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Human Urinary Composition Controls Antibacterial Activity of Siderocalin*

  • Robin R. Shields-Cutler
    Footnotes
    Affiliations
    From the Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

    Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Jan R. Crowley
    Affiliations
    Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri 63110,
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  • Chia S. Hung
    Affiliations
    From the Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

    Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri 63110
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  • Ann E. Stapleton
    Affiliations
    the Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, Washington 98195,
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  • Courtney C. Aldrich
    Footnotes
    Affiliations
    the Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, and
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  • Jonas Marschall
    Footnotes
    Affiliations
    From the Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

    the Department of Infectious Diseases, Bern University Hospital and University of Bern, 3010 Bern, Switzerland
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  • Jeffrey P. Henderson
    Correspondence
    Recipient of a Career Award for Medical Scientists from the Burroughs Welcome Fund. To whom correspondence should be addressed: Division of Infectious Diseases, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-747-0240; Fax: 314-454-8294;
    Affiliations
    From the Division of Infectious Diseases, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

    Center for Women's Infectious Disease Research, Washington University School of Medicine, St. Louis, Missouri 63110
    Search for articles by this author
  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01DK099534 and P50DK064540 from NIDDK. This work was also supported by National Center for Advancing Translational Sciences Grant UL1TR000448 and the Longer Life Foundation. Mass spectrometry was supported by United States Public Health Service Grants P41RR000954, P30DK020579, P30HL101263 and P30DK056341.
    ♦ This article was selected as a Paper of the Week.
    2 Supported by National Institutes of Health Grant AI070219.
    1 Supported by National Institutes of Health Grant T32GM007067-37 and a Monsanto Excellence Fund Graduate Fellowship.
    3 Supported by National Institutes of Health Grants U54CK000162 and K12HD001459 and the Barnes-Jewish Hospital Patient Safety and Quality Fellowship Program.
Open AccessPublished:April 10, 2015DOI:https://doi.org/10.1074/jbc.M115.645812
      During Escherichia coli urinary tract infections, cells in the human urinary tract release the antimicrobial protein siderocalin (SCN; also known as lipocalin 2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3). SCN can interfere with E. coli iron acquisition by sequestering ferric iron complexes with enterobactin, the conserved E. coli siderophore. Here, we find that human urinary constituents can reverse this relationship, instead making enterobactin critical for overcoming SCN-mediated growth restriction. Urinary control of SCN activity exhibits wide ranging individual differences. We used these differences to identify elevated urinary pH and aryl metabolites as key biochemical host factors controlling urinary SCN activity. These aryl metabolites are well known products of intestinal microbial metabolism. Together, these results identify an innate antibacterial immune interaction that is critically dependent upon individualistic chemical features of human urine.

      Introduction

      Escherichia coli is the predominant cause of urinary tract infections (UTIs),
      The abbreviations used are: UTI
      urinary tract infection
      SCN
      siderocalin
      UPEC
      uropathogenic E. coli
      ROC
      receiver operating characteristic
      2,3-DHB-AMS
      ((2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl)methyl-(2,3-dihydroxybenzoyl)sulfamate
      DA
      discriminant analysis
      LC-CNL
      LC-constant neutral loss
      ANOVA
      analysis of variance
      PCA-DA
      principal component analysis-discriminant analysis
      QC
      quality control
      ESI
      electrospray ionization
      AUC
      area under the curve
      UFLC
      ultra fast liquid chromatography.
      one of the world's most common bacterial infections (
      • Foxman B.
      The epidemiology of urinary tract infection.
      ). Compared with familiar K12 strains, uropathogenic E. coli (UPEC) secretes an expanded repertoire of siderophores, low molecular weight metal ion chelators defined by their ability to scavenge Fe(III) for nutritional purposes (
      • Henderson J.P.
      • Crowley J.R.
      • Pinkner J.S.
      • Walker J.N.
      • Tsukayama P.
      • Stamm W.E.
      • Hooton T.M.
      • Hultgren S.J.
      Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli.
      ,
      • Miethke M.
      • Marahiel M.A.
      Siderophore-based iron acquisition and pathogen control.
      ). Siderophore biosynthetic genes are dramatically up-regulated during experimental UTI, and a UPEC siderophore has been directly detected in urine from UTI patients using mass spectrometry (
      • Reigstad C.S.
      • Hultgren S.J.
      • Gordon J.I.
      Functional genomic studies of uropathogenic Escherichia coli and host urothelial cells when intracellular bacterial communities are assembled.
      ,
      • Snyder J.A.
      • Haugen B.J.
      • Buckles E.L.
      • Lockatell C.V.
      • Johnson D.E.
      • Donnenberg M.S.
      • Welch R.A.
      • Mobley H.L.
      Transcriptome of uropathogenic Escherichia coli during urinary tract infection.
      ,
      • Chaturvedi K.S.
      • Hung C.S.
      • Crowley J.R.
      • Stapleton A.E.
      • Henderson J.P.
      The siderophore yersiniabactin binds copper to protect pathogens during infection.
      ). Genes encoding the enterobactin siderophore system are ubiquitous in E. coli, whereas the nonconserved yersiniabactin, salmochelin, and aerobactin systems exist in varying combinations (
      • Henderson J.P.
      • Crowley J.R.
      • Pinkner J.S.
      • Walker J.N.
      • Tsukayama P.
      • Stamm W.E.
      • Hooton T.M.
      • Hultgren S.J.
      Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli.
      ). Expression of different siderophore types exacts varying metabolic costs that may affect each siderophore system's frequency within a population (
      • Lv H.
      • Hung C.S.
      • Henderson J.P.
      Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli.
      ).
      The evolutionary circumstances that have led UPEC to express multiple siderophore systems are incompletely understood. Differential susceptibility to sequestration by the innate immune defense protein siderocalin (SCN; also known as lipocalin 2/LCN2, neutrophil gelatinase-associated lipocalin/NGAL, or 24p3) has been proposed as a major selective pressure driving acquisition of chemically diverse, virulence-associated UPEC siderophores (
      • Fischbach M.A.
      • Lin H.
      • Zhou L.
      • Yu Y.
      • Abergel R.J.
      • Liu D.R.
      • Raymond K.N.
      • Wanner B.L.
      • Strong R.K.
      • Walsh C.T.
      • Aderem A.
      • Smith K.D.
      The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2.
      ,
      • Hoette T.M.
      • Clifton M.C.
      • Zawadzka A.M.
      • Holmes M.A.
      • Strong R.K.
      • Raymond K.N.
      Immune interference in Mycobacterium tuberculosis intracellular iron acquisition through siderocalin recognition of carboxymycobactins.
      ,
      • Abergel R.J.
      • Moore E.G.
      • Strong R.K.
      • Raymond K.N.
      Microbial evasion of the immune system: structural modifications of enterobactin impair siderocalin recognition.
      ). SCN is introduced into the urinary tract by neutrophil granules and uroepithelial cells, which up-regulate SCN >100-fold within 24 h of bladder inoculation (
      • Reigstad C.S.
      • Hultgren S.J.
      • Gordon J.I.
      Functional genomic studies of uropathogenic Escherichia coli and host urothelial cells when intracellular bacterial communities are assembled.
      ). SCN can restrict iron accessibility by binding Fe(III) complexes with high affinity within the protein’s positively charged binding site, or calyx (
      • Goetz D.H.
      • Holmes M.A.
      • Borregaard N.
      • Bluhm M.E.
      • Raymond K.N.
      • Strong R.K.
      The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.
      ,
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ). Although enterobactin is the prototypical ferric ligand in these complexes, other siderophore and non-siderophore ligands have been described and substantiated in detailed binding and crystallographic studies (
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ,
      • Holmes M.A.
      • Paulsene W.
      • Jide X.
      • Ratledge C.
      • Strong R.K.
      Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration.
      ).
      Here, we used genetic and chemical approaches to determine how UPEC siderophores and SCN interact in the chemically distinctive urinary environment associated with human UTIs. We found that human urinary constituents make enterobactin critically important for resisting SCN, in contrast to its role as an SCN target in nonurinary environments (
      • Fischbach M.A.
      • Lin H.
      • Zhou L.
      • Yu Y.
      • Abergel R.J.
      • Liu D.R.
      • Raymond K.N.
      • Wanner B.L.
      • Strong R.K.
      • Walsh C.T.
      • Aderem A.
      • Smith K.D.
      The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2.
      ,
      • Bachman M.A.
      • Oyler J.E.
      • Burns S.H.
      • Caza M.
      • Lépine F.
      • Dozois C.M.
      • Weiser J.N.
      Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.
      ,
      • Raffatellu M.
      • George M.D.
      • Akiyama Y.
      • Hornsby M.J.
      • Nuccio S.-P.
      • Paixao T.A.
      • Butler B.P.
      • Chu H.
      • Santos R.L.
      • Berger T.
      • Mak T.W.
      • Tsolis R.M.
      • Bevins C.L.
      • Solnick J.V.
      • Dandekar S.
      • Bäumler A.J.
      Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype Typhimurium for growth and survival in the inflamed intestine.
      ). Individual differences in urinary properties allowed us to chemically dissect urinary composition, revealing pH and aryl alcohol metabolites as critical SCN antibacterial activity correlates. These results point to the existence of a urinary tract-specific host-pathogen interaction system involving siderocalin, host metabolites, and bacterial enterobactin biosynthesis. These findings suggest nonantibiotic chemical therapeutic strategies that potentiate innate antibacterial defenses against urinary pathogens.

      Discussion

      Together, these results describe a biochemical network in which siderocalin, urinary aryl metabolites, and enterobactin interact to control bacterial growth. Our conclusion that SCN uses a subset of urinary metabolites as cofactors to withhold iron from E. coli emerges from an experimental strategy that used individual human differences as an independent variable; this approach informed further analyses that identified urinary pH and aryl metabolite associations with SCN activity. Consistent with these associations are the known pH and catechol requirements for ferric ion binding by SCN (
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ). Although enterobactin's role in resisting SCN-mediated growth inhibition in urine was unexpected, it is consistent with the concept that niche-specific roles drive siderophore diversity among bacterial pathogens (
      • Chaturvedi K.S.
      • Hung C.S.
      • Crowley J.R.
      • Stapleton A.E.
      • Henderson J.P.
      The siderophore yersiniabactin binds copper to protect pathogens during infection.
      ,
      • Chaturvedi K.S.
      • Hung C.S.
      • Giblin D.E.
      • Urushidani S.
      • Austin A.M.
      • Dinauer M.C.
      • Henderson J.P.
      Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic.
      ,
      • Bachman M.A.
      • Lenio S.
      • Schmidt L.
      • Oyler J.E.
      • Weiser J.N.
      Interaction of lipocalin 2, transferrin, and siderophores determines the replicative niche of Klebsiella pneumoniae during pneumonia.
      ,
      • Brock J.H.
      • Williams P.H.
      • Licéaga J.
      • Wooldridge K.G.
      Relative availability of transferrin-bound iron and cell-derived iron to aerobactin-producing and enterochelin-producing strains of Escherichia coli and to other microorganisms.
      ,
      • Luo M.
      • Lin H.
      • Fischbach M.A.
      • Liu D.R.
      • Walsh C.T.
      • Groves J.T.
      Enzymatic tailoring of enterobactin alters membrane partitioning and iron acquisition.
      ). Therefore, although enterobactin may be particularly consequential in the urinary environment examined here, SCN evasion by non-enterobactin “stealth siderophores” may dominate in nonurinary environments lacking urine's unique aryl metabolite composition.
      SCN′s documented ability to form a highly stable complex with ferric enterobactin led us to initially hypothesize that salmochelin and/or yersiniabactin biosynthesis would be necessary for growth. Our contrary observations in urine can be explained by the presence of one or more non-enterobactin urinary ligands within the SCN calyx that function as host-derived cofactors (
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ,
      • Correnti C.
      • Strong R.K.
      Mammalian siderophores, siderophore-binding lipocalins, and the labile iron pool.
      ). Previous work by Bao et al. (
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ) demonstrated that endogenous urinary ligands, including aryl alcohols as described here, do form stable ferric complexes with SCN. Because of its insolubility in aqueous environments, “free” iron exists in a labile pool, for which aryl alcohols are well suited ligands. Indeed, the higher pH associated with restrictive urine closely parallels the trends in ferric complex stability and SCN binding observed for catecholate ligands (
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ,
      • Abergel R.J.
      • Warner J.A.
      • Shuh D.K.
      • Raymond K.N.
      Enterobactin protonation and iron release: structural characterization of the salicylate coordination shift in ferric enterobactin.
      ). Because ferric complexes can form from multiple aryl alcohol chelators (e.g. three divalent catechol chelators per ferric ion) and multiple members of this class may be present in human urine, restrictive urines may arise from idiosyncratic combinations of these molecules. The sulfated aryl metabolites tracked here likely represent readily detectable markers of free aryl alcohols. Future studies will be necessary to identify which of these contribute to SCN activity.
      A proposed model for SCN function in the urinary tract is shown in Fig. 11. In this model, the host responds to UPEC by producing SCN, which (at pH >6.45) stabilizes ferric complexes of appropriate urinary metabolites in its calyx (Fig. 11, a and b). Although SCN can bind enterobactin with high affinity, this interaction may be greatly impeded when urine-derived ferric complexes occupy the calyx. In this setting, enterobactin’s remarkable ferric ion affinity may instead enable it to competitively liberate iron from SCN-bound cofactors (Fig. 11c), thus promoting bacterial growth. This ability suggests a basis for the ubiquitous expression of enterobactin observed to date in urinary E. coli isolates. Although yersiniabactin can avoid SCN binding, it is also an avid Cu(II) ligand with lower ferric affinity than enterobactin and so may be less able to liberate iron from SCN as suggested in Fig. 2b (
      • Miethke M.
      • Marahiel M.A.
      Siderophore-based iron acquisition and pathogen control.
      ,
      • Chaturvedi K.S.
      • Hung C.S.
      • Crowley J.R.
      • Stapleton A.E.
      • Henderson J.P.
      The siderophore yersiniabactin binds copper to protect pathogens during infection.
      ,
      • Perry R.D.
      • Balbo P.B.
      • Jones H.A.
      • Fetherston J.D.
      • DeMoll E.
      Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation.
      ). Similarly, although loss of salmochelin production did not affect urinary growth (Fig. 2b), we cannot rule out its potential role in liberating SCN-bound iron. Further studies are necessary to determine how ferric enterobactin avoids binding to the SCN calyx following ferric ion removal, which may be attributable to competitive binding or slow release of aferric urinary ligands. This working model (Fig. 11) suggests important areas for future investigation and opportunities for new virulence-targeting UTI therapeutic approaches distinct from broad-spectrum antibiotics.
      Figure thumbnail gr11
      FIGURE 11Proposed model of SCN antimicrobial activity in restrictive urine. a, human urine contains a complex and variable mixture of metabolites, including aryl metabolites such as catecholates. Iron is limiting, and it exists as a labile pool of low affinity complexes with numerous ligands. b, when UPEC enters the bladder, SCN is up-regulated and secreted into the bladder lumen. Elevated pH facilitates host-derived ferric-aryl complex assembly in the SCN calyx, starving UPEC of iron. c, in this context, the siderophore enterobactin is able to outcompete the SCN-bound complexes for iron, making it bioavailable to UPEC.
      The biochemical network described here raises the possibility that promoting restrictive urinary characteristics in patients may prevent or treat antibiotic-resistant E. coli UTI without incurring “collateral damage” on the vaginal or gut microbiome (
      • Foxman B.
      The epidemiology of urinary tract infection.
      ,
      • Barber A.E.
      • Norton J.P.
      • Spivak A.M.
      • Mulvey M.A.
      Urinary tract infections: current and emerging management strategies.
      ). This strategy may benefit from an enterobactin biosynthesis inhibitor such as DHB-AMS. Urinary alkalinization is readily achieved through existing interventions such as oral bicarbonate therapy. Although favorable urinary aryl metabolite profiles are associated with the highest level of SCN activity, future studies will be required to determine how to therapeutically recapitulate this in susceptible patients. The urinary aryl sulfates identified here (Table 1) and in prior studies are known to originate from the combination of diet, gut microbial metabolism, and host liver metabolism. Individual differences in these metabolites may thus have multiple origins. Of note, urinary catechols are associated with consumption of polyphenol-rich foods such as tea, coffee, wine, and cranberries (
      • Lang R.
      • Mueller C.
      • Hofmann T.
      Development of a stable isotope dilution analysis with liquid chromatography-tandem mass spectrometry detection for the quantitative analysis of di- and trihydroxybenzenes in foods and model systems.
      ,
      • van Duynhoven J.
      • Vaughan E.E.
      • Jacobs D.M.
      • Kemperman R.A.
      • van Velzen E.J.
      • Gross G.
      • Roger L.C.
      • Possemiers S.
      • Smilde A.K.
      • Doré J.
      • Westerhuis J.A.
      • Van de Wiele T.
      Metabolic fate of polyphenols in the human superorganism.
      ,
      • Gonthier M.-P.
      • Cheynier V.
      • Donovan J.L.
      • Manach C.
      • Morand C.
      • Mila I.
      • Lapierre C.
      • Rémésy C.
      • Scalbert A.
      Microbial aromatic acid metabolites formed in the gut account for a major fraction of the polyphenols excreted in urine of rats fed red wine polyphenols.
      ,
      • Pérez-Jiménez J.
      • Hubert J.
      • Hooper L.
      • Cassidy A.
      • Manach C.
      • Williamson G.
      • Scalbert A.
      Urinary metabolites as biomarkers of polyphenol intake in humans: a systematic review.
      ), suggesting that dietary strategies may be feasible.
      Mass spectrometry-based metabolomic approaches have excellent potential to resolve individual differences relevant to many disease processes (
      • Lv H.
      • Hung C.S.
      • Chaturvedi K.S.
      • Hooton T.M.
      • Henderson J.P.
      Development of an integrated metabolomic profiling approach for infectious diseases research.
      ,
      • Wang J.H.
      • Byun J.
      • Pennathur S.
      Analytical approaches to metabolomics and applications to systems biology.
      ,
      • Vinayavekhin N.
      • Homan E.A.
      • Saghatelian A.
      Exploring disease through metabolomics.
      ). Although definitive metabolite identification using these methods is often a laborious process, we were aided here by a specific ion fragmentation process (neutral loss of 80 atomic mass units) characteristic of a urinary biochemical class linked by structure to candidate SCN ligands (
      • Bao G.
      • Clifton M.
      • Hoette T.M.
      • Mori K.
      • Deng S.-X.
      • Qiu A.
      • Viltard M.
      • Williams D.
      • Paragas N.
      • Leete T.
      • Kulkarni R.
      • Li X.
      • Lee B.
      • Kalandadze A.
      • Ratner A.J.
      • et al.
      Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
      ,
      • Yi L.
      • Dratter J.
      • Wang C.
      • Tunge J.A.
      • Desaire H.
      Identification of sulfation sites of metabolites and prediction of the compounds' biological effects.
      ). This “molecular bootstrapping” approach, in which a molecular lead derived from untargeted profiling is used to inform a second round of class-specific profiling, may aid further metabolomic discovery work in UTI and other disease processes.

      Acknowledgments

      We thank Bradley Ford and Kaveri S. Parker for helpful discussions, Thomas Brett and Dan Kober for technical assistance, and Roland Strong and Jonathan Barasch for sharing siderocalin constructs.

      Author Profile

      References

        • Foxman B.
        The epidemiology of urinary tract infection.
        Nat. Rev. Urol. 2010; 7: 653-660
        • Henderson J.P.
        • Crowley J.R.
        • Pinkner J.S.
        • Walker J.N.
        • Tsukayama P.
        • Stamm W.E.
        • Hooton T.M.
        • Hultgren S.J.
        Quantitative metabolomics reveals an epigenetic blueprint for iron acquisition in uropathogenic Escherichia coli.
        PLoS Pathog. 2009; 5: e1000305
        • Miethke M.
        • Marahiel M.A.
        Siderophore-based iron acquisition and pathogen control.
        Microbiol. Mol. Biol. Rev. 2007; 71: 413-451
        • Reigstad C.S.
        • Hultgren S.J.
        • Gordon J.I.
        Functional genomic studies of uropathogenic Escherichia coli and host urothelial cells when intracellular bacterial communities are assembled.
        J. Biol. Chem. 2007; 282: 21259-21267
        • Snyder J.A.
        • Haugen B.J.
        • Buckles E.L.
        • Lockatell C.V.
        • Johnson D.E.
        • Donnenberg M.S.
        • Welch R.A.
        • Mobley H.L.
        Transcriptome of uropathogenic Escherichia coli during urinary tract infection.
        Infect. Immun. 2004; 72: 6373-6381
        • Chaturvedi K.S.
        • Hung C.S.
        • Crowley J.R.
        • Stapleton A.E.
        • Henderson J.P.
        The siderophore yersiniabactin binds copper to protect pathogens during infection.
        Nat. Chem. Biol. 2012; 8: 731-736
        • Lv H.
        • Hung C.S.
        • Henderson J.P.
        Metabolomic analysis of siderophore cheater mutants reveals metabolic costs of expression in uropathogenic Escherichia coli.
        J. Proteome Res. 2014; 13: 1397-1404
        • Fischbach M.A.
        • Lin H.
        • Zhou L.
        • Yu Y.
        • Abergel R.J.
        • Liu D.R.
        • Raymond K.N.
        • Wanner B.L.
        • Strong R.K.
        • Walsh C.T.
        • Aderem A.
        • Smith K.D.
        The pathogen-associated iroA gene cluster mediates bacterial evasion of lipocalin 2.
        Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 16502-16507
        • Hoette T.M.
        • Clifton M.C.
        • Zawadzka A.M.
        • Holmes M.A.
        • Strong R.K.
        • Raymond K.N.
        Immune interference in Mycobacterium tuberculosis intracellular iron acquisition through siderocalin recognition of carboxymycobactins.
        ACS Chem. Biol. 2011; 6: 1327-1331
        • Abergel R.J.
        • Moore E.G.
        • Strong R.K.
        • Raymond K.N.
        Microbial evasion of the immune system: structural modifications of enterobactin impair siderocalin recognition.
        J. Am. Chem. Soc. 2006; 128: 10998-10999
        • Goetz D.H.
        • Holmes M.A.
        • Borregaard N.
        • Bluhm M.E.
        • Raymond K.N.
        • Strong R.K.
        The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition.
        Mol. Cell. 2002; 10: 1033-1043
        • Bao G.
        • Clifton M.
        • Hoette T.M.
        • Mori K.
        • Deng S.-X.
        • Qiu A.
        • Viltard M.
        • Williams D.
        • Paragas N.
        • Leete T.
        • Kulkarni R.
        • Li X.
        • Lee B.
        • Kalandadze A.
        • Ratner A.J.
        • et al.
        Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex.
        Nat. Chem. Biol. 2010; 6: 602-609
        • Holmes M.A.
        • Paulsene W.
        • Jide X.
        • Ratledge C.
        • Strong R.K.
        Siderocalin (Lcn 2) also binds carboxymycobactins, potentially defending against mycobacterial infections through iron sequestration.
        Structure. 2005; 13: 29-41
        • Bachman M.A.
        • Oyler J.E.
        • Burns S.H.
        • Caza M.
        • Lépine F.
        • Dozois C.M.
        • Weiser J.N.
        Klebsiella pneumoniae yersiniabactin promotes respiratory tract infection through evasion of lipocalin 2.
        Infect. Immun. 2011; 79: 3309-3316
        • Raffatellu M.
        • George M.D.
        • Akiyama Y.
        • Hornsby M.J.
        • Nuccio S.-P.
        • Paixao T.A.
        • Butler B.P.
        • Chu H.
        • Santos R.L.
        • Berger T.
        • Mak T.W.
        • Tsolis R.M.
        • Bevins C.L.
        • Solnick J.V.
        • Dandekar S.
        • Bäumler A.J.
        Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype Typhimurium for growth and survival in the inflamed intestine.
        Cell Host Microbe. 2009; 5: 476-486
        • Murphy K.C.
        • Campellone K.G.
        Lambda Red-mediated recombinogenic engineering of enterohemorrhagic and enteropathogenic E. coli.
        BMC Mol. Biol. 2003; 4: 11
        • Datsenko K.A.
        • Wanner B.L.
        One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.
        Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 6640-6645
        • Hooton T.M.
        Uncomplicated urinary tract infection.
        N. Engl. J. Med. 2012; 366: 1028-1037
        • Matzanke B.F.
        • Ecker D.J.
        • Yang T.S.
        • Huynh B.H.
        • Müller G.
        • Raymond K.N.
        Escherichia coli iron enterobactin uptake monitored by Mössbauer spectroscopy.
        J. Bacteriol. 1986; 167: 674-680
        • Sikora A.L.
        • Wilson D.J.
        • Aldrich C.C.
        • Blanchard J.S.
        Kinetic and inhibition studies of dihydroxybenzoate-AMP ligase from Escherichia coli.
        Biochemistry. 2010; 49: 3648-3657
        • Miethke M.
        • Bisseret P.
        • Beckering C.L.
        • Vignard D.
        • Eustache J.
        • Marahiel M.A.
        Inhibition of aryl acid adenylation domains involved in bacterial siderophore synthesis.
        FEBS J. 2006; 273: 409-419
        • Lv H.
        • Hung C.S.
        • Chaturvedi K.S.
        • Hooton T.M.
        • Henderson J.P.
        Development of an integrated metabolomic profiling approach for infectious diseases research.
        Analyst. 2011; 136: 4752-4763
        • Want E.J.
        • Wilson I.D.
        • Gika H.
        • Theodoridis G.
        • Plumb R.S.
        • Shockcor J.
        • Holmes E.
        • Nicholson J.K.
        Global metabolic profiling procedures for urine using UPLC-MS.
        Nat. Protoc. 2010; 5: 1005-1018
        • Clayton T.A.
        • Baker D.
        • Lindon J.C.
        • Everett J.R.
        • Nicholson J.K.
        Pharmacometabonomic identification of a significant host-microbiome metabolic interaction affecting human drug metabolism.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14728-14733
        • Lang R.
        • Mueller C.
        • Hofmann T.
        Development of a stable isotope dilution analysis with liquid chromatography-tandem mass spectrometry detection for the quantitative analysis of di- and trihydroxybenzenes in foods and model systems.
        J. Agric. Food Chem. 2006; 54: 5755-5762
        • Bouatra S.
        • Aziat F.
        • Mandal R.
        • Guo A.C.
        • Wilson M.R.
        • Knox C.
        • Bjorndahl T.C.
        • Krishnamurthy R.
        • Saleem F.
        • Liu P.
        • Dame Z.T.
        • Poelzer J.
        • Huynh J.
        • Yallou F.S.
        • Psychogios N.
        • et al.
        The human urine metabolome.
        PloS One. 2013; 8: e73076
        • Booth A.N.
        • Masri M.S.
        • Robbins D.J.
        • Emerson O.H.
        • Jones F.T.
        • De Eds F.
        The metabolic fate of gallic acid and related compounds.
        J. Biol. Chem. 1959; 234: 3014-3016
        • Stalmach A.
        • Mullen W.
        • Barron D.
        • Uchida K.
        • Yokota T.
        • Cavin C.
        • Steiling H.
        • Williamson G.
        • Crozier A.
        Metabolite profiling of hydroxycinnamate derivatives in plasma and urine after the ingestion of coffee by humans: identification of biomarkers of coffee consumption.
        Drug. Metab. Dispos. 2009; 37: 1749-1758
        • Miller R.C.
        • Brindle E.
        • Holman D.J.
        • Shofer J.
        • Klein N.A.
        • Soules M.R.
        • O'Connor K.A.
        Comparison of specific gravity and creatinine for normalizing urinary reproductive hormone concentrations.
        Clin. Chem. 2004; 50: 924-932
        • Paragas N.
        • Kulkarni R.
        • Werth M.
        • Schmidt-Ott K.M.
        • Forster C.
        • Deng R.
        • Zhang Q.
        • Singer E.
        • Klose A.D.
        • Shen T.H.
        • Francis K.P.
        • Ray S.
        • Vijayakumar S.
        • Seward S.
        • Bovino M.E.
        • et al.
        α-Intercalated cells defend the urinary system from bacterial infection.
        J. Clin. Invest. 2014; 124: 2963-2976
        • Steigedal M.
        • Marstad A.
        • Haug M.
        • Damås J.K.
        • Strong R.K.
        • Roberts P.L.
        • Himpsl S.D.
        • Stapleton A.
        • Hooton T.M.
        • Mobley H.L.
        • Hawn T.R.
        • Flo T.H.
        Lipocalin 2 imparts selective pressure on bacterial growth in the bladder and is elevated in women with urinary tract infection.
        J. Immunol. 2014; 193: 6081-6089
        • van Duynhoven J.
        • Vaughan E.E.
        • Jacobs D.M.
        • Kemperman R.A.
        • van Velzen E.J.
        • Gross G.
        • Roger L.C.
        • Possemiers S.
        • Smilde A.K.
        • Doré J.
        • Westerhuis J.A.
        • Van de Wiele T.
        Metabolic fate of polyphenols in the human superorganism.
        Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 4531-4538
        • Marschall J.
        • Zhang L.
        • Foxman B.
        • Warren D.K.
        • Henderson J.P.
        • CDC Prevention Epicenters Program
        Both host and pathogen factors predispose to Escherichia coli urinary-source bacteremia in hospitalized patients.
        Clin. Infect. Dis. 2012; 54: 1692-1698
        • Hung C.
        • Marschall J.
        • Burnham C.-A.
        • Byun A.S.
        • Henderson J.P.
        The bacterial amyloid curli is associated with urinary source bloodstream infection.
        PLoS ONE. 2014; 9: e86009
        • Mobley H.L.
        • Green D.M.
        • Trifillis A.L.
        • Johnson D.E.
        • Chippendale G.R.
        • Lockatell C.V.
        • Jones B.D.
        • Warren J.W.
        Pyelonephritogenic Escherichia coli and killing of cultured human renal proximal tubular epithelial cells: role of hemolysin in some strains.
        Infect. Immun. 1990; 58: 1281-1289
        • Simerville J.A.
        • Maxted W.C.
        • Pahira J.J.
        Urinalysis: a comprehensive review.
        Am. Fam. Physician. 2005; 71: 1153-1162
        • Sieniawska C.E.
        • Jung L.C.
        • Olufadi R.
        • Walker V.
        Twenty-four-hour urinary trace element excretion: reference intervals and interpretive issues.
        Ann. Clin. Biochem. 2012; 49: 341-351
        • Abergel R.J.
        • Warner J.A.
        • Shuh D.K.
        • Raymond K.N.
        Enterobactin protonation and iron release: structural characterization of the salicylate coordination shift in ferric enterobactin.
        J. Am. Chem. Soc. 2006; 128: 8920-8931
        • Yi L.
        • Dratter J.
        • Wang C.
        • Tunge J.A.
        • Desaire H.
        Identification of sulfation sites of metabolites and prediction of the compounds' biological effects.
        Anal. Bioanal. Chem. 2006; 386: 666-674
        • Wikoff W.R.
        • Nagle M.A.
        • Kouznetsova V.L.
        • Tsigelny I.F.
        • Nigam S.K.
        Untargeted metabolomics identifies enterobiome metabolites and putative uremic toxins as substrates of organic anion transporter 1 (Oat1).
        J. Proteome Res. 2011; 10: 2842-2851
        • Gonthier M.-P.
        • Cheynier V.
        • Donovan J.L.
        • Manach C.
        • Morand C.
        • Mila I.
        • Lapierre C.
        • Rémésy C.
        • Scalbert A.
        Microbial aromatic acid metabolites formed in the gut account for a major fraction of the polyphenols excreted in urine of rats fed red wine polyphenols.
        J. Nutr. 2003; 133: 461-467
        • Carmella S.G.
        • La Voie E.J.
        • Hecht S.S.
        Quantitative analysis of catechol and 4-methylcatechol in human urine.
        Food Chem. Toxicol. 1982; 20: 587-590
        • Hunter J.
        Matplotlib: A 2D graphics environment.
        Comput. Sci. Eng. 2007; 9: 90-95
        • Chaturvedi K.S.
        • Hung C.S.
        • Giblin D.E.
        • Urushidani S.
        • Austin A.M.
        • Dinauer M.C.
        • Henderson J.P.
        Cupric yersiniabactin is a virulence-associated superoxide dismutase mimic.
        ACS Chem. Biol. 2014; 9: 551-561
        • Bachman M.A.
        • Lenio S.
        • Schmidt L.
        • Oyler J.E.
        • Weiser J.N.
        Interaction of lipocalin 2, transferrin, and siderophores determines the replicative niche of Klebsiella pneumoniae during pneumonia.
        mBio. 2012; 3: e00224-11
        • Brock J.H.
        • Williams P.H.
        • Licéaga J.
        • Wooldridge K.G.
        Relative availability of transferrin-bound iron and cell-derived iron to aerobactin-producing and enterochelin-producing strains of Escherichia coli and to other microorganisms.
        Infect. Immun. 1991; 59: 3185-3190
        • Luo M.
        • Lin H.
        • Fischbach M.A.
        • Liu D.R.
        • Walsh C.T.
        • Groves J.T.
        Enzymatic tailoring of enterobactin alters membrane partitioning and iron acquisition.
        ACS Chem. Biol. 2006; 1: 29-32
        • Correnti C.
        • Strong R.K.
        Mammalian siderophores, siderophore-binding lipocalins, and the labile iron pool.
        J. Biol. Chem. 2012; 287: 13524-13531
        • Perry R.D.
        • Balbo P.B.
        • Jones H.A.
        • Fetherston J.D.
        • DeMoll E.
        Yersiniabactin from Yersinia pestis: biochemical characterization of the siderophore and its role in iron transport and regulation.
        Microbiology. 1999; 145: 1181-1190
        • Barber A.E.
        • Norton J.P.
        • Spivak A.M.
        • Mulvey M.A.
        Urinary tract infections: current and emerging management strategies.
        Clin. Infect. Dis. 2013; 57: 719-724
        • Pérez-Jiménez J.
        • Hubert J.
        • Hooper L.
        • Cassidy A.
        • Manach C.
        • Williamson G.
        • Scalbert A.
        Urinary metabolites as biomarkers of polyphenol intake in humans: a systematic review.
        Am. J. Clin. Nutr. 2010; 92: 801-809
        • Wang J.H.
        • Byun J.
        • Pennathur S.
        Analytical approaches to metabolomics and applications to systems biology.
        Semin. Nephrol. 2010; 30: 500-511
        • Vinayavekhin N.
        • Homan E.A.
        • Saghatelian A.
        Exploring disease through metabolomics.
        ACS Chem. Biol. 2010; 5: 91-103