PROJECT SUMMARY / ABSTRACT Uropathogenic Escherichia coli (UPEC), the major causative agent of urinary tract infections, can colonize the bladder (cystitis) and ascend the ureter leading to infection within the kidney (pyelonephritis). Though less frequent than cystitis, pyelonephritis carries increased health risks, including hypertension, renal abscess formation, and renal scarring. Nevertheless, the mechanisms of UPEC pathogenesis during pyelonephritis have remained elusive due to lack of an optimal preclinical infection model. Published work from our group has established that males exhibit an elevated susceptibility to chronic pyelonephritis and renal abscess formation. This inherent sex bias in UTI outcomes is androgen dependent, leading us to create new published models of ascending UTI in androgen-exposed female mice that enable investigation of UPEC virulence strategies within the kidney. The UPEC secreted toxin alpha-hemolysin (HlyA) elicits cell lysis and ATP release from various mammalian cell types in vitro, but was previously shown to be dispensable during UPEC cystitis. Preliminary studies using our updated mouse model of ascending UTI demonstrated that HlyA is required for optimal UPEC colonization and associated with severe pathology and renal abscess formation in androgen-exposed hosts. This initial work is congruent with epidemiologic data showing that ~80% of clinical UPEC isolated from patients with pyelonephritis are HlyA positive, versus only 40% of cystitis isolates. Purinoreceptors (P2X) are host cell membrane channels that open upon sensing extracellular ATP and facilitate non-selective passage of small cations. Interestingly, the P2X4 isotype expressed ubiquitously throughout the renal tubular epithelium, is allosterically activated by direct interactions with testosterone; modulation of P2X4 function by testosterone increases receptor affinity for ATP, driving increased ion permeability and subsequent pore conductance. We have found that P2X receptors amplify HlyA cytotoxicity in vitro. Specifically, exogenous testosterone significantly enhances HlyA-mediated lysis of renal epithelial cells, and this effect is abolished in the presence of a non-selective P2X inhibitor. Leveraging updated models of ascending UTI, small-molecule P2X inhibitors, and our in vitro model of renal collecting duct infection, we will test the central hypothesis that UPEC alpha-hemolysin drives renal pathology during pyelonephritis, and its activity is augmented by testosterone via enhancement of P2X channel function. Completion of this work will delineate the contribution of HlyA to UPEC pathogenesis within the kidney, determine the mechanism which facilitates the sex-biased pathogenic effect of HlyA, and define the contribution of pharmacologically targetable host P2X4 channels to HlyA toxicity during pyelonephritis.