SUMMARY Significance: Urinary tract infection (UTI) is among the most prevalent urologic diseases, and it is caused primarily by uropathogenic Escherichia coli (UPEC). Bladder infection by UPEC is characterized by a transient intracellular stage during which bacteria invade superficial epithelial (facet) cells and divide within the cytosol to form multicellular communities called biofilms. After replicating in the cytosol, bacteria exit the intracellular biofilm – killing the bladder epithelial cell in the process – and disseminate to naïve facet cells or to the upper urinary tract. While in the intracellular biofilm state, bacteria evade innate immune responses and the effects of antibiotics. Similarly, in catheterized individuals, formation of biofilm on the catheter surface creates an additional protective niche for UPEC, from which it can disseminate to the bladder and seed infection. The goal of this proposal is to evaluate the potential of inhibiting biofilm by interfering with UPEC respiration. Rationale and Hypothesis: Although UPEC are facultative anaerobes, they respire oxygen during infection in the hypoxic bladder environment. Aerobic respiration and oxygen sensing have also been linked to the expression of critical UPEC virulence factors. We have previously shown that aerobic respiration is essential for UPEC to establish infection. Of the three respiratory quinol oxidases encoded by UPEC, cytochrome bd has the highest affinity for molecular oxygen, exceeding the affinity of mitochondrial cytochrome c by 1000-fold. Deletion of the cydABX genes that code for cytochrome bd, does not impart a growth defect in vitro, but leads to significant alterations in UPEC biofilm architecture, leading to higher susceptibility to antibiotics in the biofilm state. Furthermore, cydABX deletion mutants are non-motile, exhibit decreased proton motive force (pmf) and are attenuated in a murine UTI model. Finally, deletion of cydABX results in increased expression of the low affinity quinol oxidase cytochrome b0. We hypothesize that cytochrome bd can be chemically targeted to thwart biofilm formation or dissemination from a pre-formed biofilm. We further posit that cytochrome bd has a role in energizing motility. We propose two aims to test the posed hypotheses: Aims: Aim 1 will will evaluate whether targeting of cytochrome bd using known cytochrome bd inhibitors can enhance antibiotic effectiveness. Aim 2 will determine whether the decreased membrane potential of the cytochrome bd mutant imparts generalized or specific effects on pmf-dependent processes and determine how these impaired processes affect motility. Finally, using chemical inhibition of cytochrome bd we will evaluate whether loss of cytochrome bd function impairs dissemination from the biofilm. Impact: These studies will be the first to address the unique contribution of cytochrome bd on UPEC motility and will determine whether targeting bacterial respiration is a viable therapeutic or preve...