SUMMARY This proposal will elucidate how an understudied uropathogen – Pseudomonas aeruginosa – establishes acute infection and will determine whether age influences infection establishment and persistence. Urinary tract infection (UTI) is among the most prevalent urologic diseases. Pseudomonas aeruginosa accounts for ~3 million UTI annually, a number that matches Pseudomonas lung and wound infections. Yet, while the pathogenesis of Pseudomonas in the lung and wound have been – and continue to be – thoroughly investigated, there has been minimal study of the pathogenesis of Pseudomonas aeruginosa in the bladder. Moreover, Pseudomonas infections tend to occur in older individuals and afflict males and females equally. With this proposed work we aim to innovate by assigning a molecular signature that defines uropathogenic Pseudomonas and elucidating host and pathogen determinants that favor tropism of this pathogen to the aging population. Rationale and Hypothesis: All prior work on understanding Pseudomonas UTIs has been performed using model Pseudomonas strains isolated from wounds in the 1950’s. Given that the Pseudomonas genome is “open”, meaning that there is vast genomic variability among strains, we hypothesized that distinct pathogen genomic features exist in uropathogenic Pseudomonas isolates. Indeed, our isolation and sequencing of 96 uropathogenic isolates and subsequent genomic comparisons with a global cohort of 700 sequenced strains revealed specific genomic differences in one respiratory complex, ccoN between uropathogenic and other isolates. Based on preliminary data that demonstrate Pseudomonas enters the urothelial cell, we postulate that the identified ccoN mutations increase respiratory potential during acute infection. Moreover, we posit that host features in the aging bladder may influence uropathogenic Pseudomonas respiration, favoring its tropism for infecting older individuals. We propose two aims to test the posed hypotheses. Aims: Aim 1 will define how CcoN contributes to respiration within the bladder epithelial environment. In collaboration with the Dietrich group, we will create a series of specific mutations in CcoN in uropathogenic and respiratory Pseudomonas strains and compare their ability to adhere, invade and consume oxygen within the host cell cytosol. Aim 2 will determine the steps in pathogenesis followed by Pseudomonas in young versus old bladders. In parallel, differences in the immune response to infection will be measured in the old and young mice. Impact/How will the work change the field? These studies are the first to indicate a unique molecular signature that may be defining uropathogenic Pseudomonas. Evaluating the contribution of this signature to establishment and energy metabolism in vivo will provide significant insights into the kinetics of UTI caused by Pseudomonas. Understanding how pathogen genomic features may influence infection as a function of age, has the potential to change how Pseudomo...