Rise of lasR mutant Pseudomonas aeruginosa keratitis

lasR突变型铜绿假单胞菌角膜炎的兴起

• 批准号: 10437757
• 负责人: ROBERT M SHANKS
• 金额: $ 38.62万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10437757
• 关键词: Address; Alleles; Animal Model; Antibiotic Resistance; Antibiotic Therapy; Antibiotic susceptibility; Antibiotics; Awareness; Back; Bacteria; Bacterial Antibiotic Resistance; Bacterial Genome; Bacterial Infections; Blindness; Clinical; Collection; Complement; Contact Lenses; Cornea; Corneal Ulcer; Disease; Dose; Eye; Eye Infections; Frequencies; Funding; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genotype; Goals; Growth; Health Personnel; Immune response; Immune system; In Vitro; India; Infection; Inflammation; Inflammatory; Keratitis; Laboratories; Link; Lipase; Measures; Mission; Modeling; Molecular Genetics; Muramidase; Mus; Mutation; Ophthalmologist; Optometrist; Organism; Oryctolagus cuniculus; Outcome; Pathogenesis; Pathogenicity; Patient-Focused Outcomes; Patients; Phenotype; Phylogenetic Analysis; Pilum; Predisposition; Pseudomonas aeruginosa; Public Health; Publishing; Reagent; Research; Resistance; Risk; Role; Signal Transduction; Source; Steroids; Testing; Therapeutic; United States; United States National Institutes of Health; Universities; Up-Regulation; Virulence; Virulence Factors; Virulent; Vision; Visual; antibiotic tolerance; antimicrobial peptide; bacterial resistance; base; clinical diagnostics; combat; corneal scar; cystic fibrosis patients; derepression; design; diagnostic strategy; improved; in vivo; in vivo Model; innovation; knowledge of results; loss of function; mutant; next generation sequencing; novel strategies; novel therapeutic intervention; ocular surface; pathogen; personalized medicine; prevent; quorum sensing; repository; success; targeted sequencing; transcription factor; treatment strategy; trend; whole genome

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.

项目总结/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， 铜绿假单胞菌的突变体导致的视力结果比角膜炎明显更差 是由野生型铜绿假单胞菌引起的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一个巨大的储存库中， 在铜绿假单胞菌角膜炎中观察到lasR突变体显著增加的趋势 隔离在美国。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开发 预防感染引起的视力丧失。本申请的总体目标是确定 铜绿假单胞菌的lasR突变体导致更差临床结果的机制。我们的核心假设 LasR转录因子抑制了影响铜绿假单胞菌存活的基因的表达， 粗糙的眼表面，使得在突变体中上调的基因赋予增加的感染性表型。的 这项研究的基本原理是，确定LasR控制眼部毒力的机制将提供一种新的方法， 科学基础，制定治疗策略，以打击视力下降。三个具体目标是 设计来询问我们的中心假设：1）表征lasR突变的遗传基础， 在坎贝尔实验室收集临床分离株，并评估突变赋予 显性或隐性毒力表型; 2）测试特异性铜绿假单胞菌基因的重要性， 在lasR突变体中表达增加，在眼表存活中起作用， 兔隐形透镜感染模型，并诱导角膜炎症，以及3）测试lasR突变体是否 对眼表先天防御和眼科相关抗生素治疗具有提高的抗性。这 一项研究将使用先进的分子遗传学组合来操纵细菌基因组，包括临床 分离株，下一代测序，以及完善的体外和体内模型。本研究具有创新性 因为目前的诊断方法不能区分引起角膜炎的铜绿假单胞菌菌株， 除了抗生素敏感性之外，这项研究将引入一种新的方法来识别高度视力。 威胁铜绿假单胞菌分离株。这项研究还将评估几个候选毒力因子， 未对眼睛进行测试。本研究的意义基于两个方面：A）其高 阐明细菌产生致盲性眼部感染的新致病机制的潜力， 并且所得到的知识最终可以用于开发预防由于眼内炎引起的视力丧失的方法。 感染，以及B）引入一种可行的个性化药物形式，可用于警告医疗保健 工人对患者最有可能丧失视力。