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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Bacteriology and Mycology Subcommittee 2 (BM)
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University of Colorado Denver
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Jobling, Michael G (2016) The chromosomal nature of LT-II enterotoxins solved: a lambdoid prophage encodes both LT-II and one of two novel pertussis-toxin-like toxin family members in type II enterotoxigenic Escherichia coli. Pathog Dis 74:
Day, Charles A; Baetz, Nicholas W; Copeland, Courtney A et al. (2015) Microtubule motors power plasma membrane tubulation in clathrin-independent endocytosis. Traffic 16:572-90
Banerjee, Tuhina; Taylor, Michael; Jobling, Michael G et al. (2014) ADP-ribosylation factor 6 acts as an allosteric activator for the folded but not disordered cholera toxin A1 polypeptide. Mol Microbiol 94:898-912
Price, Gregory A; Holmes, Randall K (2014) Immunizing adult female mice with a TcpA-A2-CTB chimera provides a high level of protection for their pups in the infant mouse model of cholera. PLoS Negl Trop Dis 8:e3356
Price, Gregory A; McFann, Kim; Holmes, Randall K (2013) Immunization with cholera toxin B subunit induces high-level protection in the suckling mouse model of cholera. PLoS One 8:e57269
Arifuzzaman, Mohammad; Rashu, Rasheduzzaman; Leung, Daniel T et al. (2012) Antigen-specific memory T cell responses after vaccination with an oral killed cholera vaccine in Bangladeshi children and comparison to responses in patients with naturally acquired cholera. Clin Vaccine Immunol 19:1304-11
Price, Gregory A; Holmes, Randall K (2012) Evaluation of TcpF-A2-CTB chimera and evidence of additive protective efficacy of immunizing with TcpF and CTB in the suckling mouse model of cholera. PLoS One 7:e42434
Jobling, Michael G; Holmes, Randall K (2012) Type II heat-labile enterotoxins from 50 diverse Escherichia coli isolates belong almost exclusively to the LT-IIc family and may be prophage encoded. PLoS One 7:e29898
Jobling, Michael G; Yang, Zhijie; Kam, Wendy R et al. (2012) A single native ganglioside GM1-binding site is sufficient for cholera toxin to bind to cells and complete the intoxication pathway. MBio 3:
Korotkov, Konstantin V; Johnson, Tanya L; Jobling, Michael G et al. (2011) Structural and functional studies on the interaction of GspC and GspD in the type II secretion system. PLoS Pathog 7:e1002228

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