The overall goals of this proposal are to elucidate the mechanisms by which C. difficile toxins damage the intestinal tract. Purified toxins will be prepared from broth culture supernatants using sequential ion exchange chromatography, high performance liquid chromatography (HPLC), and immunoaffinity chromatography with monoclonal antibodies. The amino acid composition and sequence of purified toxins will be compared with other known bacterial enterotoxins and cytotoxins and will attempt to dissociate toxins into binding and catalytic sub-units using detergents, reducing agents and urea. The mechanisms of toxins A and B will be studied in vitro using fibroblast and intestinal cell lines. Since toxin B causes profound changes in microfilaments of fibroblasts and smooth muscle cells, we will further explore the effects of toxin B on other cytoskeletal and matrix proteins in fibroblasts as well as intestinal epithelial cells exposed to toxin B. Cytoskeletal and matrix proteins will be assayed by immunofluorescent microscopy and SDS-PAGE. The effects of toxins on intracellular calcium release in target cells will be explored. We have recently demonstrated that toxin A causes a dramatic rise in intracellular ionized Ca++ in human neutrophils. We will determine intracellular calcium concentration in other target cells including the enterocyte. These results will be correlated with measurements of phospholipase C activation and phosphorylation of cellular proteins in target cells. These studies should elucidate the cellular mechanisms activated by toxins A and B. The in vivo effects of toxins will be studied in rabbit ileal and colonic loops. We will determine the effects of purified toxins A and B, or mixtures of the two, on enterocyte morphology, cytoskeletal proteins, fluid secretion, permeability and macro-molecular synthesis. We will study the contributions of neutrophils and mast cells to the enteritis by using pharmacologic blockers of cyclooxygenase and lipoxygenase pathways, oxygen radical scavengers and antihistamines. Finally, we will study receptor binding to radiolabelled toxins A and B to target cells and brush border membranes of animal and human small and large intestine. Because infection with C. difficile appears not to cause disease in newborns and infants, we propose to measure toxin binding to enterocytes at various stages of post-natal development to determine if lack of toxin binding in early life correlates with lack of gut response.
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