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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要铜绿假单胞菌 (Pa) 是一种机会性环境细菌，是导致尽管 Pa 具有临床相关性，但医院获得性感染和慢性囊性纤维化 (CF) 肺部感染。 Pa如何从其环境储存库转变为感染人类尚不清楚。对从慢性感染患者中分离出的 Pa 的研究记录了 Pa 毒力的适应性， CF 肺环境中生物膜的形成和代谢随时间的推移而变化。我初步发现，表型与调节核苷酸环二 GMP (cdG) 的变化有关。筛选了 53 个 cdG 代谢转座子突变体库，发现了三个改变了表型的突变体正如文献报道的那样，当在模拟 CF 气道营养物质的培养基中生长时，我也发现了这种情况。观察到的表型变异性或异质性，在基因范围内单个细胞中的 cdG 浓度由我采用的荧光报告基因测量的相同的 Pa 群体，因为 cdG 在其中发挥着核心作用。调节Pa的临床相关行为，我建议建立三个cdG之间的关系在模拟营养环境的条件下，单个 Pa 细胞内的候选酶和适应性第一个目标是使用延时显微镜观察 CF 肺，我将通过研究其作用来跟进结果。这些候选者在 cdG 代谢和表型异质性以及 cdG 的适应性方面。第二个目标是，该提案将采取公正的方法来识别代谢途径，这些途径是与野生型细菌相比，三种 cdG 代谢突变体在转化为营养物质时受到差异性调节采用与评估 cdG（表型）相同的技术。 Pa中差异调节代谢途径的异质性和适应性以及cdG的影响总体而言，该提案将研究 cdG 代谢的影响。在模拟 CF 肺环境的条件下，这将提供所需的单细胞适应性。深入了解Pa如何成功感染人类。