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Harnessing microbial-derived metabolites in the urinary tract to prevent infection induced catheter encrustation

ABSTRACT:

Proteus mirabilis is a predominant cause of catheter associated urinary tract infection (CAUTI), and a key virulence factor is its urease enzyme which hydrolyzes urea to ammonia. This process results in the formation of urinary stones, facilitating catheter blockage and bacteremia. The only FDA approved urease inhibitor, acetohydroxamic acid (AHA), has side effects that limit its clinical use. Common urinary tract colonizers modulate P. mirabilis urease activity via secreted small molecules. A global metabolomics analysis of six modulatory bacterial species revealed 31 candidate urease-dampening metabolites, seven of which were verified to decrease P. mirabilis urease activity. Six metabolites decreased the activity of purified urease enzyme, suggesting a direct mechanism of action, and all seven dampened urease activity in at least two other urease-positive bacterial species. One metabolite (D-imidazole lactate) demonstrated antimicrobial activity and decreased expression of the urease operon in P. mirabilis. All pairwise combinations of metabolites and AHA were assessed for synergistic reduction in P. mirabilis urease activity. Ten combinations demonstrated significant synergistic dampening, and two were further validated to prevent catheter encrustation in an in vitro model for CAUTI. Prophylactic use of urease dampening metabolites with AHA could improve the efficacy of antimicrobial treatment against catheter biofilms.

INSTRUMENT(S): Liquid Chromatography MS - - - Q Exactive

PROVIDER: MTBLS891 | MetaboLights | 2025-08-29

REPOSITORIES: MetaboLights

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			Action	DRS
	23434 Peak Area Table.xlsx	Xlsx
	QXA15POSEAR20210709_SUNY0221VW_SALINE1_03.mzML	Mzml
	QXA15POSEAR20210709_SUNY0221VW_SALINE1_04.mzML	Mzml
	QXA15POSEAR20210709_SUNY0221VW_SALINE1_05.mzML	Mzml
	QXA15POSEAR20210709_SUNY0221VW_SALINE1_06.mzML	Mzml

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Project description:Catheter-associated urinary tract infections (CAUTIs) account for over 30% of acute nosocomial infections in the U.S. and generate $340 million in healthcare costs annually. A major causative agent of CAUTIs is Proteus mirabilis, an understudied Gram-negative pathogen noted for its ability to form urinary stones via the activity of urease. Urease mutants cannot induce stones and are attenuated in a murine UTI model, indicating this enzyme is essential to P. mirabilis pathogenesis. The ability to induce urinary stone formation requires an active urease, a nickel metalloenzyme that hydrolyzes urea. This reaction produces ammonia as a byproduct, which can serve as a nitrogen source and weak base that raises the local pH. The resulting alkalinity induces the precipitation of polyvalent cations and anions to form stones. Expression of urease genes is activated by transcriptional regulator UreR in a urea-dependent manner. Thus, urease genes are highly expressed in the urinary tract where urea is abundant (~400 mM in human urine). Production of mature urease also requires the import of nickel into the cytoplasm and its incorporation into the urease apoenzyme. Urease accessory proteins primarily acquire nickel from the Ynt transporter and facilitate the incorporation of nickel to form mature urease. P. mirabilis encodes a second, low-affinity transport system (Nik) that can provide nickel when this metal is abundant. In this study, we identified UreR as the first defined regulator of nickel transport in P. mirabilis. We also offer evidence for direct regulation of the ynt promoter by UreR. Using bioinformatics, we identified UreR-regulated urease loci in 15 Morganellaceae family species across three genera. Additionally, we located two mobilized UreR-regulated urease loci that also encode the ynt transporter, implying that UreR regulation of ynt is a conserved regulatory relationship. Our study demonstrates that UreR regulates all genes required to produce mature urease, an essential virulence factor for P. mirabilis uropathogenesis.

Project description:The enteric bacterium Proteus mirabilis is a common cause of complicated urinary tract infections. In the study, microrarrays were used to analyze P. mirabilis gene expression in vivo from experimentally infected mice. Urine was collected at 1, 3, and 7d postinfection, and RNA was isolated from bacteria in the urine for transcriptional analysis. Across 9 microarrays, 471 genes were upregulated and 82 were downregulated in vivo compared to in vitro broth culture. Genes upregulated in vivo encoded MR/P fimbriae, urease, iron uptake systems, amino acid and peptide transporters, pyruvate metabolism, and portions of the TCA cycle. Flagella were downregulated. Ammonia assimilation gene glnA (glutamine synthetase) was repressed in vivo while gdhA (glutamate dehydrogenase) was upregulated in vivo. Contrary to our expectations, ammonia availability due to urease activity in P. mirabilis did not drive this gene expression. A gdhA mutant was growth-deficient in minimal medium with citrate as the sole carbon source, and loss of gdhA resulted in a significant fitness defect in the mouse model of urinary tract infection. Unlike Escherichia coli, which represses gdhA and upregulates glnA in vivo and cannot utilize citrate, the data suggest that P. mirabilis uses glutamate dehydrogenase to monitor carbon-nitrogen balance, and this ability contributes to the pathogenic potential of P. mirabilis in the urinary tract. Voided urine from female CBA/J mice infected with Proteus mirabilis was collected and pooled in RNA stabilizing reagent (RNAprotect). Urine was collected at 1, 3, and 7 d postinfection. RNA was isolated from urine and log-phase LB cultures, converted to cDNA, and labeled with CyDye. Three arrays were completed per time point (9 arrays total). Slides were scanned with a ScanArray Express microarray scanner (Perkin Elmer) at 10 μm resolution and quantified using ScanArray Express software. Resulting data were normalized by total intensity and median spot intensities were identified using MIDAS (v. 2.22) software.

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Harnessing microbial-derived metabolites in the urinary tract to prevent infection induced catheter encrustation

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