The goal of this application is to elucidate the molecular basis for invasion and intoxication of intestinal cells by cholera toxin (CT). To induce disease, CT (and the other AB5 toxins) must breech the mucosal epithelial barrier that is normally impermeant to macromolecules by passive diffusion. CT does this as a stably folded protein complex by entering the intestinal epithelial cell after co-opting a retrograde lipid trafficking pathway from the plasma membrane (PM), through the trans Golgi, to the endoplasmic reticulum (ER). The pathway from PM to ER is a specific lipid, not a """"""""protein"""""""", sorting pathway. It is also a general pathway hijacked by all the AB5 toxins and the polyoma viruses to cause disease. Once in the ER, a portion of CT, the A1 chain, co-opts the cellular machinery that allows terminally misfolded proteins in the secretory pathway to cross the ER membrane for degradation in the cytosol, a process termed retro-translocation. These are fundamental aspects of epithelial cell function that are broadly clinically relevant and poorly understood.
In Aim 1, we will use zebrafish in forward and reverse genetic studies to elucidate molecular components involved in CT toxicity. We have recently discovered that zebrafish model all aspects of the cell biology hijacked by CT to cause disease in mammalian cells, including retrograde transport from PM to ER and retro-translocation to the cytosol.
In Aim 2, we will use fourier transform mass spectrometry to identify structural isoforms of the glycolipid receptor ganglioside GM1 that explain how the epithelial cell sorts GM1 specifically into the retrograde pathway. We will confirm the identity of these structures using synthetic GM1 isoforms in functional reconstitution experiments. We will use wild-type (wt) and mutant toxins with altered binding function to measure diffusional coefficients and clustering efficiencies of the CT-GM1 complex in the cell membrane. This will test how GM1 may couple the toxin to lipid rafts that appear to function as key trafficking platforms.
In Aim 3, we will use wt and a variety of mutant toxins for advanced imaging of live and fixed cells, and for novel biochemical in vitro vesicular transport assays to identify the intracellular compartment(s) and molecular mechanism(s) that sort the CT-GM1 complex away from the other glycolipids (and their cargos) and into the retrograde pathway. Reverse genetics in cell culture will also be used.
In Aim 4, we will use reverse genetics in cell culture and in zebrafish, and a novel in vitro protease protection assay based on a mutant toxin containing a cleavable HA-tag. The assay will biochemically model the retro-translocation reaction in order to examine the molecular components essential for this process. The significance of these studies pertains to their relevance to epithelial mucosal biology and a broad range of clinically important diseases. Such diseases are global in distribution and include acute infectious diarrheas as well as those that result from abnormal interactions with the intestinal microflora, such as IBD.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
Project #
Application #
Study Section
Gastrointestinal Mucosal Pathobiology Study Section (GMPB)
Program Officer
Hamilton, Frank A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Children's Hospital Boston
United States
Zip Code
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
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
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
Rath, Timo; Baker, Kristi; Dumont, Jennifer A et al. (2015) Fc-fusion proteins and FcRn: structural insights for longer-lasting and more effective therapeutics. Crit Rev Biotechnol 35:235-54
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
Lencer, Wayne I; DeLuca, Heidi; Grey, Michael J et al. (2015) Innate immunity at mucosal surfaces: the IRE1-RIDD-RIG-I pathway. Trends Immunol 36:401-9

Showing the most recent 10 out of 74 publications