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

7.项目总结/摘要 我们研究计划的总体目标是开发更好的预防视力的治疗策略 由于微生物角膜炎造成的损失，以及通过宿主-病原体增加我们对眼部生物学的理解 交互作用分析这项具体的研究将阐明一种新的宿主-病原体相互作用，这对细菌 眼睛表面的感染。我们关注的是一类经常引起眼部感染的细菌 和多药耐药医院感染，肠杆菌科，这是研究不足，就眼部 感染.重要的是，我们将使用最近临床分离的接触透镜相关性角膜炎 事件而不是可能不共享眼部毒力因子的非眼部实验室菌株。 使用最常引起角膜炎的肠杆菌科成员作为模型生物， 粘质沙雷氏菌，我们发现了一种新的宿主-病原体相互作用，我们将在这项研究中定义。 也就是说，细菌介导的眼细胞死亡的一个步骤是形成不同类型的膜泡。 初步的数据表明了S. marcescens需要一种细菌类型 V分泌系统，一种溶细胞素，以及一种保守但未研究的应激调节因子，我们称之为GumB。 利用我们最先进的显微镜设备，分子工具，可调表达系统， 眼感染模型，我们准备完成我们的研究目标。我们的目标是：1）解决 假设涉及溶细胞素蛋白ShlB和ShlA的V型分泌系统是必需的， 足以在人眼细胞中诱导膜泡。这将带来一个全新的 细菌影响角膜细胞的机制; 2）确定GumB 调节滤过泡形成和对角膜上皮细胞的细胞毒性。这一目标将揭示 GumB作为一种毒力因子调节因子，是一种保守的细菌蛋白质，在许多种属中发现， 与眼部宿主-病原体相互作用; 3）使用兔建立GumB在眼部毒力中的作用 接触性透镜角膜炎模型。这项工作将表明GumB是一种新的眼毒力主调节因子 这些因素，并将确定这些因素。这些研究有可能发现一种全新的机制 细菌通过它影响人体细胞和角膜疾病。长期的影响是， 角膜炎至少部分是由于这种宿主-病原体相互作用。该机制的解决办法可以 为更有效的感染治疗奠定基础，例如通过化学抑制 研究的蛋白质。

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.