Bacteroides fragillis are the leading causes of anaerobic bacteremia and intraabdominal abscesses. Certain strains of B. fragilis termed enterotoxigenic B. fragillis (ETBF) are linked epidemiologically to diarrheal illnesses in animals, children and adults. The only recognized virulence factor of ETBF is a secreted 20 kDa zinc-dependent metalloprotease toxin termed the B. fragillis toxin (BFT). BFT has sequence homology to eukaryotic matrix metalloproteases (MMPs) of the metzincin family. By cell-surface proteolysis, MMPs regulate eukaryotic cell signal transduction and function. Our data reveal that BFT treatment of intestinal epithelial cells in vitro results acutely in cleavage of the extracellular domain of E-cadherin (the key intercellular adhesion protein of the intestinal epithelial cell zonula adherens) and stimulation of several signal transduction pathways (Beta-catenin, tyrosine kinase, nuclear factor-KB and mitogen-activated kinases) yielding cellular cytoskeletal, physiologic and nuclear activation sequelae. Thus, we postulate BET is a prokaryotic mimic of the eukaryotic MMPs. Our preliminary data also suggest that BET binds to a sialic acid-containing glycoprotein. Based on our data, we postulate that BFT binds to a specific cell receptor, possibly a receptor tyrosine kinase. Activation of a tyrosine kinase(s) contributes to E-cadherin cleavage and induces expression and secretion of the proinflammatory chemokine, interleukin-8, by intestinal epithelial cells. E-cadherin cleavage results in: 1) reorganization of the apical cytoskeleton of intestinal epithelial cells leading to reduced barrier function and chloride secretion; and 2) T cell factor (TCF)-dependent Beta-catenin nuclear signaling resulting in c-Myc induction and cellular proliferation. The precise cellular substrate for BET is not clear but is postulated to be E-cadherin or the cellular receptor for BFT. Our long range goal is to define in detail the cellular mechanism of action of BFT. We now propose to identify the very early steps in the mechanism of action of BET and to define the functional domains of the BET protein.
Our specific aims are: 1) to determine the impact of the intercellular adhesion protein, E-cadherin, in mediating the mechanism of action of BET. The E-cadherin cleavage site will be identified and mutated such that it is resistant to BFT-initiated proteolysis. The BET biologic activities attributable to E-cadherin cleavage will be unambiguously identified; 2) to investigate the functional domains of BFT including the catalytic domain, the minimal protein domain yielding BET biologic activity and the function of cysteines in the proprotein toxin domain; and 3) to establish that BFT binds specifically to a cellular receptor and to identify the BFT receptor. These studies will advance our understanding of the mechanism of action of a novel and potent bacterial toxin and may provide a molecular rationale for human investigations to further define the morbidity of ETBF colonization and disease.
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