chromosome, whereas exotoxins are usually encoded by plasmid or bacteriophage DNA. The toxicity of endotoxins is low in comparison with that of exotoxins. All endotoxins produce the same generalized effects of fever and hypotension (shock), although the endotoxins of some organisms are more effective than others (Figure 3). Endotoxins are weakly antigenic and are not used as antigens in any available vaccine.
Fig3. Mode of action of endotoxin. Endotoxin is the most important cause of septic shock, which is characterized primarily by fever, hypotension, and disseminated intravascular coagulation (DIC). Endotoxin causes these effects by activating three critical processes: (1) activating macrophages to produce interleukin-1 (IL-1), tumor necrosis factor (TNF), and nitric oxide; (2) activating the alternative pathway of complement; and (3) activating tissue factor, an early component of the coagulation cascade.
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 host cell 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 macro phage is then activated, resulting in the synthesis of cytokines such as IL-1, TNF, and nitric oxide (see later and Figure 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.
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 become negative). This occurs because septic shock is mediated by cytokines (e.g., TNF and IL-1) that continue to act even after the bacteria are no longer viable.
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 long-chain polysaccharide on the exterior portion of endotoxin is called the somatic (O) antigen. It is an important antigen of many gram-negative bacteria and is composed of 3 to 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. There are many types of E. coli based on their O antigens, the most important of which is E. coli O-157, the cause of hemolytic uremic syndrome. 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 6) include the following:
(1) Fever due to the release of IL-1 (endogenous pyrogen) and IL-6 by macrophages, 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 damaged endothelial cells. Tissue factor interacts with circulating coagulation factors, causing widespread clotting within capillaries.
(4) Activation of the alternative pathway of the complement cascade, resulting in recruitment of neutrophils, inflammation, and tissue damage.
(5) Activation of macrophages, increasing their phagocytic ability, and activation of many B cell clones, increasing anti body production. (Endotoxin is a polyclonal activator of B cells, but not T cells.)
Table6. Effects of Endotoxin
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 organ ism, 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. TNF is the central mediator because purified recombinant TNF reproduces the effects of endotoxin and antibody against TNF blocks the effects of the endotoxin. Endo toxin also induces macrophage migration inhibitory factor, which also plays a role in the induction of septic shock.
Note that while TNF mediates detrimental effects when in large amounts, in small amounts has beneficial effects (e.g., causing an inflammatory response to the presence of a microbe). 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; 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.
Table7. Beneficial and Harmful Effects of TNF
Endotoxins can cause fever in patients if present in intra venous fluids. In the past, intravenous fluids were sterilized by autoclaving, which killed any organisms present but released endotoxins, which are not heat inactivated. These fluids are now sterilized by filtration, which physically removes organisms without releasing endotoxin.
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. Attempts to treat septic shock with hemoperfusion to adsorb endotoxin or by administering anti bodies specific to lipid A and TNF have been mixed.
