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Chapter 27 : Pathogenicity of Toxins

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Abstract:

Pathogenic strains of cause diarrhea and colitis in humans through the release of two protein exotoxins, toxin A and toxin B. This chapter reviews the mechanisms whereby these toxins exert their cytotoxic, enterotoxic, and proinflammatory effects. Antibiotic therapy is the main risk factor for infection. infection is caused by ingestion of spores. infection results in a broad spectrum of clinical manifestations ranging from asymptomatic carriage to severe, life threatening pseudomembranous colitis. Intestinal injury and inflammation result from the effects of toxins. -associated diarrhea and colitis are usually treated with oral metronidazole or vancomycin to eradicate vegetative forms of the bacterium. toxin A and toxin B are the major known virulence factors of . These AB-type toxins share similar domains including an N-terminal enzymatic domain responsible for the toxicity of the molecule and a C-terminal binding domain composed of repeating sequences. The observation that neonates and infants are frequently colonized by toxigenic but remain asymptomatic suggests that the expression or glucosylation of human intestinal receptors for toxins is not complete before the age of 2 years. The translocation of toxin A and toxin B into the cytosol is a prerequisite for their cytotoxic activity. Some bacterial toxins, including diphtheria toxin, translocate from the endosomal compartment to the cytosol following receptor-mediated endocytosis.

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27

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Image of FIGURE 1
FIGURE 1

Pathogenesis of -associated diarrhea. The pathogenesis of -associated diarrhea and colitis involves an initial disruption of the normal colonic bacterial flora by antibiotic treatment. This allows colonization with if the individual is exposed to an environment where spores are sufficiently abundant. Pathogenic strains of release toxins A and B, which cause colonic mucosal injury and inflammation. An early anamnestic immune response to toxin A can be detected in asymptomatic carriers but not in those who develop diarrhea and colitis. (From reference , with permission.)

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 2
FIGURE 2

Pseudomembranous colitis. (A) The macroscopic features of pseudomembranous colitis, the most severe form of infection, are shown in a colectomy specimen. The colonic mucosa is covered by characteristic yellow and raised pseudomembranes (arrows) containing fibrin, cellular debris, and neutrophils. These lesions have a patchy distribution but become confluent as the inflammation and ulceration progress in severity and extent. (B) Colonoscopic biopsy specimen from a patient with pseudomembranous colitis showing focal ulceration (lower arrow) surmounted by a “summit” or “volcano” lesion of pseudomembranous exudate (upper arrow). (Hematoxylin-and eosin-stained tissue section, original magnification ×55.) (From reference , with permission.)

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 3
FIGURE 3

The pathogenicity locus (PaLoc) is a 19.6-kb segment carrying five genes () including the genes encoding toxin A () and toxin B (). TcdD appears to activate toxin A and toxin B transcription by forming a complex with RNA polymerase that binds to and promoter regions. Whereas the function of TcdC is unknown, TcdE may regulate toxin release through its ability to disrupt the bacterial cytoplasmic membrane. (Figure based on data from references , and .)

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 4
FIGURE 4

Structure of toxins. Toxin A and toxin B are AB-type toxins and share three similar domains: (i) an N-terminal enzymatic domain responsible for cytotoxicity that carries a conserved tryptophan residue (Trp-102) probably involved in binding to UDPglucose; (ii) a central, major hydrophobic region of 172 amino acids that is highly conserved and may act as a transmembrane domain to facilitate exit from endosomes; and (iii) a Cterminal binding domain composed of contiguous repeating units also known as CROPs. Toxin A carries 30 CROPs, whereas toxin B carries 19. They include sequences of 50 amino acids (represented in gray) and 21 amino acids (represented in white). The monoclonal antibody PCG4, which neutralizes toxin A enterotoxicity in animal models, recognizes epitopes on toxin A CROPs which are illustrated by horizontal bars.

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 5
FIGURE 5

Rho protein cycle and the consequences of Rho glucosylation. (A) Rho proteins are signaling molecules that function as molecular switches. Rho GTPases function as a molecular switch through a GTP-binding/GTPase cycle regulated by three classes of proteins: (i) GEFs, which stimulate the exchange of GTP for GDP; (ii) GAPs, which catalyze GTP hydrolysis; and (iii) GDIs, which block the dissociation of GDP from Rho proteins, inhibit intrinsic and GAP-catalyzed GTP hydrolysis, and regulate the subcellular localization of Rho proteins. In the cytosol, Rho proteins are bound to GDI in the GDP-bound, inactive state. In response to upstream signals, Rho is dissociated from GDI. The geranylgeranylated moiety of Rho is then inserted into the plasma membrane lipid bilayer, a step essential for Rho function. Moreover, a GEF catalyzes nucleotide exchange and Rho activation. In its active membrane-associated state, Rho interacts with downstream effectors to induce coordinated signals that regulate cytoskeletal structure and gene transcription. Rho inactivation occurs as GAPs catalyze GTP hydrolysis. The GDP-bound form is then extracted from the membrane by Rho-GDI. (B) toxin-induced glucosylation blocks Rho cycling, effector coupling, and downstream signaling. Toxins A and B are UDP-glucose hydrolases and glucosyltransferases. After hydrolyzing UDP-glucose, both toxins catalyze the transfer of the glucose moiety to a conserved threonine residue (Thr-35 or Thr-37) of Rho proteins when released from GDI. Since glucosylated Rho cannot bind GDI-1, glucosylated Rho cannot be extracted by GDI from the plasma membrane, and this interrupts Rho cycling. Furthermore, the glucose moiety prevents Rho interaction with downstream effectors, resulting in cytoskeleton collapse and inhibition of signaling.

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 6
FIGURE 6

Morphological features of toxin-induced cytotoxic effect in cultured cells. CHO cells were incubated overnight in the absence (A and C) or in the presence (B and D) of 30 fM toxin B. (A and B) Cell morphology as observed by phase-contrast microscopy. Toxin exposure caused cell rounding and retraction of the cell body (B). (C and D) Cells were stained with fluorescein isothiocyanate-labeled phalloidin, which binds to actin microfilaments and reveals stress fibers. In toxin B-treated cells, the stress fibers have disaggregated (D). Toxin A causes identical effects (not shown).

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 7
FIGURE 7

toxin effects on intestinal epithelial cells. Toxin A and toxin B bind to specific extracellular receptors expressed at the luminal surface of intestinal epithelial cells. After toxin internalization and transfer into the cytosol, the enzymatic domain catalyzes the glucosylation of Rho proteins. Inactivated Rho proteins cause perijunctional actin changes leading to loss of transepithelial electrical resistance and increased paracellular permeability. Toxin exposure also triggers the release of inflammatory mediators including IL-8 and reactive oxygen species that may in turn activate lamina propria cells. Whether this inflammatory response results from Rho glucosylation or Rho-independent mechanisms, such as receptormediated signaling, is not clear.

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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Image of FIGURE 8
FIGURE 8

Serum IgG anti-toxin A antibody response and clinical outcome of infection. Patients with nosocomial diarrhea were studied prospectively and serum IgG anti-toxin A antibody concentrations were measured by enzyme-linked immunosorbent assay at regular intervals. A correlation was observed between the IgG response and the clinical outcome of infection. Asymptomatic carriers mounted an early anamnestic response to toxin A. By contrast, no significant increase in serum IgG to toxin A was found in patients who suffered recurrent diarrhea. In those who had a single episode of diarrhea, anti-toxin A IgG was generally increased on day 12 of their first episode. Thus, a serum antibody response to toxin A during infection is associated with protection against symptoms or against recurrent diarrhea. (Figure based on data from references and .)

Citation: Warny M, Kelly C. 2003. Pathogenicity of Toxins, p 503-524. In Hecht G (ed), Microbial Pathogenesis and the Intestinal Epithelial Cell. ASM Press, Washington, DC. doi: 10.1128/9781555817848.ch27
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