The long range goals of this project are to understand the structure and function of cholera toxin at the molecular level and apply that knowledge for development of improved vaccines against cholera. During the current project period we will use site-directed and regional mutagenesis to analyze the structure-function relationships of the A and B polypeptides of cholera toxin (CT),analyze the mechanisms by which Vibrio cholerae excretes CT as and extracellular protein, and construct chimeric cholera toxin neoantigens that express additional protective antigens of V. cholerae to test for mucosal immunogenicity and adjuvanticity. We recently constructed a fusion protein consisting of alkaline phosphatase and the A2 domain of CT and demonstrated that it can assemble in vivo with native CT-B to form functional chimeric holotoxin. This finding provides the basis for a novel method to construct chimeric cholera toxins as vehicles for delivery of foreign antigens to mucosal surfaces in a highly immunogenic form. Our mutational analysis of CT-B will characterize structural features required for formation of B pentamers, assembly of holotoxin, and binding to ganglioside GM1. Our studies of CT-A will analyze structural features required for holotoxin assembly, catalytic activity and stimulation by ADP ribosylation factors. Mutant CTs that we generate will be screened with previously characterized monoclonal anti-CT and anti-LT antibodies to identify structural changes in CT-A or CT-B that affect expression of conformationally dependent epitopes. We recently cloned a gene (excA) that is required for translocation of CT across the outer membrane of V. cholerae. We will analyze the mechanism of CT excretion by characterizing the excA gene product and determining if additional exc genes are required for excretion of CT. We will also test whether exc gene products can function as protective immunogens against cholera. Ct is one of the most potent mucosal immunogens known, and it stimulates the mucosal immune response to other antigens administered simultaneously. The molecular basis for these properties of CT will be analyzed by comparing the mucosal immune responses of mice to wild type CT and mutant CTs deficient in specific functions (e.g., ganglioside GM1-binding activity of CT-B or enzymatic activity of CT-A), and chimeric CT neoantigens will be tested for their potency as multivalent mucosal immunogens.