Determining the role of cyclic-di-GMP in Pseudomonas aeruginosa's transition to the nutritional environment of the cystic fibrosis lung

确定环二 GMP 在铜绿假单胞菌向囊性纤维化肺营养环境转变中的作用

• 批准号: 10640093
• 负责人: Roxanne Morris
• 金额: $ 3.26万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-09 至 2025-03-08
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640093
• 关键词: Acute; Aerobic; Affect; Bacteria; Behavior; Biology; Carbon; Caring; Cells; Characteristics; Chronic; Cystic Fibrosis; Data; Development; Environment; Enzymes; Exhibits; Gene Expression; Generations; Genetic Transcription; Growth; Heterogeneity; Human; Individual; Infection; Interruption; Knock-out; Libraries; Life Style; Literature; Lung infections; Measures; Metabolic; Metabolic Pathway; Metabolism; Methods; Microbial Biofilms; Microscopy; Modeling; Multi-Drug Resistance; Noise; Nosocomial Infections; Nucleotides; Nutrient; Nutrient availability; Nutritional; Outcome; Oxygen; Pathway interactions; Patients; Periodicity; Persons; Phenotype; Physiology; Play; Population; Pseudomonas aeruginosa; Pulmonary Cystic Fibrosis; Regulatory Pathway; Reporter; Reporting; Role; Second Messenger Systems; Source; Surface; Techniques; Testing; Time; Translating; Translations; Variant; Virulence; Virulence Factors; antimicrobial; cell behavior; cell growth; cell motility; chronic infection; clinically relevant; cofactor; cystic fibrosis airway; cystic fibrosis patients; diguanylate cyclase; environmental change; experience; fitness; follow-up; improved; innovation; insight; mutant; novel strategies; opportunistic pathogen; pathogen; phosphoric diester hydrolase; response; success; time use; transcriptome

Project Summary Pseudomonas aeruginosa (Pa) is an opportunistic, environmental bacterium that is a leading cause of hospital acquired infections and chronic cystic fibrosis (CF) lung infections. Despite Pa’s clinical relevance, how Pa transitions from its environmental reservoir to infect humans is not well understood. Studies of Pa isolated from chronically infected patients have documented adaptations in Pa virulence, biofilm formation, and metabolism over time in the CF lung environment. Many of these characteristics, or phenotypes, are associated with changes in cyclic di-GMP (cdG), a regulatory nucleotide. Preliminarily, I have screened a library of 53 cdG metabolism transposon mutants and found three that have altered phenotypes when grown in media that mimics the nutrients in the CF airway. As is reported in the literature, I have also observed phenotypic variability, or heterogeneity, of cdG concentrations in individual cells within genetically identical Pa populations as measured by a fluorescent reporter I adapted. As cdG plays a central role in regulating clinically relevant behaviors of Pa, I propose to establish the relationship between the three cdG enzyme candidates and fitness within individual Pa cells in conditions that model the nutrient environment of the CF lung using time lapse microscopy in the first aim. I will follow up on the result by investigating the role of these candidates in cdG metabolism and phenotypic heterogeneity and of cdG on fitness. In a second aim, this proposal will take an unbiased approach to identity metabolic pathways that are differentially regulated in the three cdG metabolism mutants vs. wild-type bacteria upon transition into nutrient environment of the CF lung. Deploying the same techniques as were used to evaluate cdG, the phenotypic heterogeneity and correlation to fitness of differentially regulated metabolic pathways and impact of cdG in Pa will be determined at the single cell level. Overall, this proposal will investigate the impact of cdG metabolism on single cell fitness in conditions that model the CF lung environment. This, in turn, will provide needed insight into how Pa successfully infects humans.

项目摘要 铜绿假单胞菌（Pa）是一种机会性环境细菌，是导致大肠杆菌感染的主要原因。 医院获得性感染和慢性囊性纤维化（CF）肺部感染。尽管爸爸的临床相关性， Pa如何从其环境宿主转变为感染人类尚不清楚。 对从慢性感染患者分离的Pa的研究已经证明了Pa毒力的适应， 生物膜形成和CF肺环境中随时间的代谢。其中许多特征，或 表型与环二GMP（cdG）（一种调节核苷酸）的变化相关。实际上，我有 筛选了53个cdG代谢转座子突变体的文库，发现了3个表型改变的突变体 当在模拟CF气道中的营养物的培养基中生长时。正如文献中所报道的那样，我也 观察到的表型变异性，或异质性的cdG浓度在个别细胞内的遗传 相同的Pa群体，如通过荧光报告分子I所测量的。由于cdG在以下方面发挥着核心作用： 调节Pa的临床相关行为，我建议建立三个cdG之间的关系 酶候选物和适应性在单个Pa细胞内的条件下， 在第一个目标中使用时间推移显微镜观察CF肺。我将根据调查结果， 这些候选人在cdG代谢和表型异质性以及cdG对适应性的影响。 在第二个目标中，该提案将采取无偏见的方法来识别代谢途径， 在三种cdG代谢突变体中与野生型细菌相比， CF肺的环境。使用与用于评估cdG相同的技术， Pa差异调节代谢途径的异质性和与适应性的相关性以及cdG的影响 将在单个细胞水平上确定。总体而言，本提案将研究cdG代谢的影响 在模拟CF肺环境的条件下对单细胞适应性的影响。反过来，这将提供所需的 了解爸爸是如何成功感染人类的