The goal of this proposal is to elucidate the endocytic and transcellular pathways taken by cholera toxin (CT) in polarized intestinal epithelia, and the interaction of toxin with endogenous components of membrane traffic. We have recently used the human intestinal cell line, T84, as an in vitro model of intestinal epithelium to study the action of CT on polarized intestinal epithelial cells. We have shown that two sequential events involving vesicular traffic of CT-containing membranes are essential for apical CT action. However, the exact route and the molecular mechanisms that allow the CT-A subunit to gain access to basolateral adenylate cyclase are not known. In addition, CT strongly enhances the mucosal immune response to orally delivered antigens, especially when antigens are also covalently bound to the B-subunit of the toxin, but whether vesicular transport of CT is important in this phenomenon remains unknown. First, we propose to examine whether toxin passage through acidic organelles, trans Golgi, or retrograde transport to Golgi cisternae or endoplasmic reticulum is necessary for the reductive cleavage and translocation of the A-subunit. We will also determine whether entry into transcytotic vesicles is necessary for the enzymatic activation of basolateral adenylate cyclase. A combination of biophysical, biochemical, and morphologic approaches will be used. The mechanism(s) by which CT assures its own delivery to subcellular compartments necessary for toxin action will be examined: l) in intact cells using recombinant toxin with point mutations in the """"""""targeting sequence"""""""" KDEL; 2) in CT-specific vesicle fractions using antibodies to the regulatory GTP-binding protein ADP-ribosylating Factor (ARF); and 3) in T84 cell monolayers permeabilized at the basolateral membrane. Finally, we will test the ability of CT to affect the endocytosis and possible transepithelial transport of model antigens by altering membrane traffic across epithelial cells. For these studies, T84 monolayers mounted in modified Ussing chambers and an established in vivo model for the quantitative assessment of transepithelial transport and the mucosal immune response to antigens covalently linked to the CT-B-subunit or other """"""""carrier"""""""" proteins and particles will be used. The significance of these studies is emphasized by the prevalence of toxigenic secretory diarrhea's which account for nearly 20% of the identifiable diarrhea worldwide and the clear ability of oral CT to act on the mucosal immune system. Elucidating the mechanism(s) and endogenous components involved in the interaction between CT and intestinal epithelia may lead to further clarification of these fundamental aspects of epithelial cell biology. Such information can enhance the development of novel strategies for oral immunization and tissue specific drug delivery for use in intestinal diseases such as IBD (inflammatory bowel disease).
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