Rise of lasR mutant Pseudomonas aeruginosa keratitis

项目摘要/摘要 美国国立卫生研究院资助的类固醇治疗角膜溃疡研究发现，由自然发生的LasR引起的角膜炎 铜绿假单胞菌突变导致的视力结果明显比角膜炎差 由野生型铜绿假单胞菌引起。所有来自印度的分离株都是在2006-2010年间获得的。使用 匹兹堡大学坎贝尔实验室的巨大储存库，该实验室自1992年以来一直分离出一种 关注的趋势是观察到在铜绿假单胞菌角膜炎中LasR突变显著增加 在美国的分离株。这是一个潜在的公共卫生问题，这项研究将有助于 与眼科医生和验光师沟通。这项研究的长期目标是发展 预防感染导致视力丧失的方法。此应用程序的总体目标是确定 铜绿假单胞菌LasR突变导致临床预后不良的机制。我们的中心假设 LasR转录因子抑制影响铜绿假单胞菌存活的基因在 严酷的眼表，使得基因在突变体中上调，从而增加了传染性表型。这个 这项研究的基本原理是，识别LasR控制眼睛毒力的机制将提供一种 为制定对抗视力丧失的治疗策略提供科学依据。三个具体目标是 旨在询问我们的中心假设：1)描述LasR突变的遗传基础 坎贝尔实验室的临床分离株收集，并评估突变带来的程度 显性或隐性毒力表型；2)测试特定的铜绿假单胞菌基因的重要性 在LasR突变体中表达增加，在眼表存活中发挥作用，建立角膜感染 建立兔隐形眼镜感染模型，并在诱导角膜炎症时，3)检测LasR是否突变 对眼表天然防御和眼科相关抗生素治疗的抵抗力增强。这 这项研究将结合先进的分子遗传学来操纵细菌基因组，包括临床 分离，下一代测序，以及建立良好的体外和体内模型。本研究具有创新性。 由于目前的诊断方法没有区分引起角膜炎的铜绿假单胞菌菌株， 除了抗生素敏感性，这项研究将介绍一种新的方法来识别高度视力 具有威胁性的铜绿假单胞菌。这项研究还将评估几个候选毒力因素，这些因素具有 没有经过眼睛方面的测试。这项研究的意义基于两个方面：a)其高度 有可能阐明细菌用来建立致盲眼部感染的新的致病机制， 由此得到的知识最终可以用来开发预防由于视力下降而导致的视力丧失的方法 感染，以及B)采用一种可行的个性化药物形式，可用于提醒保健 工作人员向最有可能丧失视力的患者提供服务。

Project Summary / Abstract The NIH funded Steroids for Corneal Ulcers Study found that keratitis caused by naturally occurring lasR mutants of the bacteria Pseudomonas aeruginosa resulted in significantly worse vision outcomes than keratitis caused by wild-type P. aeruginosa. All the isolates, from India, were obtainted between 2006-2010. Using the vast repository at the University of Pittsburgh Campbell Laboratory, which has isolates back to 1992, a concerning trend was observed that lasR mutants have dramatically increased among P. aeruginosa keratitis isolates in the United States. This is a potential public health problem that this study would help to communicate to ophthalmologists and optometrists. The long-term goal of this research is to develop approaches to prevent vision loss caused by infections. The overall objective of this application is to determine the mechanism by which lasR mutants of P. aeruginosa cause worse clinical outcomes. Our central hypothesis is that the LasR transcription factor inhibits expression of genes that influence P. aeruginosa survival on the harsh ocular surface, such that genes upregulated in the mutant confer an increased infectivity phenotype. The rationale for this study is that identifying the mechanisms by which LasR controls ocular virulence will provide a scientific basis to develop therapeutic strategies to combat vision loss. Three specific aims have been designed to interrogate our central hypothesis: 1) to characterize the genetic basis of lasR mutations among the clinical isolate collection at the Campbell Laboratory, and evaluate the extent to which the mutations confer dominant or recessive virulence phenotypes; 2) to test the importance of specific P. aeruginosa genes that have increased expression in lasR mutants for a role in ocular surface survival, establishing corneal infection in a rabbit contact lens infection model, and in inducing corneal inflammation, and 3) to test whether lasR mutants have elevated resistance to ocular surface innate defenses and ophthalmically relevant antibiotic therapy. This study will use a combination of advanced molecular genetics to manipulate bacterial genomes including clinical isolates, next generation sequencing, and well-established in vitro and in vivo models. This study is innovative because current diagnostic approaches do not differentiate between P. aeruginosa strains that cause keratitis, other than antibiotic susceptibility, and this study will introduce a new approach to identify highly vision threatening P. aeruginosa isolates. This study will also evaluate several candidate virulence factors that have not been tested with respect to the eye. The significance of this study is based on two items: A) its high potential to elucidate new pathogenic mechanisms that bacteria wield to establish blinding ocular infections, and the resulting knowledge can ultimately be used to develop approaches to prevent vision loss due to ocular infections, and B) introducing a feasible form of personalized medicine that can be used to alert health care workers to patients most at risk for vision loss.