Characterization of pathogen nutrient acquisition and transport systems required during UTI

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Project Abstract Urinary tract infection (UTI) represents a substantial burden in the United States generating over 11 million clinic visits and costing $3.5 billion annually. Uropathogenic Escherichia coli (UPEC) is the causative organism for 80% of uncomplicated UTI cases that are currently treated with antibiotics; it is becoming increasingly evident that other treatments are needed. These treatments will be especially important for the 4 million U.S. women who suffer recurrent UTI and are given long term antibiotic regimens, which in turn fuels increasing antibiotic resistance. There is currently a major gap in the understanding of how UPEC obtain the nutrients needed to rapidly replicate inside the host, as well as knowledge of specific growth compounds that promote successful colonization. The urinary tract is a harsh and nutrient-restricted environment; therefore, bacterial pathogens must adapt their nutrient uptake and corresponding metabolic pathways to best utilize available resources. In contrast to E. coli in the intestinal tract, UPEC in the bladder is thought to utilize amino acids as a primary carbon source. Guided by preliminary data, this proposed study will work toward the long-term goal of achieving a better understanding of UPEC biology to encourage the development of improved UTI treatments. My central hypothesis is that specific UPEC transport systems are required during UTI to facilitate metabolic adaptation to the host urinary tract environment and allow for infection to occur. This hypothesis will be tested by conducting two Specific Aims: 1) delineate transport systems that are crucial infection-specific fitness factors for growth in human urine, and 2) identify transport systems that serve as fitness factors during UTI in vivo. This proposed study will characterize critical UPEC transport systems and identify metabolic pathways that are dependent on the substrate being transported during infection. We have previously constructed, identified, and ordered the required transposon mutants needed for this study. Under the first aim, transporter mutants will be grown in human urine and nutrient-rich media to compare, and subsequently eliminate, mutants with generalized growth defects to identify those with only growth defects in urine. Additionally, gene expression profiles will be compared to identify the metabolic shifts that occur when essential nutrients are eliminated from the milieu. Under the second aim, the well-established CBA/J murine model of ascending UTI will be utilized to identify transport systems that serve as host-specific fitness factors during UTI in vivo (e.g., required during UTI but not required for growth in human urine). The contribution of individual transporters will be assessed and ranked in an unbiased manner through a novel co-challenge technique utilizing qPCR to quantify levels of mutant bacteria among small subpopulations of similar mutants. Phenotypic assays will be performed on select transport mutants to elucidate the mechanisms of action contributing to in vivo fitness defects. The proposed research is significant because it will provide insight into how UPEC acquire and utilize vital nutrients that allows for the metabolic flexibility needed to successfully cause UTI.