Diarrhea remains the leading cause of death of children under the age of 5 worldwide, and toxigenic diarrheas (such as those caused by cholera toxin, CT) account for nearly 20% of identifiable diarrheas. Determining cellular mechanisms of CT action may aid in the development of improved therapies and preventative measures against CT and the closely related E. coli heat-labile toxin, LT. The overall goal of this proposal is to elucidate the structure and function of detergent-insoluble glycolipid membrane microdomains (DIGs) in the transport of cholera toxin into model human intestinal epithelia (T84 cells). Our first specific aim is to determine the role DIGs play in targeting CT into polarized cell compartments necessary for signal transduction (as evidenced by a C1- secretory response that can be measured electrically). Anthrax edema toxin (AT) elicits a C1- secretory response from T84 cells similar to that of CT, although with different kinetics. However, AT does not bind via DIGs to the cell surface. Chimeric toxins matching cell-binding and enzymatic units from CT and AT will be used to determine if entry to the cell via DIGs is critical to CT's ability to elicit T84 cell secretory responses. The ability to elicit a secretory response as well as the kinetics of secretion will yield information about the importance of cell entry via DIGs to CT action upon polarized epithelia. The second specific aim is to identify membrane proteins associated with CT-specific DIGs. We propose to accomplish this by first characterizing the role of actin in maintaining DIG attachment to our model system, Triton X- 100 extracted T84 monolayers. Secondly, we plan to investigate CT- specific DIG components by chemical cross-linking and co- immunoprecipitation of proteins closely associated with CT and its receptor ganglioside GM1.
