Saturday, December 21, 2024
12/21/2024
Uropathogenic bacteria must acquire nutrients present in urine or those released by an inflamed or damaged epithelium to successfully colonize the urinary tract. Our long term goal is to understand the contribution of each virulence factor and core gene that allows uropathogens to colonize, persist, and damage the urinary tract. Our overall objective is to identify and study essential transporters required by uropathogenic Escherichia coli (UPEC), a cause of uncomplicated urinary tract infection (UTI), and Proteus mirabilis, a cause of catheter- associated UTI, for growth and survival in the human urinary tract. Both E. coli and P. mirabilis are members of the Enterobacteriaceae, are motile, produce numerous and distinct fimbriae with which they mediate adherence to the uroepithelium, secrete hemolysins and proteases, and elaborate siderophores and heme-binding proteins to capture iron from the host. While we have previously studied all of these classes of virulence determinants, we now propose to focus on transport systems as critical functions for the survival of these two pathogens. In this proposal, we will advance the central hypothesis that uropathogenic E. coli and P. mirabilis differentially employ transport systems, unique to each species, to acquire peptides/amino acids and sugars, respectively, to establish fitness advantages in the urinary tract. We will test this hypothesis by carrying out the following specific aims: 1) Identify specific transport systems required for the development of urinary tract infection by uropathogenic Escherichia coli and Proteus mirabilis. 2) Determine the kinetic parameters of unique and common transporters that contribute to the virulence of uropathogenic Escherichia coli and Proteus mirabilis in the urinary tract. In the first aim, we will identify genes from among 643 identified transporter genes of E. coli and 386 identified transport genes of P. mirabilis, that when mutated result in a fitness defect in the mouse model of ascending UTI. We will use a Tn-seq screen of ordered transposon libraries of both species in the mouse model of UTI, expression data from in vivo RNA-seq transcriptomes, transurethral cochallenge of mice with clean deletion mutants vs the wild type strain, and urine metabolomes to select transporters for further study. In the second aim, prioritized transporters will be characterized for growth in filter-sterilized human urine, minimal medium containing the known or suspected substrate, and Biolog assays to confirm substrates and identify transporters that share the same substrate. Kinetics of substrate uptake [KT (affinity for substrate) and Vmax (rate of transport)] will be measured using radiolabeled substrates. Finally, a hierarchy of transporters will be established for UPEC and P. mirabilis. The expected outcome of conducting these aims will be to characterize key transport systems and their substrates of our two most troublesome uropathogens. The positive impact will include identification of novel targets of antimicrobial therapy.
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