Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera. To induce disease, CT must bind ganglioside GM1 on the host cell apical membrane, enter the cell by endocytosis, and then somehow cross the cell to activate adenylyl cyclase on the cytoplasmic surface of the basolateral membrane. The investigator published data show that CT may move retrograde through Golgi and ER before arrival at the basolateral membrane, and that sorting into this pathway may depend on the lipid-based membrane anchor provided by the toxin's receptor GM1. GM1 concentrates CT in detergent-insoluble glycolipid-rich apical membrane microdomains (DIGs or """"""""lipid rafts""""""""). The applicant hypothesizes that GM1 endows CT with a lipid-based sorting motif that specifies association with DIGs and trafficking into the apical endosome, Golgi cisternae, ER, or transcytotic pathway. It is also not known how the toxin's enzymatic A-subunit enters the cytosol of host cells. Since CT must enter the ER for bioactivity, the investigator hypothesizes that C2 opportunistically utilizes the ER associated degradation system (ERAD) to unfold and cross the membrane via a protein translocase, possibly sec61p. To test these ideas, the applicant will define whether the ceramide domain of GM1 specifies the selective association with DIGs and toxin action in polarized T84 cells. The PI will use toxin variants deficient in clustering GM1 to test if cross-linking individual gangliosides is a prerequisite for association with DIGs or toxin function. The PI will examine raft dependence on membrane cholesterol by using beta-methyl-cyclodextrin, heterogeneity in GM1 content by using a CT variant attenuated in binding GM1, and functional association with the cortical cytoskeleton by membrane fractionation, disruption of actin filaments, and depletion of cholesterol. To test whether GM1 specifies toxin sorting into Golgi and ER, as opposed to the endosome-lysosomal or direct transcytotic pathway, the intracellular itinerary of CT (that binds GM1) and the closely related E. coli toxin LTIIb (that binds ganglioside GD1a) will be systematically compared. Toxin entry into the Golgi or ER, will be defined by microscopy and by exploiting the trans-Golgi specific transfer of sulfate and the ER specific transfer of N-linked oligosaccharides to label CT in these compartments. The mechanism of toxin-unfolding and dislocation from the ER to cytosol will be examined in vitro using purified ER lumenal and membrane proteins, and in intact T84 cells through the use of selected toxin variants lacking sites for proteolytic nicking, ubiqitination and the cysl87-199 disulfide bond in the toxin's enzymatic A-subunit.
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