Clostridium difficile is a common and relentless pathogen which is the basis for a substantial proportion of infectious gastrointestinal disease in the United States and other countries. Although seldom lethal by itself, C. difficile represents a significant health risk, particularly among those in their very early or very elderly years because of its tenacity, or propensity for relapse and recurrence. A complete understanding the pathogenetic mechanisms of C. difficile would have an important impact on the public health and on healthcare-related economics; as such, the problem is certainly worthy of the attention. This application is for the renewal of an award held by the principal investigator for several years. C. difficile reeks its havoc by elaboration of two unique toxins, labelled A and B. These toxins gain entrance into colonocytes by binding to cell surface receptors; thereafter, endocytosis facilitates cellular entry and subsequent attenuation of intracellular machinery. Specifically, the intracellular targets of toxins A and B appear to be the GTP-binding Rho proteins which regulate the actin cytoskeleton. A substantial portion of our understanding of these processes is consequent to data collected by the investigator's laboratory over the past decade. This application proposes to continue this investigation along three distinct fronts. First, purification and cloning of the receptors for Toxin A and B will be carried out using human tissue. This laboratory has recently carried out similar work in the rabbit, demonstrating that sucrase-isomaltase is the primary cell surface receptor for Toxin A in this species. Characterization of the human receptors is a logical extension of this work. Second, epithelial cell transduction mechanisms activated by toxin binding will be determined. In this series of experiments, the investigators will extend their preliminary observations regarding the effect of toxin binding on colonocyte Ca flux, with particular emphasis on the roles of protein tyrosine kinase and protein kinase C in this processes. Third, the means by which toxins gain access to its site of action within colonocytes will be investigated. Specific emphasis will be placed on receptor mediated endocytosis and vesicular transport in toxin internalization.
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