EXCEED THE SPACE PROVIDED Vibrio cholerae strains that do not produce cholera toxin induce a more inflammatory diarrhea than normal cholera disease, implicating other potent toxins in the pathogenesis of cholera Several accessory toxins of V cholerae are purported to account for this reactogenic response One of these factors is the newly discovered VcRtxA toxin of V cholerae VcRtxA is a large protein toxin that is a unique member of the RTX family Production of this toxin has been evolutionarily conserved by Vibrio sp indicating that maintenance of this large toxin is essential for virulence or survival in the environment Its cytotoxicity has been further shown to function by a novel mechanism This toxin causes depolymerization of actin stress fibers in both polarized and non-polarized cell lines by a unique pathway Concurrent with depolymerization, the actin molecules become covalently linked together into dimers, trimers, and higher order multimers This observation suggests that covalent crosslinking of actin by the toxin drives actin depolymerization This unusual reaction distinguishes VcRtxA from all other bacterial toxins that cause actin depolymerization Research performed under this grant proposal will investigate further the novel biochemical properties of this important virulence factor The VcRtxA toxin is the largest single polypeptide protein toxin ever described, however, it is unclear whether the full 4545 amino acid protein is the toxic moiety The size of the toxic moiety and potential post-translational modifications will by identified by examining biochemical properties of the active form of purified toxin The catalytic activity will then be investigated and the role of a putative catalytic domain in this reaction will be assessed tt will then be established whether the toxin is active at the membrane or within the cytoplasm of the target cells, and the role of a putative actin binding domain will be characterized PERFORMANCE SITE ========================================Section End===========================================
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