PROJECT SUMMARY/ABSTRACT Uropathogenic Escherichia coli (UPEC) is the main etiological agent of urinary tract infections (UTI) and primarily affects otherwise healthy women. A third of women diagnosed with a UTI will experience a recurrent UTI (rUTI) in the following months, with some women suffering as many as six or more rUTIs per year. Further, more than 60% of rUTIs are caused by the same strain of E. coli that caused the initial infection, arguing that host- associated UPEC reservoirs are recalcitrant to antibiotic treatment and can seed rUTIs. One major reservoir for E. coli is the gastrointestinal tract (GIT). At the time of a UTI, the causal E. coli strain is most often the predominant E. coli strain in the GIT, and the same strain can persist within the GIT even after antibiotic therapy. A healthy GIT microbiota maintains homeostasis with the host immune system and prevents colonization by bacterial pathogens. Ironically, antibiotic treatments meant to clear the UTI can also disrupt the microbiota and expose individuals to an increased risk of colonization by bacterial pathogens in the GIT. Previous studies performed with Salmonella enterica serovar Typhimurium have demonstrated that the type VI secretion system (T6SS) is critical for colonization and infection within the mouse gut. Interestingly, UPEC cystitis isolate, UTI89, possesses two T6SS; and my data reveals that deletion of either T6SS cluster decreases the relative GIT fitness of UTI89 in our streptomycin (STM) mouse model. Moreover, comparative genomics data analyses of diverse E. coli strains reveal that a specific subtype of T6SS is enriched in clade B2 E. coli (the predominant clade causing UTIs in the western hemisphere), suggesting that B2 E. coli may share functions and gene content that promote GIT colonization. Despite numerous studies demonstrating the GIT to be an important reservoir for UPEC in humans, UPEC determinants critical for productive GIT colonization have yet to be fully investigated. I hypothesize that UPEC possess an arsenal of fitness factors and gene regulatory programs that enhance GIT colonization and persistence increasing UTI susceptibility and rUTIs. To test this, I will use: i) the established prototypical human cystitis isolate, UTI89, and clinically relevant UPEC and E. coli isolates; ii) complementary in vivo and in vitro experimentation; and iii) robust computational and bioinformatic analyses. Aim 1 will assess the relative fitness of T6SS mutants in our STM model, compare GIT microbiota profiles and inflammatory responses, and determine the cellular targets of the T6SS. Aim 2 will assess the relative fitness of 30 diverse E. coli clinical isolates from healthy and rUTI patients in our STM model, compare their transcriptional profiles and genetic content using newly developed multi-omic analyses, and perform targeted deletions to confirm phenotypes in our STM model. This two-pronged research strategy provides both a targeted (Aim 1) and unbi...