Pathogensis of Bacterial corneal infection

描述（由申请人提供）：该资助申请的广泛，长期目标是了解分子和细胞宿主对细菌病原体的反应，这些病原体是角膜感染的重要原因。具体而言，本申请将重点关注铜绿假单胞菌和金黄色葡萄球菌引起的感染。这两种病原体是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微生物疗法的重要手段，并精心制作了大量的毒力因子，这些毒力因子导致角膜损伤和视力丧失。这些感染的治疗需要减少细菌数量，并允许宿主有足够的和有益的炎症反应来清除病原体，而不会对角膜造成损害。对于铜绿假单胞菌，大多数感染抓伤小鼠眼睛的细菌都存在于细胞内，并且进入需要与囊性纤维化跨膜传导调节因子（CFTR）结合。这导致炎症的开始，包括促炎基因的转录因子的激活、促炎分子的合成以及主要由PMN组成的细胞流入的开始，其将清除病原体，但也可能对角膜造成损伤。中性粒细胞内流也由TH 17调节性T细胞网络控制，这也将在这些研究中进行研究。为了确定CFTR如何协调炎症，我们将分析感染铜绿假单胞菌的野生型CFTR细胞产生的特异性效应物，将其与缺乏CFTR的细胞进行比较，并在角膜感染的动物模型中验证CFTR依赖性因子在细菌清除和角膜病理学中的作用。对于鼠伤寒沙门氏金黄色葡萄球菌，最近急剧增加的耐甲氧西林的S。金黄色葡萄球菌（MRSA），特别是产生Pantone-Valentine杀白细胞素（PVL）的菌株，使得这种病原体作为严重眼部疾病的原因而受到严重关注。PVL和PVL抗体（通常在正常人血清中发现）的作用将在组织培养和使用等基因S.本发明的方法包括使用PVL表达阳性或阴性的金黄色葡萄球菌菌株，以及用PVL组分免疫小鼠以分析抗体如何调节感染过程。这些研究也将为杀白细胞素在链球菌发病机制中的一般作用提供信息。金黄色角膜感染。对MRSA菌株的进一步研究将扩展到S.金黄色葡萄球菌感染，利用聚-N-乙酰葡糖胺（PNAG）表面多糖作为缀合物疫苗的活性组分，以改善感染的后果。将在角膜损伤的鼠模型中评价主动接种以及使用多克隆抗体和全人单克隆抗体的被动转移研究，以确定PNAG是否是S.金黄色角膜感染这些研究将有助于我们深入了解角膜感染的细菌毒力机制和有效的宿主防御机制，并为S.金黄色葡萄球菌，可能非常有效。

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.