Thesis

Genetic analysis of persister formation in the respiratory pathogen pseudomonas aeruginosa

Persister cells are a small percentage of the bacterial population (0.01-10%) that survive antibiotic therapy along with other stressors. They do this without undergoing any genetic change, only phenotypic variation. The exact mechanisms of persister cell formation are still not completely understood. We are in the process of identifying genes important for persister cell formation in a highly virulent respiratory pathogen, Pseudomonas aeruginosa. This organism is a major health concern, especially since antibiotic resistance is on the rise. We found high levels of differentially gene expression in persister cells through RNA sequencing. Interestingly, we found more genes were actually upregulated compared to downregulated. These results indicate that previously held ideas about persister cell dormancy are debatable. We quantified persister cell formation in selected mutants by directly evaluating the number of colony forming units (CFUs). Selected hits included pyocin synthesis genes, where mutants failed to form any persister cells. We characterized the role of two genes, PA3819 and gshA, through assays of physiological and biochemical functions including pyocyanin production, biofilm formation, and motility of both mutants in order to characterize the role of these genes within the cell. The knowledge derived from these studies will help identify better targets for treatment of P. aeruginosa acute infections, chronic infections, and post-treatment relapses.

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