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By developing a detailed understanding of the role of intimin in the pathogenesis of EHEC, the proposed experiments may lead to therapeutic strategies designed to prevent colonization at one of the earliest steps or to minimize the intestinal absorption of toxin after infection has been established.
Enterohemorrhagic E. coli (EHEC) is an important cause of diarrheal disease and renal failure in the U.S. EHEC colonizes the mucosa of the large bowel, and the extraintestinal manifestations of EHEC infection result from the absorption across the epithelium of Shiga-like toxin (Stx) produced by intestinal EHEC. During attachment to colonic epithelium, the bacterium disrupts the host cell cytoskeleton and forms a highly organized cytoskeletal structure underneath the bound bacterium, termed an attaching and effacing (AE) lesion. Intimin, a bacterial outer membrane protein that mediates tight host cell attachment, is required for AE lesion formation and full virulence. Experimental infection with EHEC expressing intimin from enteropathogenic E. coli (EPEC), a pathogen that infects a different intestinal site, suggested that intimin influences tissue tropism. We postulate that: (1) intimin plays a central role in determining the site of colonization; and that (2) intimin-mediated cytoskeletal disruption of intestinal epithelial cells facilitates delivery of Stx to extraintestinal sites. To characterize the features of intimin that influence tissue tropism and promote Stx translocation, the following questions will be addressed: 1. Does an EHEC strain that expresses EPEC intimin under appropriate regulatory controls demonstrate altered tissue tropism? The EHEC chromosomal eae coding sequence will be specifically replaced by the EPEC eae coding sequence, and the effect of this alteration on tissue tropism will be assessed. 2. What domain of intimin influences the site of intestinal colonization? The region of EPEC intimin responsible for the differences in tissue tropism that we expect to find in Aim 1 will be identified by analyzing strains that express hybrid EHEC/EPEC intimin proteins. 3. Does the ability to generate robust AE lesions correlate with mucosal damage and/or toxin translocation? Isogenic EHEC strains differing in their ability to generate AE lesions will be characterized for differences in mucosal damage and in translocation of toxin across intestinal epithelium.