Identification of bacterial small molecule inhibitors of Proteus mirabilis urease activity

项目总结/文摘

Project Summary/Abstract Patients with indwelling urinary catheters have nearly double the mortality rate compared to non-catheterized patients, in part due to increased colonization by multidrug-resistant organisms, catheter-associated urinary tract infections (CAUTI), and secondary bacteremia. The Gram-negative bacterium Proteus mirabilis is a predominant cause of both CAUTI and bacteremia, particularly with long-term catheterization. This bacterium has long been recognized as a problematic colonizer of the urinary tract due to its potent urease enzyme, which hydrolyzes the urea in urine to carbon dioxide and ammonia. Bacterial urease activity ultimately increases urine pH, induces precipitation of polyvalent ions, and causes painful catheter encrustation, blockage, and urinary stones (urolithiasis). In human patients and animal infection models, P. mirabilis urease activity elicits bladder obstruction and renal damage and greatly facilitates the development of bacteremia. However, disrupting urease activity abrogates development of urolithiasis and dramatically reduces the incidence of bacteremia, making urease a promising target for treating or preventing P. mirabilis CAUTI sequelae. CAUTI is also frequently polymicrobial, and we have demonstrated that co-infection of P. mirabilis with other uropathogens modulates the risk of urolithiasis and bacteremia in a urease-dependent manner. Specifically, Enterococcus faecalis and Providencia stuartii increase the incidence of urolithiasis and bacteremia by enhancing P. mirabilis urease activity, while Morganella morganii and Enterobacter aerogenes decrease infection severity by dampening P. mirabilis urease activity. In all cases, modulation of P. mirabilis urease activity is mediated by as-yet unidentified factors that are secreted, smaller than 3 kDa, and heat-stable. We therefore hypothesize that cell-free supernatants from urease-modulatory species can be exploited to identify membrane-permeable small molecules that regulate activity of the cytoplasmic urease enzyme in P. mirabilis and that have strong potential to be developed into a non-antibiotic approach for treating or preventing P. mirabilis CAUTI sequelae. In Aim 1, we will determine the mechanism of action of urease modulation by cell-free supernatants focusing on i) direct interaction with the P. mirabilis urease enzyme and ii) indirect effects on production of urease structural subunits and the ratio of catalytically-active urease holoenzyme to apoenzyme. In Aim 2, we will conduct a global metabolomics study to identify small molecule signatures unique to cell-free supernatants from species that dampen P. mirabilis urease activity. Candidate small molecules revealed through this study will then be purchased, synthesized, isolated, or enriched for to verity the mechanism by which they decrease P. mirabilis urease activity. The proposed approach represents both a conceptual and technical innovation in the search for urease inhibitors, and has the potential to change the paradigm of P. mirabilis urease regulation. The knowledge gained will support a future R01 aimed at assessing the therapeutic potential of urease-dampening small molecules and developing a pre-clinical regimen to reduce risk of CAUTI sequelae in animal infection models.