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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI031940-12
Application #
6435001
Study Section
Bacteriology and Mycology Subcommittee 2 (BM)
Program Officer
Hall, Robert H
Project Start
1992-02-01
Project End
2007-01-31
Budget Start
2002-02-01
Budget End
2003-01-31
Support Year
12
Fiscal Year
2002
Total Cost
$432,697
Indirect Cost
Name
University of Colorado Denver
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
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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