Cholera toxin (CT) produced by Vibrio cholerae is the virulence factor responsible for the massive secretory diarrhea seen in Asiatic cholera. To induce disease, CT must bind ganglioside GM1 on the host cell apical membrane, enter the cell by endocytosis, and then somehow cross the cell to activate adenylyl cyclase on the cytoplasmic surface of the basolateral membrane. The investigator published data show that CT may move retrograde through Golgi and ER before arrival at the basolateral membrane, and that sorting into this pathway may depend on the lipid-based membrane anchor provided by the toxin's receptor GM1. GM1 concentrates CT in detergent-insoluble glycolipid-rich apical membrane microdomains (DIGs or """"""""lipid rafts""""""""). The applicant hypothesizes that GM1 endows CT with a lipid-based sorting motif that specifies association with DIGs and trafficking into the apical endosome, Golgi cisternae, ER, or transcytotic pathway. It is also not known how the toxin's enzymatic A-subunit enters the cytosol of host cells. Since CT must enter the ER for bioactivity, the investigator hypothesizes that C2 opportunistically utilizes the ER associated degradation system (ERAD) to unfold and cross the membrane via a protein translocase, possibly sec61p. To test these ideas, the applicant will define whether the ceramide domain of GM1 specifies the selective association with DIGs and toxin action in polarized T84 cells. The PI will use toxin variants deficient in clustering GM1 to test if cross-linking individual gangliosides is a prerequisite for association with DIGs or toxin function. The PI will examine raft dependence on membrane cholesterol by using beta-methyl-cyclodextrin, heterogeneity in GM1 content by using a CT variant attenuated in binding GM1, and functional association with the cortical cytoskeleton by membrane fractionation, disruption of actin filaments, and depletion of cholesterol. To test whether GM1 specifies toxin sorting into Golgi and ER, as opposed to the endosome-lysosomal or direct transcytotic pathway, the intracellular itinerary of CT (that binds GM1) and the closely related E. coli toxin LTIIb (that binds ganglioside GD1a) will be systematically compared. Toxin entry into the Golgi or ER, will be defined by microscopy and by exploiting the trans-Golgi specific transfer of sulfate and the ER specific transfer of N-linked oligosaccharides to label CT in these compartments. The mechanism of toxin-unfolding and dislocation from the ER to cytosol will be examined in vitro using purified ER lumenal and membrane proteins, and in intact T84 cells through the use of selected toxin variants lacking sites for proteolytic nicking, ubiqitination and the cysl87-199 disulfide bond in the toxin's enzymatic A-subunit.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK048106-12
Application #
6897776
Study Section
General Medicine A Subcommittee 2 (GMA)
Program Officer
Hamilton, Frank A
Project Start
1993-09-01
Project End
2006-04-30
Budget Start
2005-04-01
Budget End
2006-04-30
Support Year
12
Fiscal Year
2005
Total Cost
$337,725
Indirect Cost
Name
Children's Hospital Boston
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02115
Luong, Phi; Hedl, Matija; Yan, Jie et al. (2018) INAVA-ARNO complexes bridge mucosal barrier function with inflammatory signaling. Elife 7:
Garcia-Castillo, Maria Daniela; Chinnapen, Daniel J-F; Te Welscher, Yvonne M et al. (2018) Mucosal absorption of therapeutic peptides by harnessing the endogenous sorting of glycosphingolipids. Elife 7:
Pyzik, Michal; Rath, Timo; Kuo, Timothy T et al. (2017) Hepatic FcRn regulates albumin homeostasis and susceptibility to liver injury. Proc Natl Acad Sci U S A 114:E2862-E2871
Garcia-Castillo, Maria Daniela; Chinnapen, Daniel J-F; Lencer, Wayne I (2017) Membrane Transport across Polarized Epithelia. Cold Spring Harb Perspect Biol 9:
Thiagarajah, Jay R; Chang, Jeffrey; Goettel, Jeremy A et al. (2017) Aquaporin-3 mediates hydrogen peroxide-dependent responses to environmental stress in colonic epithelia. Proc Natl Acad Sci U S A 114:568-573
Nelms, Bradlee D; Waldron, Levi; Barrera, Luis A et al. (2016) CellMapper: rapid and accurate inference of gene expression in difficult-to-isolate cell types. Genome Biol 17:201
Saslowsky, David E; Thiagarajah, Jay R; McCormick, Beth A et al. (2016) Microbial sphingomyelinase induces RhoA-mediated reorganization of the apical brush border membrane and is protective against invasion. Mol Biol Cell 27:1120-30
Raghunathan, Krishnan; Wong, Tiffany H; Chinnapen, Daniel J et al. (2016) Glycolipid Crosslinking Is Required for Cholera Toxin to Partition Into and Stabilize Ordered Domains. Biophys J 111:2547-2550
Lencer, Wayne I (2016) Opening CFTR in the Intestine: Flushing on Demand. Cell Mol Gastroenterol Hepatol 2:256
Lexmond, Willem S; Rufo, Paul A; Fiebiger, Edda et al. (2015) Electrophysiological Studies into the Safety of the Anti-diarrheal Drug Clotrimazole during Oral Rehydration Therapy. PLoS Negl Trop Dis 9:e0004098

Showing the most recent 10 out of 77 publications