Project description:The kidney and bladder expresses a variety of powerful defense mechanisms to limit urinary infection including iron scavenging, a process called "nutritional immunity". We previously demonstrated that bladder and kidney epithelia generates the archetype of the urinary iron defense, a lipocalin called NGAL which binds Enterochelin, a bacterial siderophore. However, because urinary bacteria can evade the bacteriostatic effects of NGAL by modifying Enterochelin and by producing additional siderophores, other mechanisms of "nutritional immunity" are anticipated. To study the defense in the kidney, we used a cell type specific method of RNA isolation in mouse (adapted from Gay et al, Oregon). A promiscuous form of phosphoribosyltransferase (UPRT) was cloned into the Rosa26 locus using a floxed-stop design which we activated in a cell specific fashion with Atp6v1b1-Cre. Thiouracil was introduced 12hr and 24hrs after urinary tract infection. Isolation of RNA directly from intercalated revealed synthetic (Npas1-Bmal1-Alas), transport (Hrg1, Flvcr1) and metabolic enzymes (Hmox1) of heme metabolism. Hmox1 was detected in the kidney with a luciferase based reporter (Contag et al, Stanford) after infection. CO production was detected in vivo and in AtpCre-mTmG FACS isolated intercalated cells in vitro using a novel metal-based probe synthesized at Columbia (adapted from Liu et al, China). Urinary bacteria (UPEC) with heme transport mutations were not competitive in the colonization of the kidney but conversely were markedly stimulated by iron, suggesting bacterial-host competition for heme capture and metabolism. In fact, infection upregulated both heme synthetic and metabolic genes, suggesting that CO production is induced by infection. Exposure to CO terminated the growth of UPEC. In sum, we have identified an unusual iron trafficking system in the collecting ducts that mirrors the nutritional requirements of UPEC to achieve pyelonephritis. Many of these components are specific to the intercalated cells, consistent with the notion that these cells defend the urinary tract from infection.

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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Urinary Microbiome

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