Type 1 pili, the preeminent virulence determinant in Escherichia coli-associated cystitis, promote binding to the bladder surface and facilitate the colonization and invasion of the epithelium. In their intracellular residence, E. coli amass into communal formations with biofilm-like properties termed intracellular bacterial communities (IBC) and then disperse and flux from the infected cell into the lumen of the bladder. Upon fluxing, type 1 pili may again mediate adherence to naive epithelial cells, reinitiating invasion and IBC formation. Because of the central role of type 1 pili in uropathogenesis, understanding the regulation of these fibers is essential for a complete molecular description of the infection and ultimately to identify novel targets for therapeutics. Type 1 pili are regulated by two recombinases, FimB and FimE, that together dynamically modulate the expression of the fibers through phase variation whereby the promoter region is inverted into ON and OFF positions. Preliminary studies have identified FimX, a third site-specific recombinase that, despite the absence of FimB and FimE, produces phase variation and the expression of type 1 pili and supports full virulence. Ablation of FimX and the other two recombinases renders the organism avirulent. We hypothesize that FimX, as regulator of type 1 pili expression, is a central virulence determinant in E. coli urinary tract infections. In Aim 1 of this proposal, we will use a combination of molecular epidemiology, genetics, and animal modeling to characterize the prevalence of fimX and study the in vivo role of FimX as a regulator of type 1 pili in E. coli cystitis. In Aim 2, through the use of genetics and biochemistry, we will study the structure-function relationships of FimX and its target DNA sequences. Given the central role of the Fim recombinases in regulating type 1 pili expression, understanding their functions and constraints may yield novel points for treatment and prevention in urinary tract infections.