Clostridium difficile: C difficile is established as the most common nosocomial enteric pathogen, causing pseudomembranous colitis, antibiotic-associated colitis and antibiotic-associated diarrhea. The most important defence against this opportunistic pathogen is the normal colonic microflora, although the microbial species responsible for C difficile and the mechanisms whereby their growth is suppressed are still not understood. Disruption of the normal ecosystem by antibiotics can result in colonization by C difficile, which, if of the right pathotype, causes diarrhea or, more seriously, pseudomembranous colitis. Production of proteolytic and hydrolytic enzymes and capsule, expression of fimbriae and flagella, chemotaxis and adhesion to gut receptors, may all play a part in the pathogenesis of C difficile-induced disease by facilitating colonization or by directly contributing to tissue damage. However, toxins A and B are thought to be the primary virulence determinants of this pathogen in the context of antibiotic-associated gastrointestinal disease. The molecular organization, and control of expression of toxins A and B are now being unravelled. Toxins A and B belong to a group of clostridial toxins designated as large (A 308 kDa; B 269 kDa) clostridial cytotoxins, which have homologous protein sequences, and modify small GTP-binding proteins of the Ras superfamily by their N-terminal glycosyl-transferase activity. This results in disruption and reorganization of cytoskeletal actin and cell rounding.
How do these toxins act in vivo, and can we interpret the disease process in terms of their activities? Both toxins are cytotoxic in vitro. B is approximately 1000-fold more cytotoxic to cultured cells than A, yet a substantial body of evidence shows that A is vastly more toxic to rabbit intestine than B, which has little effect on animal mucosae in vivo on its own. The effect of A on the rabbit terminal ileum was drastic, with near total destruction of villi and production of bloody proteinaceous fluid. In the colon, epithelia became detached, and hemorrhage was tissue localized: fluid was watery and nonproteinaceous. In immunized animals, tissue hemorrhage was prevented but epithelial desquamation or fluid secretion in the rabbit colon was not. In experimental infection of hamsters, virulence of C difficile strains correlated with the degree of production in vivo of A rather than B. Later studies with human biopsies showed that toxin A binds directly to human colonocytes, causing them to detach, and induces production of the inflammatory cytokine IL-8. Toxin A also killed lamina propria macrophages and T cells, which, these authors suggested, could be the basis of C difficile-induced suppression of immune responses. Thus, a powerful case can be marshalled to implicate toxin A as the major effector in C difficile diarrheal disease and of the colitis that is characteristic of C difficile infections.
However, as in all overly neat syntheses, there are awkward, ill-fitting pieces. Others claim that in an in vitro Ussing chamber study, the human colon was 10 times more sensitive to toxin B than to toxin A. A cytotoxigenic strain of C difficile has been described that does not produce toxin A, but causes classic hemorrhage and bloody fluid accumulation in rabbit ileal loops, and diarrhea in hamsters. Perhaps the biggest unsolved puzzle is that both these toxins have exactly the same enzymatic activity that results in the glycosylation of the same serine residue in target proteins, but yet they have very different biological properties. Attempts to explain this discrepancy have been made as follows. In cell culture systems, cytotoxicity correlated with the efficiency of toxin binding to cells. Moreover, although the specific glucosyltransferase activity of A was 100 times less than that of B, toxin A did modify an additional substrate. While it is difficult to see how this explains the dramatic effect(s) of toxin A on intact mucosae, it does point to the possibility that the primary biological effects are unrelated to glucosyltransferase enzyme activity but are due to some other mechanism. Branka et al purport to demonstrate that the main effect of toxin A is to upregulate the secretion of IL-8 from colonocytes and downregulate the exocytosis of mucin. This would result in the recruitment of inflammatory and immune cells, with consequential indirect mucosal damage. The depression of mucin secretion could well be explained as a secondary effect in terms of the enzymatic activity of the toxin, which by disruption of the cytoskeleton would impair exo-cytosis. It would not be surprising to find that the explanation of differential histotoxicity lies in the C-terminal repeat sequences (which carry cell recognition and signal transducing?) sites in the larger A protein or, that these proteins are differentially multifunctional.