INTESTINAL DISEASE--ENTEROCYTE TOXIN INTERACTION

Children'S Hospital Boston, Boston MA

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Abstract

DESCRIPTION: The goal of this proposal is to elucidate the cellular mechanisms which target cholera toxin (CT) and E. coli heat labile toxin (LT) into basolaterally directed transcytotic pathway of polarized intestinal epithelia. Transcytosis of macromolecules across the intestinal barrier plays an essential role in the absorption of selected nutrients, in the secretion of polymeric immunoglobulins, and in antigen delivery to the mucosal immune system. Very little is known, however, about the membranes or cellular mechanisms that mediate these processes. CT opportunistically enters a transcytotic pathway in polarized human intestinal T84 cells, and provides an experimental model to examine the details of this endogenous pathway. Recombinant Type I and Type IIb labile toxins will be used to ascertain the specific role of the cell surface receptor, ganglioside GM1, in targeting CT into the endosome and Golgi complex of T84 monolayers. Whether GM1-rich membrane domains (`caveolae') may mediate this transport will be examined. A combination of biophysical, biochemical, and morphologic approaches will be used. Chimera toxins mixing the B-subunits of Type I and Type II LT will be constructed to confirm experimental findings. Whether contact with apical membranes, or passage through endosomes and trans Golgi are sufficient for proteolytic activation of CT A-subunits will be determined using recombinant wt and mutant protease-resistant CT. Whether toxin entry into Golgi cisternae is a prerequisite for membrane translocation of the A-subunit will be examined by morphology using monolayers permeabilized on the basolateral membrane. Interaction between CT and the regulatory GTP-binding protein ADP-ribosylating Factor (ARF) will be examined by protein chemistry in "caveolae" and Golgi fractions, and by function in T84 cell monolayers permeabilized at the basolateral membrane. Finally, the role of microtubules in endocytosis, retrograde Golgi transport, and trafficking through the basolaterally directed transcytotic pathway will be examined biophysically and morphologically. The significance of these studies is emphasized by the prevalence of toxigenic secretory diarrhea's worldwide, and the clear ability of oral CT to breach the epithelial barrier and act on the mucosal immune system. Elucidating the cellular components involved in such transport may lead to further clarification of these fundamental aspects of epithelial cell biology. Such information may enhance the development of novel strategies for oral immunization and tissue specific drug delivery for use in a variety of intestinal diseases.