Clostridium difficile, the causative agent of antibiotic-associated diarrhea and colitis, is now recognized as one of the major enteric pathogens. C. difficile is an anaerobic bacillus which releases several protein toxins that mediate tissue damage in the colon of infected animals or humans. The major toxin, toxin B, is a heat labile protein which causes rounding of cells growing in monolayer culture. This toxin is also demonstrable in the stools of patients with antibiotic-associated diarrhea and colitis, and presumably mediates the tissue injury (colitis) which accompanies infection with this pathogen. The overall goals of this proposal are to purify C. difficile toxin B and determine its mechanism of action in vitro and in vivo. The hypothesis to be tested in this project is that toxin B interferes with the structure and function of the cytoskeleton, causing cell rounding, and that this in turn causes inhibition of macromolecular synthesis and eventually death of the cell. The purified toxin will be characterized as regards composition, molecular weight, sub-unit structure and structure-activity relationships. The purified toxin will then be used to determine the mechanism of cell rounding, more specificially its effects on cytoskeletal and matrix proteins. The syntehsis of actin, fibronectin, myosin and other filament proteins will be studied in fibroblast and smooth muscle cells exposed to graded doses of pure toxin B. The effect of toxin B on macromolecular synthesis will be quantitated using 3H-leucine and 3H-thymidine as precursors for protein and DNA syntehsis. The significance of these studies relates to the importance of C. difficile as a human pathogen. By providing a better understanding of how toxin B works it should be possible to define the pathophysiology of one of the commonest and most important enteric infections in man. From a basic science viewpoint, these studies should provide insights into the relationship of cytoskeleton and matrix proteins to cell shape, synthetic function and proliferation.
