Bacteroides fragilis are the leading causes of anaerobic bacteremia and intra-abdominal abscesses. In addition, certain strains of B.fragilis which produce an ca. 20 kDa heat-labile metalloprotease toxin (termed enterotoxigenic B.fragilis or ETBF) are associated with diarrheal disease in young children and may also be associated with some extraintestinal B. fragilis infections. Purified B. fragilis toxin (BFT) stimulates secretion in animal intestine and is proposed to be a key virulence protein of ETBF strains. The long range goal of this laboratory is to determine the mechanisms by which ETBF cause diarrheal disease. To this end, the investigators initiated studies in the first granting period to understand the action of BFT on intestinal epithelial cells and to clone the toxin gene. This work revealed that ETBF strains produce two highly related, but distinct, isoforms of BFT (termed BFT-1 and BFT-2) which are the products of two distinct toxin genes (termed bft-1 and bft-2). Human ETBF strains produce only one of these toxins; no strain producing both toxins has yet been identified. Furthermore, the bft genes are flanked by ca. 12 kb of DNA largely unique to the ETBF strains (i.e., not present in nontoxigenic strains) suggesting these organisms possess a putative 'pathogenicity island'. BFT alters the morphology and function of intestinal epithelial cells in a strikingly polar manner and, counter-intuitive to conventional theories of pathogenesis, the activity of the toxin is greatest at the basolateral membrane of the cells, not the apical membrane. BFT dramatically rearranges, but does not cleave, actin in intestinal epithelial cells. However, E-cadherin, a protein on the lateral membranes of intestinal cells responsible for cell-to-cell adhesion, is cleaved by BFT. The investigators hypothesize that proteolysis of E-cadherin triggers a cascade of intracellular events including destabilization of catenins, and actin rearrangement leading to the polar morphologic and functional sequelae observed in response to BFT. In this grant, their primary goal will be to test their hypothesis that E-cadherin proteolysis accounts for the BFT-induced pathophysiologic outcomes that they identified in the first granting period.
The Specific Aims are: 1) To characterize the role of E-cadherin proteolysis in the mechanism of action of BFT on intestinal epithelial cells.; 2) To determine the domains of BFT important for functional activity.; and 3) To define the putative pathogenicity island of ETBF strains. This three-pronged approach will advance our understanding of the molecular pathogenesis of ETBF infections and will serve to delineate in much greater detail the cellular mechanism of action of BFT.
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