Project Summary/Abstract Urinary tract infection (UTI) is one of the most common infections worldwide and a leading cause of morbidity and healthcare expenditures across all ages. Older adults and individuals with catheters have a particularly high risk of UTI sequelae, including kidney infection, permanent renal damage, and urosepsis. The average mortality rate for urosepsis is 20-40%, but ranges as high as 66% for patients with catheter- associated UTI (CAUTI). Catheterization also promotes polymicrobial colonization and infection by a wide range of understudied microbes, including multidrug-resistant organisms that threaten the utility of last- resort antibiotics, and there is a substantial gap in knowledge regarding the host-microbe interactions that impact progression from initial colonization to urosepsis. The urothelium is the site of initial host-pathogen interactions in the urinary tract, a major component of which is the glycosaminoglycan (GAG)-rich glycocalyx that coats the apical surface of the umbrella cells. The GAG layer protects the urothelium in part by shielding bacterial surface receptors and contributing to immune surveillance. However, our preliminary data demonstrate that two of the most common CAUTI pathogens, Proteus mirabilis and Enterococcus faecalis, encode six putative GAG-degrading enzymes (GAGases). Our initial study of two of these GAGases found that they facilitate bladder colonization, kidney infection, and urosepsis during experimental infection, and one allowed P. mirabilis to use GAGs as a carbon source. We hypothesize that the six GAGases facilitate CAUTI and urosepsis by supporting bacterial growth, urothelial invasion, and immune modulation, and that differences in GAG composition will influence CAUTI susceptibility and severity. In Aim 1, we will conduct a comprehensive examination of the GAG composition throughout the urinary tract in mice, a three- dimensional urine-tolerant human urothelial model, and urine specimens from c