Pathogenesis of microbial anterior eye diseases

描述（由申请人提供）：该资助申请的广泛，长期目标是了解微生物因素以及分子和细胞宿主对病原体的反应，这些病原体是前眼感染的重要原因。这些见解应该导致预防和治疗策略，可以在临床上评估预防或治疗这些严重感染。具体而言，该提案将集中于由铜绿假单胞菌引起的感染以及引起角膜炎和结膜炎的大量病原体，这些病原体表达广泛保守的表面多糖聚-N-乙酰葡糖胺（PNAG）。PNAG不被铜绿假单胞菌表达，但被与眼部感染相关的大多数主要革兰氏阳性和革兰氏阴性细菌生物体表达，包括金黄色葡萄球菌、葡萄球菌、金黄色葡萄球菌和金黄色葡萄球菌。表皮链球菌、肺炎链球菌、S.化脓性链球菌、流感嗜血杆菌和淋病奈瑟氏菌以及引起角膜炎的严重真菌，包括镰刀菌属，尼日尔曲霉和白色念珠菌。对于铜绿假单胞菌，我们已经发现这种微生物在体外和在实验性感染刮伤的小鼠眼睛期间通过含有囊性纤维化跨膜传导调节因子（CFTR）的脂筏进入角膜上皮细胞，并且大部分细菌细胞是细胞内的。CFTR的细菌配体是细胞表面脂多糖的外核寡糖。在目前资助的提案中，我们进一步定义了角膜中由铜绿假单胞菌-CFTR相互作用激活的一些分子和细胞因子，导致破坏性炎症反应，并深入分析了铜绿假单胞菌抗体预防角膜炎病理和感染的能力。我们开始了一项严格的研究，毒力和免疫S。金黄色葡萄球菌角膜感染，包括表达Panton-Valentine杀白细胞素（PVL）的亚群，并建立了针对PNAG表面多糖的免疫治疗以预防或治疗S.金黄色角膜炎在接下来的几年中，我们将通过使用角膜炎和结膜炎模型进一步分析抗体介导的对PNAG表达生物体的免疫力以预防前眼感染的能力来扩展我们的长期目标，其中一种发生在无血管组织中，另一种发生在血管化的眼组织中。 这些对比设置将使我们能够研究、比较和对比有效免疫所需的宿主细胞辅因子，这可以使用缺乏成熟免疫系统的转基因和无菌小鼠进行。此外，为了继续研究铜绿假单胞菌角膜感染的发病机制，我们将使用铜绿假单胞菌饱和转座子（Tn）库的高通量测序分析，我们最近建立了该库作为研究宿主-病原体相互作用的主要方法，以研究角膜炎发病机制中涉及的细菌和宿主因素。实现这些目标将为在人类眼部感染中测试针对PNAG的免疫疗法提供临床前依据，并将对铜绿假单胞菌角膜炎的发病机理的了解扩展到迄今为止未达到的精确度和细节水平。

DESCRIPTION (provided by applicant): The broad, long term goals of this grant application are to understand the microbial factors and the molecular and cellular host responses to pathogens that are significant causes of infections in the anterior eye. These insights should lead to prophylactic and therapeutic strategies that can be clinically assessed for preventing or treating these serious infections. Specifically, this proposal will focus on infections caused by Pseudomonas aeruginosa and the plethora of pathogens causing keratitis and conjunctivitis that express the broadly conserved surface polysaccharide poly-N-acetyl glucosamine (PNAG). PNAG is not expressed by P. aeruginosa but is expressed by most major gram-positive and gram-negative bacterial organisms associated with eye infections including Staphylococcus aureus, S. epidermidis, Streptococcus pneumoniae, S. pyogenes, Hemophilus influenzae and Neisseria gonorrhoeae as well as by serious fungal causes of keratitis including Fusarium spp., Aspergillus niger and Candida albicans. For P. aeruginosa, we have found that this microbe enters corneal epithelial cells both in vitro and during experimental infection of the scratched-injured mouse eye via lipid rafts containing the cystic fibrosis transmembrane conductance regulator (CFTR), and that a large proportion of the bacterial cells are intracellular. The bacteril ligand for CFTR is the outer core oligosaccharide of the cell surface lipopolysaccharide. In the currently-funded proposal, we further defined some of the molecular and cellular factors activated by the P. aeruginosa-CFTR interaction in the cornea leading to destructive inflammatory responses and also analyzed in depth the ability of antibody to P. aeruginosa to prevent pathology and infection in keratitis. We initiated a rigorous study of virulence and immunity to S. aureus corneal infection including the subset that express the Panton-Valentine Leukocidin (PVL) and established the potential for immunotherapy against the PNAG surface polysaccharide to prevent or treat of S. aureus keratitis. In the ensuing years we will extend our long term goals by further analyzing the ability of antibody-mediated immunity to PNAG-expressing organisms to prevent anterior eye infections using models of keratitis and conjunctivitis, one occurring in an avascular tissue and the other in a vascularized ocular tissue. These contrasting settings will allow us to investigate, compare and contrast the host cellular co-factors needed for effective immunity which can be conducted using transgenic and germ-free mice lacking matured immune systems. Additionally, to continue our investigations into the pathogenesis of P. aeruginosa corneal infection we will use high- throughput sequencing analysis of a saturated transposon (Tn) bank of P. aeruginosa that we have recently established as a major approach to the study of the host-pathogen interaction to investigate bacterial and host factors involved in the pathogenesis of keratitis. Accomplishing these goals will provide the pre-clinical justification for testing of immunotherapies to PNAG in human ocular infections and extend the insight into the pathogenesis of P. aeruginosa keratitis to a heretofore unachieved level of precision and detail.