application) The essential first stage of typical E. coli O157:H7 infection is colonization of the intestinal tract. To date, only one bacterial factor has been shown to mediate intestinal adherence by O157:H7, the outer membrane protein intimin which was originally discovered in the Principal Investigator's laboratory. However, a variety of observations indicate that additional intestinal colonization factors may play a role in disease due to E. coli O157:H7 and Shiga toxin-producing E. coli of serotypes other than O157:H7 The overall goal of this project is to investigate the mechanisms by which E. coli O157:H7 and non-O157:H7 enterohemorrhagic E. coli colonize the intestine. Our approach will be focused in four specific aims. Three of the specific aims will focus on characterization of three newly discovered potential colonization factors of EHEC. Two of these factors have homology to previously described adhesins in other enteric pathogens and the third factor has no obvious homology to previously described adhesins. For each of these factors we will construct isogenic non-polar mutations in the structural gene and test the mutants for decreased adherence in differential intestinal epithelial cell lines, freshly harvested human intestinal epithelial cells, and in a gnotobiotic piglet model of disease. The demonstrated involvement of any of the three novel colonization factors in intestinal adherence would provide new targets for vaccine development.
The fourth aim will focus on a novel regulatory mechanism of the best-characterized EHEC colonization factor, intimin. We recently showed that in vitro expression of the intimin adhesin and the type III secretion system encoded on the LEE pathogenicity island is positively regulated by autoinducer molecules expressed by normal flora E. coli strains, i.e., quorum sensing. We propose to test whether quorum sensing is active in vivo by constructing an O157:H7 derivative that no longer responds to quorum sensing and testing it for colonization in a pig model. If quorum sensing is active in vivo, this would open up a new area for potential therapeutic intervention based on inhibiting the quorum sensing mechanism, thereby decreasing expression of intestinal colonization factors.
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