Approximately 1.5 billion cases of diarrhea cause 4 million deaths annually in children under 5 years old, and 5-7 million cases of cholera cause about100,000 deaths. Cholera toxin (CT) from Vibrio cholerae causes the massive watery diarrhea of cholera. Enterotoxigenic E. coli (ETEC) cause up to 20 percent of diarrheal disease in developing countries, and produce heat-labile enterotoxins called LTI and LTII that are closely related to CT in structure and function. The best current vaccines against cholera provide only moderate protection for short periods of time and are not licensed in the United States, and there are no vaccines for human use against ETEC. CT and related enterotoxins are potent immunogens and mucosal adjuvants, and they are also used widely as tools to investigate the role of heterotrimeric G proteins in signal transduction, the role of gangliosides in endocytosis and vesicular trafficking, the mapping and/or ablation of neural pathways, and many other cell functions. We study the structure and function of CT and use LTI and LTII in comparative studies to explore the molecular basis for functional differences between them. Our long term goals are to elucidate the molecular basis for biological activities of CT and related enterotoxins, and to use that knowledge to design novel structure-based vaccines and therapeutics to prevent or treat enterotoxic diarrheas. CT, LTI or LTII are also being studied widely as vaccine components, adjuvants or immunomodulators to prevent or treat diseases unrelated to enterotoxic diarrheas. Important issues concerning structure and function of CT that are not yet understood include identifying and characterizing: conformational changes that activate the catalytic capacity of CT-A1 after nicking and reduction of CT holotoxin; motifs on CT-A1 that determine its interactions with Gsalpha/beta/gamma as a substrate for ADP ribosylation and with ADP-ribosylation factors (ARFs) as stimulators of catalytic activity; features of CT-A and CT-B that enable them to assemble spontaneously into CT holotoxin; mechanisms by which binding of enterotoxins to plasma membrane receptors determines their trafficking within target cells; and pathway(s) by which CT-A1 is translocated from the ER to the cytoplasm to reach its intracellular target and cause toxicity. During the next project period we will use a wide variety of novel methods from microbiology, genetics, biochemistry, cell biology and structural biology to investigate these important current issues concerning the structure and function of cholera toxin.