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

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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.