Bacterial Factors that Regulate Ocular Host-Pathogen Interactions and Microbial Keratitis

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7. Project Summary / Abstract The overall goal of our research program is to develop better treatment strategies for the prevention of vision loss due to microbial keratitis, as well as increasing our understanding of ocular biology through host-pathogen interaction analysis. This specific study will elucidate a novel host-pathogen interaction important for bacterial infection of the ocular surface. We are focusing on a class of bacteria that frequently cause ocular infections and multidrug resistant hospital infections, the Enterobacteriaceae, that is understudied with respect to ocular infections. Importantly, we will be using recent clinical isolates derived from contact lens associated keratitis event rather than non-ocular laboratory strains that may not share ocular virulence factors. Using the member of the Enterobacteriaceae that most commonly causes keratitis as a model organism, Serratia marcescens, we have uncovered a novel host-pathogen interaction that we will define in this study. Namely, a step in bacteria-mediated ocular cell death is the formation of a distinct type of membrane bleb. Preliminary data indicates the mechanism underlying bleb induction by S. marcescens requires a bacterial type V secretion system, a cytolysin, and a conserved but unstudied stress regulator we are calling GumB. Taking advantage of our state of the art microscopy facility, molecular tools, tunable expression systems, and ocular infection models, we are poised to complete our research goals. Our aims will: 1) address the hypothesis that a type V secretion system involving cytolysin proteins ShlB and ShlA is necessary and sufficient to induce membrane blebs in human ocular cells. This will introduce a completely new mechanism by which bacteria influence corneal cells; 2) determine the mechanism by which GumB regulates bleb formation and cytotoxicity to corneal epithelial cells. This aim will uncover the role of GumB as a virulence factor regulator, which is a conserved bacterial protein found in many species associated with ocular host-pathogen interactions; 3) establish the role of GumB in ocular virulence using a rabbit contact lens keratitis model. This work will show that GumB is a novel master regulator of ocular virulence factors, and will identify those factors. These studies have the potential to discover an entirely new mechanism by which bacteria impact human cells and corneal disease. The long-term implication is that CL-associated keratitis is due, at least in part, to this host-pathogen interaction. The resolution of this mechanism could establish the foundation for more effective infection treatments, such as through chemical inhibition of the proteins studied here.