The hypothesis of this proposal is that following the attachment of enteropathogenic E. coli to intestinal cells, specific signal transduction pathways are activated which induce changes in intestinal epithelial function including active ion transport, barrier function and recruitment of inflammatory cells. The long-term objective is to understand the mechanisms by which EPEC induces alterations in host intestinal epithelial physiology.
Specific aims are: 1). To investigate the influence of EPEC on intestinal secretion and absorption. This will involve ion flux studies in Ussing chambers using T-84 cells. Preliminary data suggests that EPEC infection decreases the ISC, but increases chloride secretion. EPEC mutant strains, differing in expression of adherence or signaling factors, and enzyme inhibitors will be used to define the role of specific EPEC genetic factors and signal cascades. 2). To elucidate the role of IL-8 in EPEC-stimulated PMN transmigration and to determine the pathways through which EPEC regulates IL-8 expression. The investigators have demonstrated that epithelial cells secrete IL-8 in response to EPEC. EPEC regulation of the IL-8 gene transcription will be studied. Electrophoretic shift assays will examine the roles of transcription factors, in particular, NFKB. 3). To determine the effects of individual EPEC proteins (bundlin, intimin, eaeB) on specific epithelial functions and determine the signaling pathways involved. EPEC will be fractionated and the effects of these fractions on function will be examined. Specific proteins of interest will be purified and studied in functional assays.
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