The major virulence factor of enterohemorrhagic Escherichia coli 0157:H7 is a family of potent cytotoxins known as Shiga toxin, Shiga-like toxin or verocytotoxin. Data from a variety of in vitro, animal, and human studies support the concept that Shiga toxins are crucial for the development of the HUS and hemorrhagic colitis caused by this pathogen. However, there are few data concerning other potential virulence factors expressed by E. coli 0157:H7. Previous work in this laboratory has identified an outer membrane protein known as intimin, encoded by the eaeA gene, which is involved in intestinal colonization by this pathogen. We have recently discovered several bacterial proteins that are secreted into the culture supernatant. These proteins, which are not Shiga toxins and are not produced by non-pathogenic E. coli, produce a strong serum antibody response in patients with HUS. We hypothesize that these proteins play an important role in the pathogenesis of disease due to E. coli 0157:H7. A multi-faceted approach is proposed to investigate these proteins. The genes encoding these proteins will be cloned and sequenced and the distribution of these genes among other Shiga toxin-producing E. coli will be studied. An ELISA assay will be developed for serodiagnosis and seroepidemiology studies and a large collection of well-characterized patient sera will be screened with this ELISA. Correlations of protein secretion patterns, patient immune response patterns and clinical outcome will be sought to study whether these proteins or the response to them can be used as a marker for clinical outcome. Using purified proteins and isogenic E. coli 0157:H7 strains specifically mutated in genes encoding these proteins, the effect of these proteins on cultured intestinal and renal cells will be studied and potential interactions with Shiga toxin will be examined in these in vitro systems. Isogenic strains mutated in these genes will also be studied in animal models to examine the contribution of these proteins to intestinal and renal manifestations of disease due to E. coli 0157:H7. Together, these studies should yield a more complete picture of disease due to EHEC and offer the possibility of new insights into pathogenesis, improved serodiagnostic methodologies, and identification of potential protective antigens that may be used for development of vaccines or immunotherapeutic agents.
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