Identification of Rab11 as a target of edema factor and dominant-negative mutants of anthrax lethal factor

Anthrax is a fatal disease caused by the spore-formulating bacterium, Bacillus anthracis. The potential for anthrax as a bioterrorist weapon raises concern, and is still common in the developing world. Antibiotic treatment of anthrax is often not effective, because toxins released in the host bloodstream can cause death even after clearance of the bacterium. Thus, it is important to further develop our knowledge of how anthrax causes its deadly effects as well as develop treatments targeting the toxins themselves. The anthrax toxin edema factor (EF) is a calmodulin-dependent adenylate cyclase and lethal factor (LF) is a zinc metalloprotease, which cleaves and inactivates MAPKKs. Like many other bacterial toxins, their activity targets conserved cellular components, making it possible to study their activity using an invertebrate model such as Drosophila. Using UAS-EF and UAS-LF transgenic lines, an extensive interaction screen with candidate UAS lines was used to identify novel targets for toxin activity. EF and LF showed synergistic interaction with Rab11 GTPase, a recycling endosome regulator, and its exocyst binding partner Sec15. Also, loss of other exocyst components Sec5, Sec15, and Exo70 enhanced the severity of the LF phenotype. Two other candidates, CG5745 and CG17282, disrupt the exocyst through their action on Sec15 and possibly Rab11. Increased knowledge of toxin activity in the long run may prove to be useful in rational drug design. Furthermore, as a more direct approach to treatment development, I conducted a genetic screen, which generated two dominant negative mutants of lethal facto

Cholera Toxin Disrupts Barrier Function by Inhibiting Exocyst-Mediated Trafficking of Host Proteins to Intestinal Cell Junctions

Cholera toxin (CT), a virulence factor elaborated by Vibrio cholerae, is sufficient to induce the severe diarrhea characteristic of cholera. The enzymatic moiety of CT (CtxA) increases cAMP synthesis in intestinal epithelial cells, leading to chloride ion (Cl(-)) efflux through the CFTR Cl(-) channel. To preserve electroneutrality and osmotic balance, sodium ions and water also flow into the intestinal lumen via a paracellular route. We find that CtxA-driven cAMP increase also inhibits Rab11/exocyst-mediated trafficking of host proteins including E-cadherin and Notch signaling components to cell-cell junctions in Drosophila, human intestinal epithelial cells, and ligated mouse ileal loops, thereby disrupting barrier function. Additionally, CtxA induces junctional damage, weight loss, and dye leakage in the Drosophila gut, contributing to lethality from live V. cholerae infection, all of which can be rescued by Rab11 overexpression. These barrier-disrupting effects of CtxA may act in parallel with Cl(-) secretion to drive the pathophysiology of cholera