Pathogenesis of microbial anterior eye diseases

描述（由申请人提供）：本资助申请的广泛，长期目标是了解微生物因素以及分子和细胞宿主对前眼感染的重要原因病原体的反应。这些见解应该导致预防和治疗策略，可以临床评估预防或治疗这些严重感染。具体而言，本提案将重点关注铜绿假单胞菌引起的感染以及引起角膜炎和结膜炎的过多病原体，这些病原体表达广泛保守的表面多糖聚n -乙酰氨基葡萄糖（PNAG）。铜绿假单胞菌不表达PNAG，但与眼部感染相关的大多数革兰氏阳性和革兰氏阴性细菌都表达PNAG，包括金黄色葡萄球菌、表皮葡萄球菌、肺炎链球菌、化脓性葡萄球菌、流感嗜血杆菌和淋病奈瑟菌，以及导致角膜炎的严重真菌，包括镰刀菌、黑曲霉和白色念珠菌。对于铜绿假单胞菌（P. aeruginosa），我们发现这种微生物在体外和实验中通过含有囊性纤维化跨膜传导调节剂（CFTR）的脂筏进入擦伤损伤小鼠眼睛的角膜上皮细胞，并且很大比例的细菌细胞在细胞内。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，我们进一步定义了一些由P. aeruginosa- cftr相互作用激活的角膜中导致破坏性炎症反应的分子和细胞因子，并深入分析了P. aeruginosa抗体预防角膜炎病理和感染的能力。我们启动了对金黄色葡萄球菌角膜感染的毒力和免疫的严格研究，包括表达PNAG表面多糖（PVL）的亚群，并确定了针对PNAG表面多糖进行免疫治疗以预防或治疗金黄色葡萄球菌角膜炎的潜力。在接下来的几年里，我们将通过进一步分析抗体介导的免疫对表达pnag的生物体的能力来扩展我们的长期目标，使用角膜炎和结膜炎模型来预防前眼感染，一种发生在无血管组织中，另一种发生在血管化的眼部组织中。

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.