The overarching goal of this application is to elucidate the molecular basis for retro-translocation of the cholera toxin (CT) A1-chain from within the lumen of the endoplasmic reticulum (ER) of host cells to the cytosol. A second goal is to determine if host cells innately sense the toxin in the ER or during the process of retro-translocation so as to induce an inflammatory or other host defense response. CT produced by Vibrio cholerae breeches the intestinal epithelial barrier and enters host epithelial cells to cause disease. CT belongs to the AB family of toxins, where the A component is enzymatically active and the B component is responsible for binding the membrane and mediating entry of the toxin into the cell;traveling from the plasma membrane to the Golgi and ER. After arrival in the ER, a portion of the A-subunit is unfolded and dissociated from the B-subunit by the ER chaperone PDI, targeted to a protein conducting channel, and transported to the cytosol. Shiga toxin and ricin follow a similar pathway. Each of these toxins has evolved structurally to exploit the normal quality control function of the ER that identifies and degrades terminally misfolded proteins in the biosynthetic pathway;the process is termed ER associated degradation (ERAD) or retro-translocation.
In Aim 1, we will examine three steps in the retro-translocation reaction: targeting the PDI-A1-chain complex to the ER lumenal membrane, transporting the A1-chain through the protein-conducting channel, and the driving force for this reaction. Our approach will be to prepare a mutant toxin predicted to be reversibly trapped as a retro-translocation intermediate in intact cells so as to identify the proteins that interact with the A1-chain;and that can be applied in a cell-free model using ER proteoliposomes. Reverse genetics using shRNA technology will be used to test dependence on certain proteins known to be required for retro-translocation.
In Aim 2, we will use mutant toxins lacking all enzymatic activity, deficient in retrotranslocation, or unable to bind GM1 to test if CT induces ER-stress or some other signal for sensing the bacterial protein in the ER or in the process of co-opting ERAD. Because CT is a prominent bacterial factor that signals the host in regulating intestinal physiology and adaptive mucosal immunity, we want to know how signal transduction by CT in the inflammatory response is explained. The significance of these studies pertains to their relevance to epithelial mucosal biology and a broad range of clinically important diseases, including acute infectious diarrheas and inflammatory bowel disease. CT is the virulence factor responsible for the massive diarrhea seen in Asiatic cholera. Cholera remains prevalent in many parts of Asia, Africa and Latin America and outbreaks can occur in areas affected by natural disasters, wars and famines .
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