Toxins produced by bacteria are generally classified into two groups: endotoxin, which is present in the outer membrane of gram-negative rods, and toxins that are secreted, such as enterotoxins and exotoxins. Enterotoxins and exotoxins are often classified by mechanisms of action and the impact on host cells and they are discussed in more detail below. The primary features of the two groups are listed in Table 1.

Table1. Characteristics of Exotoxins and Endotoxins (Lipopolysaccharides)

A. Exotoxins

Many gram-positive and gram-negative bacteria produce exotoxins of considerable medical importance. Some of these toxins have had major roles in world history. For example, tetanus caused by the toxin of C tetani killed as many as 50,000 soldiers of the Axis powers in World War II; the Allied forces, however, immunized military personnel against tetanus, and very few died of that disease. Vaccines have been developed for some of the exotoxin-mediated diseases and continue to be important in the prevention of disease. These vaccines— called toxoids—are made from exotoxins, which are modified so that they are no longer toxic. Many exotoxins consist of A and B subunits (often referred to as binary toxins or type III toxins). The B subunit generally mediates adherence of the toxin complex to a host cell and aids entrance of the exotoxin into the host cell. The A subunit provides the toxic activity. Examples of some pathogenetic mechanisms associated with exotoxins are given below. Other toxins of specific bacteria are discussed in the chapters covering those bacteria.

C diphtheriae is a gram-positive rod that can grow on the mucous membranes of the upper respiratory tract or in minor skin wounds. Strains of C diphtheriae that carry a lysogenic, temperate corynebacteriophage (β-phage or ω-phage) with the structural gene for the toxin are toxigenic and produce diphtheria toxin and cause diphtheria. Many factors regulate toxin production; when the availability of inorganic iron is the factor limiting the growth rate, then maximal toxin production occurs. The toxin molecule is secreted as a single polypeptide molecule (molecular weight [MW], 62,000). This native toxin is enzymatically degraded into two fragments, A and B, linked together by a disulfide bond. Fragment B (MW 40,700) binds to specific host cell receptors and facilitates the entry of fragment A (MW 21,150) into the cytoplasm. Fragment A inhibits peptide chain elongation factor EF-2 by catalyzing a reaction that attaches an adenosine diphosphate–ribosyl group to EF-2, yielding an inactive adenosine diphosphate–ribose–EF-2 complex. Arrest of protein synthesis disrupts normal cellular physiologic functions. Diphtheria toxin is very potent.

C tetani is an anaerobic gram-positive rod that causes tetanus. C tetani from the environment contaminates wounds, and the spores germinate in the anaerobic environment of the devitalized tissue. Infection often is minor and not clinically apparent. The vegetative forms of C tetani produce the toxin tetanospasmin (MW 150,000) that is cleaved by a bacterial protease into two peptides (MW 50,000 and 100,000) linked by a disulfide bond. The toxin initially binds to receptors on the presynaptic membranes of motor neurons. It then migrates by the retrograde axonal transport system to the cell bodies of these neurons to the spinal cord and brainstem. The toxin diffuses to terminals of inhibitory cells, including both glycinergic interneurons and γ-aminobutyric acid (GABA)–secreting neurons from the brainstem. The toxin degrades synaptobrevin, a protein required for docking of neurotransmitter vesicles on the presynaptic membrane. Release of the inhibitory glycine and GABA is blocked, and the motor neurons are not inhibited. Spastic paralysis results. Extremely small amounts of toxin can be lethal for humans. Tetanus is totally preventable in immunologically normal people by immunization with tetanus toxoid.

C botulinum causes botulism. This anaerobic, gram positive spore-forming organism is found in soil or water and may grow in foods (eg, canned, vacuum packed) if the environment is appropriately anaerobic. An exceedingly potent toxin (the most potent toxin known) is produced. It is heat labile and is destroyed by sufficient heating. There are seven distinct serologic types of toxin. Types A, B, E, and F are most commonly associated with human disease. The toxin is very similar to tetanus toxin, with a 150,000 MW protein that is cleaved into 100,000-MW and 50,000-MW proteins linked by a disulfide bond. Botulinum toxin is absorbed from the gut and binds to receptors of presynaptic membranes of motor neurons of the peripheral nervous system and cranial nerves. Proteolysis, by the light chain of botulinum toxin, of target proteins in the neurons inhibits the release of acetylcholine at the synapse, resulting in lack of muscle contraction and flaccid paralysis.

Spores of C perfringens are introduced into wounds by contamination with soil or feces. In the presence of necrotic tissue (an anaerobic environment), spores germinate and vegetative cells can produce several different toxins. Many of these are necrotizing and hemolytic and—together with distention of tissue by gas formed from carbohydrates and interference with blood supply—favor the spread of gas gangrene. The alpha toxin of C perfringens is a lecithinase that damages cell membranes by splitting lecithin to phosphorylcholine and diglyceride. Theta toxin also has a necrotizing effect. Collagenases and DNAses are produced by clostridiae as well.

Some S aureus strains growing on mucous membranes (eg, the vagina in association with menstruation) or in wounds, elaborate toxic shock syndrome toxin-1 (TSST-1), which causes toxic shock syndrome. The ill ness is characterized by shock, high fever, and a diffuse red rash that later desquamates; multiple other organ systems are involved as well. TSST-1 is a superantigen (also referred to as a type I toxin), and superantigens do not need to enter cells to cause their potent cellular disruption. TSST-1 stimulates most T-cells by binding directly to MHC-II and T-cell receptors. The net result is production of large amounts of the cytokines interleukin-2 (IL-2), interferon γ, and tumor necrosis factor (TNF). The major clinical manifestations of the disease appear to be secondary to the effects of the cytokines. The systemic effects of TSST-1 are due to the massive cytokine stimulation. Some strains of group A β-hemolytic streptococci produce pyrogenic exotoxins A and C. Rapidly progressive soft tissue infection by streptococci that produce the pyrogenic exotoxin A has many clinical manifestations similar to those of staphylococcal toxic shock syndrome. The pyrogenic exotoxins A and C are also superantigens that act in a manner similar to TSST-1.

Type II toxins are proteins that typically affect cell mem branes facilitating invasion by the pathogen secreting them.

B. Exotoxins Associated with Diarrheal Diseases and Food Poisoning

 Exotoxins associated with diarrheal diseases are frequently called enterotoxins and many belong to the type III toxin family. Characteristics of some important enterotoxins are discussed as follows.

V cholerae has produced epidemic diarrheal disease (cholera) in many parts of the world and is another toxin-produced disease of historical and current importance. After entering the host via contaminated food or drink, V cholerae penetrates the intestinal mucosa and attaches to microvilli of the brush border of gut epithelial cells. V cholerae, usually of the serotype O1 (and O139), can produce an enterotoxin with a MW of 84,000. The toxin consists of two subunits—A, which is split into two peptides, A1 and A2 , linked by a disulfide bond, and B. Subunit B has five identical peptides and rapidly binds the toxin to cell membrane ganglioside molecules. Subunit A enters the cell membrane and causes a large increase in adenylate cyclase activity and in the concentration of cAMP. The net effect is rapid secretion of electrolytes into the small bowel lumen, with impairment of sodium and chloride absorption and loss of bicarbonate. Life-threatening massive diarrhea (eg, 20–30 L/ day) can occur, and acidosis develops. The deleterious effects of cholera are due to fluid loss and acid–base imbalance; treatment, therefore, is by electrolyte and fluid replacement.

Some strains of S aureus produce enterotoxins while growing in meat, dairy products, or other foods. In typical cases, the food has been recently prepared but not properly refrigerated. There are at least seven distinct types of the staphylococcal enterotoxin. After the preformed toxin is ingested, it is absorbed in the gut, where it stimulates vagus nerve receptors. The stimulus is transmitted to the vomiting center in the central nervous system. Vomiting, often projectile, results within hours. Diarrhea is less frequent. Staphylococcal food poisoning is the most common form of food poisoning. S aureus enterotoxins are superantigens.

Enterotoxins are also produced by some strains of Y enterocolitica, Vibrio parahaemolyticus, Aeromonas species, and other bacteria, but the role of these toxins in pathogenesis is not as well defined. The enterotoxin produced by C perfringens is discussed in Chapter 11.

C. Lipopolysaccharides of Gram-Negative Bacteria

The LPS (endotoxin) of gram-negative bacteria are bacterial cell wall components that are often liberated when the bacteria lyse. The substances are heat stable, have MWs between 3000 and 5000 (lipooligosaccharides, LOS), and several million (lipopolysaccharides) can be extracted (eg, with phenol-water). They have three main regions (see Figure 1). The lipid A domain is the region recognized by the immune system and is the component that is responsible for cytokine stimulation (see below). The other two components are an oligosaccharide core and an outermost O-antigen polysaccharide.

Fig1. lipopolysaccharide structure. A: The lipopolysaccharide from Salmonella. This slightly simplified diagram illustrates one form of the LPS. Abe, abequose; Gal, galactose; GlcN, glucosamine; Hep, heptulose; KDO, 2-keto-3-deoxyoctonate; Man, mannose; NAG, N-acetylglucosamine; P, phosphate; Rha, l-rhamnose. Lipid A is buried in the outer membrane. B: Molecular model of an Escherichia coli lipopolysaccharide. The lipid A and core polysaccharide are straight; the O side chain is bent at an angle in this model. (Reproduced with permission from Willey VM, Sherwood LM, Woolverton CJ: Prescott, Harley, and Klein’s Microbiology, 7th ed. McGraw-Hill, 2008. © The McGraw Hill Companies, Inc.)

The pathophysiologic effects of LPS are similar regard less of their bacterial origin except for those of Bacteroides species, which have a different structure and are less toxic. LPS in the bloodstream is initially bound to circulating proteins, which then interact with receptors on macrophages, neutrophils, and other cells of the reticuloendothelial system. Proinflammatory cytokines such as IL-1, IL-6, IL-8, TNF-a, and other cytokines are released, and the complement and coagulation cascades are activated. The following can be observed clinically or experimentally: fever, leukopenia, and hypoglycemia; hypotension and shock resulting in impaired perfusion of essential organs (eg, brain, heart, kidney); intravascular coagulation; and death from massive organ dysfunction.

Injection of LPS produces fever after 60–90 minutes, the time needed for the body to release IL-1. Injection of IL-1 produces fever within 30 minutes. Repeated injection of IL-1 produces the same fever response each time, but repeated injection of LPS causes a steadily diminishing fever response because of tolerance partly caused by reticuloendothelial blockade and partly caused by IgM antibodies to LPS.

Injection of LPS produces early leukopenia, as does bacteremia with gram-negative organisms. Secondary leukocytosis occurs later. The early leukopenia coincides with the onset of fever caused by liberation of IL-1. LPS enhances glycolysis in many cell types and can lead to hypoglycemia.

Hypotension occurs early in gram-negative bacteremia or after injection of LPS. There may be widespread arteriolar and venular constriction followed by peripheral vascular dilation, increased vascular permeability, decrease in venous return, lowered cardiac output, stagnation in the microcirculation, peripheral vasoconstriction, shock, and impaired organ perfusion and its consequences. Disseminated intra vascular coagulation (DIC) also contributes to these vascular changes.

LPS is among the many different agents that can activate the alternative pathway of the complement cascade, precipitating a variety of complement-mediated reactions (eg, anaphylatoxins, chemotactic responses, membrane damage) and a drop in serum levels of complement components (C3, C5–C9).

Disseminated intravascular coagulation is a frequent complication of gram-negative bacteremia and can also occur in other infections. LPS activates factor XII (Hageman factor)—the first step of the intrinsic clotting system—and sets into motion the coagulation cascade, which culminates in the conversion of fibrinogen to fibrin. At the same time, plasminogen can be activated by LPS to plasmin (a proteolytic enzyme), which can attack fibrin with the formation of fibrin split products. Reduction in platelet and fibrinogen levels and detection of fibrin split products are evidence of DIC. Heparin can sometimes prevent the lesions associated with DIC.

LPS causes platelets to adhere to vascular endothelium and occlusion of small blood vessels, causing ischemic or hemorrhagic necrosis in various organs.

Endotoxin levels can be assayed by the limulus test: A lysate of amebocytes from the horseshoe crab (limulus) gels or coagulates in the presence of 0.0001 μg/mL of endotoxin. This test is rarely used in clinical laboratories because it is difficult to perform accurately.

D. Peptidoglycan of Gram-Positive Bacteria

The peptidoglycan of gram-positive bacteria is made up of cross-linked macromolecules that surround the bacterial cells (see Figure 2-15). Vascular changes leading to shock may also occur in infections caused by gram positive bacteria that contain no LPS. Gram-positive bacteria have considerably more cell wall–associated peptidoglycan than do gram-negative bacteria. Peptidoglycan released during infection may yield many of the same biologic activities as LPS, although peptidoglycan is invariably much less potent than LPS.

Fig2. Components and structure of peptidoglycan. A: Chemical structure of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM); the ring structures of the two molecules are glucose. Glycan chains are composed of alternating subunits of NAG and NAM joined by covalent bonds. Adjacent glycan chains are cross-linked via their tetrapeptide chains to create peptidoglycan. B: Interconnected glycan chains form a very large three-dimensional molecule of peptidoglycan. The β1→4 linkages in the backbone are cleaved by lysozyme. (Reproduced with permission from Nester EW, Anderson DG, Roberts CE, Nester MT: Microbiology: A Human Perspective, 6th ed. McGraw-Hill; 2009.)

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