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Our long-term goal is to develop new therapeutic strategies to combat diarrhea in human and livestock caused by food borne pathogens.<P> The objective of this particular application is to test the central hypothesis that bacterial enterotoxins precondition the host intestinal epithelium for colonization by the population of bacteria from which the toxins are expressed. We have formulated this hypothesis based on our preliminary findings that suggest i) heat-labile enterotoxin (LT)-positive strains bind the IPEC-J2 porcine intestinal cell line with higher affinity than do LT-negative strains, and ii) bacterial adhesion to intact epithelial cells does not proceed at a rate consistent with the typical kinetics of receptor-adhesin interactions. <P>Preconditioning of the host cell by diffusible enterotoxin may play a significant role in enhancing the ability of enteric bacteria to adhere to the host. The similarities in both intestinal colonization and enterotoxin secretion mechanisms between E. coli and other enteric pathogens suggest that development of novel therapeutic strategies against enterotoxins will have broad utility.
Non-Technical Summary: Escherichia coli is a significant source of food borne disease and a leading cause of worldwide malnutrition and infant mortality. Bacterial toxins appear to have a role in assisting bacteria in efficient colonization of their host. The applicant is studying these bacterial toxins and expressing them in an inactivated state to provide prophylactic protection against E. coli. <P> Approach: We will test our central hypothesis and accomplish the overall objective of this application by pursuing the following specific aims: Specific Aim1. Characterize the role of the heat-labile enterotoxin (LT) in preconditioning the intestinal epithelium for bacterial colonization. Our working hypothesis is that pretreatment of the porcine intestinal epithelial cell line IPEC-J2 with LT will enhance the kinetics and magnitude of ETEC adherence. To test this hypothesis, we will incubate IPEC-J2 cells with purified LT, followed by experimental challenge with well-characterized ETEC strains. After challenge, we will quantify bacterial adherence. Specific Aim 2. Quantify the colonizing ability of an ETEC strain expressing a catalytically inactive LT mutant (R192G) compared to an isogenic strain expressing native LT. Our working hypothesis is that catalytically inactive LT, which fails to stimulate host secretory activity, will also fail to enhance bacterial intestinal colonization. To test this hypothesis, we will incubate IPEC-J2 cells with a catalytically inactive LT mutant or with an isogenic strain expressing native LT. After challenge, we will quantify bacterial adherence. Specific Aim 3. Determine the extent to which the ETEC heat-stable enterotoxin STa and the EHEC Shiga toxin 2 (Stx2), which act through different enzymatic mechanisms than LT, enhance bacterial colonization to intestinal epithelial cells. Our working hypothesis is that the ability of enterotoxins to enhance colonization is independent of their specific enzymatic activity. To test this hypothesis, we will incubate IPEC-J2 cells with an ETEC strain that expresses STa and compare bacterial adherence to an isogenic ETEC strain that does not express STa. We will extend these studies to examine EHEC Stx2 by determining the extent of correlation between the magnitude of Stx2 expression and the ability of EHEC to adhere to Caco-2 cells.