Pathogensis of Bacterial corneal infection

描述(由申请人提供)：这项资助申请的广泛、长期目标是了解作为角膜感染重要原因的细菌病原体的分子和细胞宿主反应。具体地说，这一应用将集中在铜绿假单胞菌和金黄色葡萄球菌引起的感染上。这两种病原体是导致严重角膜感染的最常见原因之一。这两种病原体的菌株已经获得了抵抗抗菌治疗的重要手段，并详细阐述了导致角膜损伤和视力丧失的大量毒力因子。这些感染的治疗方法需要减少细菌数量，并允许宿主对清除病原体有足够和有用的炎症反应，而不会对角膜造成损害。对于铜绿假单胞菌，大多数感染划痕损伤小鼠眼睛的细菌都在细胞内发现，进入细胞需要与囊性纤维化跨膜传导调节因子(CFTR)结合。这会导致炎症的启动，包括激活促炎基因的转录因子，合成促炎分子，以及启动细胞内流，主要由PMN组成，这既可以清除病原体，也可以对角膜造成损害。PMN的内流也受TH17调节性T细胞网络的控制，这也将在这些研究中进行研究。为了确定CFTR如何协调炎症，我们将分析携带野生型CFTR的细胞感染铜绿假单胞菌后产生的特定效应器，将这些效应器与缺乏CFTR的细胞进行比较，并在角膜感染的动物模型中验证CFTR依赖因子在细菌清除和角膜病理中的作用。对于金黄色葡萄球菌来说，最近耐甲氧西林金黄色葡萄球菌(MRSA)的急剧增加，特别是产生潘通-瓦伦丁杀白素(PVL)的菌株，使这种病原体成为严重眼病的重要原因。PVL的作用以及在正常人血清中常见的PVL抗体，将在组织培养和使用PVL表达阳性或阴性的同源金黄色葡萄球菌菌株的小鼠感染模型中进行检测，并用PVL成分免疫小鼠，以分析抗体如何调节感染过程。这些研究还应有助于了解杀白素在金黄色葡萄球菌角膜感染发病机制中的一般作用。对MRSA菌株的进一步研究将扩展到金黄色葡萄球菌感染候选疫苗的可能性，利用聚N-乙酰氨基葡萄糖(PNAG)表面多糖作为结合疫苗的活性成分，以改善感染的后果。在小鼠角膜损伤模型中，将评估主动疫苗接种以及使用多克隆抗体和完全人类单抗的被动转移研究，以确定PNAG是否是金黄色葡萄球菌角膜感染免疫治疗的基本靶点。建议的研究将扩展我们对细菌毒力和角膜感染有效宿主防御机制的洞察，并为高效的金黄色葡萄球菌疫苗方法提供临床前数据。与公共卫生相关：眼表(角膜)感染是世界上导致视力和视力丧失的最重要原因。细菌病原体是这些感染的重要原因，其中最常见的是铜绿假单胞菌和金黄色葡萄球菌。这项应用将研究这些微生物如何对角膜造成损害，从而导致视力丧失，并评估包括疫苗在内的干预措施，这些干预措施可用于预防或治疗这些感染，并将对角膜的损害降至最低，从而对个人视力造成损害。

DESCRIPTION (provided by applicant): The broad, long term goals of this grant application are to understand the molecular and cellular host responses to bacterial pathogens that are significant causes of corneal infection. Specifically, this application will focus on infections caused by Pseudomonas aeruginosa and Staphylococcus aureus. These two pathogens are among the most common causes of serious corneal infections. Strains of both pathogens have acquired significant means to resist antimicrobial therapies and elaborate a large armamentarium of virulence factors that contribute to corneal damage and loss of visual acuity. Therapies for these infections need to both reduce bacterial numbers and allow the host to have an adequate and helpful inflammatory response to clear pathogens without causing damage to the cornea. For P. aeruginosa, most of the bacteria infecting scratch-injured mouse eyes are found inside of cells, and entry requires binding to the cystic fibrosis transmembrane conductance regulator (CFTR). This leads to initiation of inflammation, including activation of transcription factors for pro-inflammatory genes, synthesis of the pro-inflammatory molecules, and initiation of the cellular influx, primarily composed of PMNs, that will both clear the pathogen but can also cause damage to the cornea. PMN influx is also controlled by the TH17 regulatory T cell network, which will also be investigated in these studies. To determine how CFTR coordinates inflammation, we will analyze the specific effectors produced by cells with wild-type CFTR that are infected with P. aeruginosa, compare these with cells lacking CFTR, and validate in animal models of corneal infection the role of the CFTR-dependent factors in bacterial clearance and corneal pathology. For S. aureus, the recent dramatic increase in methicillin-resistant S. aureus (MRSA), particularly strains elaborating the Pantone-Valentine leukocidin (PVL), makes this pathogen a significant concern as a cause of serious eye disease. The role of PVL, and antibody to PVL which is found commonly in normal human sera, will be examined in tissue culture and murine models of infection using isogenic S. aureus strains positive or negative for PVL expression, as well as immunizing mice with the PVL components to analyze how antibody modulates the course of infection. These studies should also be informative about the general role leukocidins have in the pathogenesis of S. aureus corneal infections. Further studies on MRSA strains will extend to the potential of a candidate vaccine for S. aureus infections, utilizing the poly-N-acetyl glucosamine (PNAG) surface polysaccharide as the active component of a conjugate vaccine, to ameliorate the consequences of infection. Active vaccination, as well as passive transfer studies using both polyclonal antibodies and a fully human monoclonal antibody, will be evaluated in the murine model of corneal injury to determine if PNAG is a rationale target for immunotherapy of S. aureus corneal infection. The proposed studies should ex- tend our insights into the mechanisms of bacterial virulence and effective host defense for corneal infections and provide pre-clinical data for vaccine approaches to S. aureus that could be highly effective. PUBLIC HEALTH RELEVANCE: Infections of the eye surface (cornea) are the most significant cause of loss of vision and visual acuity in the world. Bacterial pathogens are very important causes of these infections, and among the most common are Pseudomonas aeruginosa and Staphylococcus aureus. This application will study how these microbes cause damage to the cornea that can result in vision loss and evaluate interventions, including vaccines, that could be used to prevent or treat these infections and minimize damage to the cornea and thus to an individual's eyesight.