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Little is known about Zn homeostasis in Yersinia pestis, the plague bacillus. The Znu ABC transporter is essential for zinc (Zn) uptake and virulence in a number of bacterial pathogens. Bioinformatics analysis identified ZnuABC as the only apparent high-affinity Zn uptake system in Y. pestis. Mutation of znuACB caused a growth defect in Chelex-100-treated PMH2 growth medium, which was alleviated by supplementation with submicromolar concentrations of Zn. Use of transcriptional reporters confirmed that Zur mediated Zn-dependent repression and that it can repress gene expression in response to Zn even in the absence of Znu. Virulence testing in mouse models of bubonic and pneumonic plague found only a modest increase in survival in low-dose infections by the znuACB mutant. Previous studies of cluster 9 (C9) transporters suggested that Yfe, a well-characterized C9 importer for manganese (Mn) and iron in Y. pestis, might function as a second, high-affinity Zn uptake system. Isothermal titration calorimetry revealed that YfeA, the solute-binding protein component of Yfe, binds Mn and Zn with comparably high affinities (dissociation constants of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM, respectively), although the complete Yfe transporter could not compensate for the loss of Znu in in vitro growth studies. Unexpectedly, overexpression of Yfe interfered with the znu mutant's ability to grow in low concentrations of Zn, while excess Zn interfered with the ability of Yfe to import iron at low concentrations; these results suggest that YfeA can bind Zn in the bacterial cell but that Yfe is incompetent for transport of the metal. In addition to Yfe, we have now eliminated MntH, FetMP, Efe, Feo, a substrate-binding protein, and a putative nickel transporter as the unidentified, secondary Zn transporter in Y. pestis. Unlike other bacterial pathogens, Y. pestis does not require Znu for high-level infectivity and virulence; instead, it appears to possess a novel class of transporter, which can satisfy the bacterium's Zn requirements under in vivo metal-limiting conditions. Our studies also underscore the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.
Zinc (Zn) is an essential trace metal required to preserve the biological function and/or structural integrity of numerous enzymes and proteins in all eukaryotic and prokaryotic cells (3, 49, 99). Procuring sufficient Zn to sustain growth during mammalian infection is a considerable challenge for bacterial pathogens (56). Serum levels of Zn are in the micromolar range, and the metal's bioavailability is restricted further because it is tightly bound to proteins and not freely exchangeable (35, 85, 88, 103). In addition, as with iron (Fe), mammals sequester Zn systemically and locally in an attempt to deprive invading pathogens of this critical micronutrient (35, 85, 88, 103). Bacteria, therefore, must depend upon the expression of high-affinity Zn uptake systems to compete successfully with the mammalian host for this metal. Although Zn is essential, high concentrations are toxic because of its proclivity to occupy ligand sites intended for other transition metals, such as Mn and Fe (39); consequently, bacteria must strictly control intracellular Zn levels to avoid disruption of physiological processes (49). Two major mechanisms by which Zn homeostasis is achieved are metal effluxers and regulation of Zn uptake systems.
The discovery of the cluster 9 (C9) family of transition metal ATP-binding cassette (ABC) transporters significantly advanced our understanding of bacterial Zn metabolism (19). The function of the C9 family was revealed primarily through genetic studies in which the growth defects of mutants under metal-limiting conditions were reversed by supplementation with Zn (27, 77) or Mn (8, 27, 59). Bioinformatic analyses of the C9 solute-binding protein (SBP) components, which capture metals within the periplasmic space and ferry them to the cytoplasmic membrane-bound permease complex, revealed a bimodal clustering pattern appearing to correlate with experimentally proven metal specificities (19). One subcluster contains transporters dedicated to the acquisition of Zn (i.e., the “Znus”), whereas the other (“non-Znus”) contains transporters primarily responsible for the acquisition of Mn, and in some cases, Fe. Although most SBPs appear to have specificity for a single metal ligand, there is in vitro and in vivo experimental evidence that some non-Znu C9 SBPs possess specificities for multiple metals. The relevance of Zn uptake and the Znu system in particular is highlighted by a number of reports demonstrating that pathogens lacking this transporter are markedly attenuated in virulence (1, 15, 24, 42, 57, 64, 67, 74, 90, 104, 107).
Y. pestis epidemic strains cause bubonic, pneumonic, and septicemic plague in humans and animals. This facultative, Gram-negative bacterium primarily infects rodents and their associated fleas and is an obligate parasite alternating between its flea vectors and mammalian hosts. After infection from a flea bite, bacteria spread via the lymphatic system to a regional lymph node where they grow to high levels. From the swollen lymph nodes (buboes), bacteria spread through the blood to the liver and spleen, where they again grow to high levels before initiating a sustained septicemia required for acquisition by naive fleas and completion of the enzootic cycle. In humans, infection of the lungs (secondary pneumonic plague) can lead to respiratory droplet spread to another individual (primary pneumonic plague) (41, 53, 80). Unlike the three epidemic biotypes/biovars of Y. pestis (antiqua, medievalis, and orientalis), two other related groups (Microtus and Pestoides; endemic/enzootic strains) are avirulent or highly attenuated in humans but still cause bubonic plague in some animals (4, 109).
Iron transport systems in Y. pestis have been extensively characterized and the Ybt siderophore-dependent system has been shown to be essential for the pathogenesis of bubonic plague and to be important for the pathogenesis of pneumonic plague (31, 37). A second system, Yfe, transports both Fe and Mn; Fe uptake by this system is inhibited by both Mn and Zn. Although to a lesser extent than ybt mutations, a mutation in the Yfe ABC transporter also affects the virulence of Y. pestis in mouse models of bubonic plague, as well as septicemic plague (10, 11, and unpublished observations). In the present study, we show that ZnuABC is a primary high-affinity Zn uptake system in Y. pestis during in vitro growth that is repressed by Zur under Zn-replete growth conditions. However, the ability of our Y. pestis znu mutant to respond to submicromolar Zn and its near retention of full virulence strongly support the presence of a second high-affinity Zn transporter not identified by current bioinformatic analysis. Along these lines, herein we have eliminated seven potential transporters as the unidentified Zn transporter. For one of these, Yfe, we made the intriguing observation that its SBP, YfeA, binds Zn in vitro and in the bacterial cell although the transporter does not appear to be capable of importing the metal. In concert with other published studies of C9 transporters (26, 50, 59, 60, 67, 90), our demonstration of Zn binding by YfeA suggests that Zn binding may be a general property of non-Znu C9 SBPs. A general Zn-binding trait by C9 SBPs underscores the need for bacterial cells to balance binding and transporter specificities within the periplasm in order to maintain transition metal homeostasis.
The bacterial strains used in the present study are listed in Table Table11 . From glycerol stocks (12), Escherichia coli strains were grown in LB and Y. pestis strains were grown on Congo red (CR) agar (95) before being transferred to tryptose blood agar base (TBA) slants. The formation of red colonies on CR plates indicates that the strain has retained the 102-kb pgm locus, which encodes the Ybt iron transport system, the Hms biofilm system, and other genes; the pgm locus can be lost spontaneously in vitro at a rate of 10−5 (14, 33).
All glassware used for Zn- and/or Fe-restricted growth studies was soaked overnight in ScotClean (OWL Scientific, Inc.) to remove contaminating metals and copiously rinsed in deionized water. For growth studies on Zn or Fe acquisition, Y. pestis cells were harvested from TBA slants and grown in a chemically defined medium, PMH2, which had been extracted prior to use with Chelex-100 resin (Bio-Rad Laboratories). Zn contamination of Chelex-100-treated PMH2 was determined to be 0.52 ± 0.24 μM (CNAL Labs). The study by Gong et al. (43) has an error in the published buffer concentrations; PIPES and HEPES should be 50 mM for PMH2 and PMH, respectively (43, 94).
Y. pestis strains also were cultivated in Chelex-100-treated PMH2 supplemented with ZnCl2 or FeCl3 to various concentrations. Unless indicated otherwise, cultures were aerated (200 rpm) with culture volumes ~10 to 20% of flask volume. For analysis of Zn effects on Fe utilization during microaerophilic growth, cells were grown in Chelex-100-treated PMH2 with 10 μM ferrozine, a ferrous chelator, and were not shaken; the culture volumes were ≥50% of flask capacity. Growth through two transfers (ca. 6 to 8 generations) was used to acclimate cells to PMH2 and various Zn or Fe conditions prior to use in all experimental studies. Growth of the cultures was monitored by determining the optical density at 620 nm (OD620) with a Genesys5 spectrophotometer (Spectronic Instruments, Inc.). Where appropriate, ampicillin (Ap; 50 to 100 μg/ml), chloramphenicol (15 to 30 μg/ml), kanamycin (Km; 50 μg/ml), spectinomycin (100 μg/ml), or streptomycin (50 μg/ml) were added to the media.
Reporter plasmids with znuA::lacZ or znuC::lacZ fusions were electroporated into Y. pestis KIM6+ (znu+ zur+), KIM6-2077+ (Δznu zur+), and KIM6-2078+ (znu+ zur::kan). Cells were acclimated to growth at 37°C in Chelex-100-treated PMH2 as described above with zinc and iron supplementation as indicated. Cells were harvested during early exponential phase. β-Galactosidase activities from whole-cell lysates were measured spectrophotometrically with a Genesys 5 spectrophotometer (Spectronic Instruments) after cleavage of o-nitrophenyl β-d-galactopyranoside, and the results are expressed in Miller units (72). Assays used three replicate samples, and the results are means from two or more independent cultures.
Plasmids (Table (Table1)1) were purified by alkaline lysis and transformed into E. coli strains by standard calcium chloride transformation or electroporated into Y. pestis cells as previously described (6, 32). DNA restriction endonucleases, T4 DNA ligase, calf intestinal alkaline phosphatase, digoxyigenin labeling, Klenow, and PCR amplifications were used according to the manufacturer's specifications. Constructed mutations were confirmed by PCR or restriction enzyme digests. Sequences of PCR amplicons used for cloning genes were confirmed by DNA sequencing.
Construction of the ΔznuBC2077 suicide vector pSucZnu3.5 and Y. pestis KIM6-2077+ (Pgm+ Znu− [ΔznuBC2077]) have been previously described (50). The ΔznuBC2077 mutation removes the entire promoter region between znuA and znuC, all of znuC, and most of znuB (Fig. (Fig.1A).1A). Although the znuA open reading frame (ORF) is intact, there is no promoter for expression of the gene. Thus, this mutation likely causes loss of expression of the entire znuACB locus. The ΔznuBC2077 mutation was introduced into Y. pestis strains with different backgrounds by electroporation of pSucZnu3.5 (Table (Table1).1). Recombinant Y. pestis mutants with the desired allelic exchange were identified by colony blot using a 420-bp digoxigenin-labeled EcoRI/HindIII fragment from within the deleted region. Colonies showing no hybridization were confirmed by Southern blot hybridization with a 875-bp digoxigenin-labeled BamHI/EcoRI fragment upstream of the deletion. One recombinant from each Y. pestis background was selected and designated KIM6-2077 (Pgm− [Δpgm; Ybt− Hms−] Znu− [ΔznuBC2077]) and KIM6-2077.1+ (Pgm+ Znu− [ΔznuBC2077] Yfe− [ΔyfeAB2031.1]) (Table (Table11).
To generate ΔmntH2122 Y. pestis backgrounds for analysis of the znu mutation, suicide plasmid pΔMntH (50) (Table (Table1)1) was electroporated into KIM6-2077+, KIM6-2077.1+, and KIM6-2031.1+. Recombinant Y. pestis mutants with the desired allelic exchange were identified by PCR using primers Yp MntH 5′ SalI and YpMntH 3′ SpeI (see Table S1 in the supplemental material) and designated KIM6-2077.2+ (Pgm+ Znu− [ΔznuBC2077] MntH− [ΔmntH2122]), KIM6-2077.3+ (Pgm+ Znu− [ΔznuBC2077] Yfe− [ΔyfeAB2031.1] MntH− [ΔmntH2122]), and KIM6-2122.1+ (Pgm+ Znu+ Yfe− [ΔyfeAB2031.1] MntH− [ΔmntH2122]) (Table (Table11).
A zur::kan insertion mutation (Fig. (Fig.1B)1B) was constructed by isolating a 1.63-kb XbaI/SalI fragment from pZur3, digesting the isolated fragment with Eco47III (which cuts within the zur ORF). A 1.26-kb HincII fragment isolated from pUC4K containing a kanamycin resistance cassette (kan), and the two fragments from pZurR3 were ligated into pSUC1, generating zur::kan suicide vector pZur4 (Table (Table1).1). pZur4 was introduced into Y. pestis strains by electroporation. Recombinant Y. pestis mutants with the desired allelic exchange were identified by PCR using the primers zur3.3 and zur5.3 (see Table S1 in the supplemental material) and designated KIM6-2078+ (Pgm+ Zur− [zur::kan2078]) and KIM6-2078 (Pgm− [Δpgm; Ybt− Hms−] Zur− [zur::kan2078]) (Table (Table11).
A fetM::kan2163.2 mutation was constructed in Y. pestis using red recombinase with pKD4 as a template (22) for PCR primers y2370forF and y2370revR (see Table S1 in the supplemental material). The amplicon was electroporated into KIM6(pKD46)+ and KIM6-2077(pKD46)+, and aliquots of the electroporation mixtures were plated on TBA-Km plates. Recombinant colonies were screened for the fetM::kan2163.1 mutation by PCR using primers y2370forC and y2370revC (see Table S1 in the supplemental material). This mutation removes 1,908 bp of the 1,919-bp gene, replacing them with a kan cassette. Mutants were selected and designated KIM6-2163.2 (fetM::kan2163.2) and KIM6-2077.4 (ΔznuBC2077 fetM::kan2163.2) (Table (Table1).1). The fetMP (y2370 and y2368) genes are encoded within the pgm locus.
A Δy2842::kan2186 mutation was constructed in Y. pestis using red recombinase with pKD4 as a template (22) for PCR primers y2842-KMI and y2842-KMII (see Table S1 in the supplemental material). The amplicon was electroporated into KIM6(pKD46)+ and KIM6-2077(pKD46)+, and aliquots of the electroporation mixtures were plated on TBA-Km plates. Recombinant colonies were screened for the Δy2842::kan2186 mutation by PCR and mutations confirmed by PCR using primers y2842-ck and KM-2 (see Table S1 in the supplemental material). This mutation replaces 846 bp of the 1,134-bp gene with a kan cassette. Mutants were selected and designated KIM6-2186+ (Pgm+ Znu+ Δy2842::kan2186) and KIM6-2077.5+ (Pgm+ Znu− [ΔznuBC2077] Δy2842::kan2186) (Table (Table11).
A Δy1245::kan2187 mutation was constructed in Y. pestis using red recombinase with pKD4 (22) as a template for PCR primers y1245-KMI and y1245-KMII (see Table S1 in the supplemental material). The amplicon was electroporated into KIM6-2077(pKD46)+, and aliquots of the electroporation mixture were plated on TBA-Km solid medium. Recombinant colonies were screened for the Δy1245::kan2187 mutation by PCR and mutations confirmed by PCR using primers y1245-3 and KM-2 (see Table S1 in the supplemental material). This mutation replaces 780 bp of the 1,053-bp gene with a kan cassette. A single mutant was selected and designated KIM6-2077.6+ (Pgm+ Znu− [ΔznuBC2077] Δy1245::kan2187) (Table (Table11).
Screening of a Sau3AI Y. pestis genomic library (78) failed to identify a zur clone. Consequently, we isolated Y. pestis KIM6+ genomic DNA, digested with BamHI and Asp718 and collected ~4- to ~8-kb fragments. These fragments were ligated into the BamHI/Asp718 sites of pWSK129. A zur clone was identified by PCR using primers zur3.3 and zur5.3 and designated pZur1. A 1,653-bp EcoRV/EcoRI fragment from pZur1 was subcloned into pWSK129 and the zur-containing plasmid designated pZur3 (Table (Table11).
Y. pestis fetMP (y2370 and y2368, respectively) were PCR amplified from KIM6+ genomic DNA using primers Fet Fwd and Fet Rev (see Table S1 in the supplemental material) with Phusion DNA polymerase (Finnzymes). The amplicon was digested with BamHI and ligated into BamHI/EcoRV sites of pWSK129, generating pWSKFetMP (Table (Table1).1). Restriction enzyme digests and sequencing (ACGT, Inc.) confirmed that no PCR errors were present in fetMP. A 2.95-kb KpnI/XbaI fragment from pWSKfetMP was ligated into these sites in pBAD24, generating pBADFetMP (arabinose-inducible expression of fetMP) (Table (Table11).
The Y. pestis efeUOB (y2450-y2452) locus was PCR amplified from KIM6+ genomic DNA using primers Efe Fwd and Efe Rev (see Table S1 in the supplemental material) with Phusion DNA polymerase (Finnzymes). The amplicon was digested with BamHI and ligated into BamHI/EcoRV sites of pWSK129, generating pWSKEfeUOB (Table (Table1).1). Restriction enzyme digests and sequencing (ACGT, Inc.) confirmed that no PCR errors were present in efeUOB. A 3.78-kb KpnI/XbaI fragment from pWSKEfeUOB was ligated into these sites in pBAD24, generating pBADEfeUOB (arabinose-inducible expression of efeUOB) (Table (Table11).
The 255-bp region between znuA and znuC containing the divergent promoters for this locus was amplified by PCR using the primers znuC.3 and znuC.5 (see Table S1 in the supplemental material). The amplicon was digested with Asp718 and ligated into pEU730. Positive clones were identified by XhoI digestion. Orientation of the znu promoter region was determined by digestion with EcoRV. znuA::lacZ and znuC::lacZ fusion reporters were identified and designated pEUZnu1 and pEUZnu2, respectively (Table (Table1).1). Sequencing confirmed that no PCR errors were present in the promoter region (Elim Biopharmaceuticals).
A portion of the yfeA gene was PCR amplified from the plasmid pYfe (50) by using the primers 5′ yfeA-NdeI and 3′ yfeA-EcoRI (see Table S1 in the supplemental material). The PCR product, encoding amino acids 44 to 323 of YfeA, was digested with NdeI and EcoRI and ligated into the corresponding sites of the expression vector pET28a (EMD Chemicals, Darmstadt, Germany). In the expression construct pET28a::YfeA-His, the YfeA ORF starts with residues ENNPS, immediately after the signal sequence, and contains an N-terminal histidine tag, followed by a thrombin cleavage site. Nucleotide sequencing confirmed the yfeA sequence (Beckman Coulter Genomics, Danvers, MA).
E. coli BL21(DE3)/pLysS cells expressing YfeA-His were grown in 1 liter of Luria broth at 37°C to an OD600 of 0.6 to 0.8, induced for 3 h with 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside), and then harvested by centrifugation at 6,000 × g for 15 min at 4°C. The cell pellet was resuspended in 4 ml of 50 mM Tris (pH 8.2), 500 mM NaCl, 100 μl of protease inhibitor cocktail (PIC; Sigma-Aldrich, St. Louis, MO), and 250 μg of lysozyme (Sigma-Aldrich) and then stored at −20°C. After thawing the suspension on ice, the volume was increased to 20 ml by the addition of 16 ml of 50 mM Tris (pH 8.2), 200 mM NaCl, and 0.1% Elugent (EMD Chemicals). After 30 min on ice, the supernatant was cleared of cell debris by centrifugation at 18,000 × g for 30 min at 4°C and subsequently applied to a Superflow Ni-NTA (Qiagen, Valencia, CA) immobilized-metal affinity chromatography (IMAC) column, which had been equilibrated with 25 mM Tris (pH 7.5), 500 mM NaCl, 20 mM imidazole, and 10% glycerol (equilibration buffer). YfeA-His was eluted with the equilibration buffer supplemented with 250 mM imidazole. To excise the N-terminal histidine tag, IMAC-pure YfeA-His with 100 U of thrombin was dialyzed overnight at 4°C against 25 mM Tris (pH 7.5), 500 mM NaCl, and 10% glycerol. YfeA was then subjected to an additional round of IMAC to remove uncleaved protein. Thrombin-cleaved YfeA was pooled and dialyzed overnight at 4°C against 50 mM Tris (pH 7.5) and 10 mM EDTA (anion-exchange buffer). The final purification step of YfeA involved anion-exchange chromatography using Macro-Prep HighQ Support resin (Bio-Rad, Hercules, CA) equilibrated with anion-exchange buffer. YfeA eluted in the flowthrough, whereas contaminating proteins remained bound to the resin. Protein concentration was determined by measuring A280 in 20 mM sodium phosphate (pH 6.5) and 6 M guanidine hydrochloride using a molar extinction coefficient of 43,890 M−1 cm−1 (29).
Contaminating metals were removed from all plasticware and glassware by washing with 2% trace metal grade nitric acid (Thermo Fisher Scientific, Waltham, MA), followed by multiple washes with 18.2 MΩ water. Dialysis tubing was treated with 10 mM EDTA and washed with metal-free water. YfeA was demetallated by dialyzing the protein against 0.1 M sodium acetate (pH 6.5 [acetate buffer]), supplemented with 10 mM EDTA; to remove the ETDA, the protein then was extensively dialyzed with the acetate buffer, to which was added 10 g of the cation-chelating resin Chelex-100 (Bio-Rad)/liter (26). Prior to isothermal titration calorimetry (ITC) experiments, the sample cell, automated-injection syringe of the VP-ITC unit (MicroCal), and the sample-loading syringe were demetallated by washing them with 10 mM EDTA, followed by several washes with 18.2 MΩ water. All metal-binding studies were performed at 20°C; samples were filtered to remove particulate matter and degassed at 20°C. Binding studies were performed with 30 10-μl volumes of 500 μM Mn or Zn acetate dissolved in acetate buffer (see above). Samples were injected at 5-min intervals with a 30-s injection time into 42 μM YfeA in the same buffer at a stir-rate of 320 revolutions per min. Binding data were analyzed using the software Origin v.7.0 (MicroCal).
A portion of the yfeA gene encoding YfeA without its signal sequence was PCR amplified from pYfe3 (10) using the primers yfeA12 and yfeA2 (see Table S1 in the supplemental material). The resulting PCR product and expression vector pQE70 were digested with BamHI and SphI and ligated to generate pQEYfeA which, upon IPTG induction in E. coli K12TB1, expresses a mature form of YfeA with a C-terminal His tag (YfeA-H6; Table Table1).1). DNA sequencing (Elim Biopharmaceuticals) found no errors in the recombinant yfeA sequence. After a 3-h induction with 0.5 mM IPTG, K-12TB1(pQEYfeA) cells were harvested, lysed, and YfeA-H6 was purified by Ni-NTA chromatography (Qiagen). Antisera against YfeA-H6 were generated in rabbits by Animal Pharm Services.
For Western blots, equal protein concentrations of Y. pestis whole-cell extracts were separated on 12% (wt/vol) polyacrylamide gels containing sodium dodecyl sulfate and immunoblotted onto polyvinylidene fluoride membranes (Immobilon-P; Millipore). Blots were processed using a modified procedure of Towbin et al. (98) as previously described (31, 38, 43). Immunoreactive proteins were detected using horseradish peroxidase-conjugated protein A and the ECL Western blot detection reagent (GE Healthcare) and exposure on Kodak Biomax Light film.
Construction and testing of potentially virulent strains was performed in a CDC-approved BSL3 laboratory following Select Agent regulations using procedures approved by the University of Kentucky Institutional Biosafety Committee. All animal care and experimental procedures were conducted in accordance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, Public Health Service policy, and the U.S. Government Principals for the Utilization of and Care for Vertebrate Animals in Teaching, Research, and Training and approved by the University of Kentucky Institutional Animal Care and Use Committee. The University of Kentucky Animal Care Program is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, Inc.
Y. pestis strains were transformed with pCD1Ap by electroporation (36, 38, 43), plasmid profiles analyzed, and transformant phenotypes determined on CR agar (95) and magnesium-oxalate plates (52). After growth at 37°C in PMH2 with or without 2.5 mM CaCl2, culture supernatants were tested for LcrV secretion by Western blotting with polyclonal antisera against histidine-tagged LcrV (38).
Subcutaneous (s.c.) infection and intranasal (i.n.) instillation of mice have been previously described (31). Briefly, for s.c. infections, cultures of Y. pestis cells were grown in heart infusion broth (HIB; Becton Dickinson) at 26°C and resuspended in mouse isotonic phosphate-buffered saline (PBS). 0.1 ml of 10-fold serially diluted bacterial suspensions were injected s.c. into groups of four 6- to 8-week-old female Swiss-Webster mice (Hsd::ND4). For i.n. infections, cells were grown at 37°C in HIB with 4 mM CaCl2 to prevent full induction of Lcr in vitro and were similarly diluted in mouse isotonic PBS. Portions (20 μl) of the bacterial suspension were administered to the nares (~5-μl doses alternating between the two nostrils) of mice sedated with ketamine and xylazine. Inocula delivered via the i.n. and s.c. routes were enumerated by plating serial dilutions on TBA plates containing Ap (50 μg/ml); colonies were counted after 2 days of incubation at 30°C (31). Mice were observed daily for 2 weeks and 50% lethal dose (LD50) values were calculated according to the method of Reed and Muench (87).
A survival analysis (Kaplan-Meier) method was used to estimate the time to death in a 14-day period between different subgroups (61) using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA). Subgroups were defined first by the route of infection (s.c. or i.n.) and then by the infectious dose used for each group of mice. Differences between low-, mid-, and high-dose groups were deemed significant if survival analysis P values were <0.05. For other comparisons we used either a paired or unpaired Student t test or the equivalent nonparametric methods (i.e., Mann-Whitney) (61).
The sequenced Y. pestis KIM10+ genome (25) was used to search for Zn transport and regulatory systems. KIM10+ lacks two virulence plasmids (pCD1 and pPCP1) and was derived from KIM6+ (Table (Table1)1) (82). Three-dimensional models of Y. pestis YfeA and ZnuA were predicted by the SWISS-MODEL server (5). YfeA and ZnuA structural representations were based on MntC of Synechocystis sp. strain 6803 (PDB code: 1XVL ) and ZnuA of E. coli (PDB code: 2PS3 ), respectively. The molecular imaging program PyMOL (Schrödinger, New York, NY) was used to generate the structural graphics.
Y. pestis KIM10+ locus tags y2249, y2250, y2251, and y3862 were annotated as znuA, znuC, and znuB (Fig. (Fig.1A)1A) and pitA, respectively, based on similarities of the predicted amino acid sequences to E. coli ZnuABC and PitA (25). We did not identify any ORFs with significant similarity to ZupT (46, 90) or the periplasmic SBP ZinT. ZinT binds Zn with high affinity and is important during severe Zn depletion in E. coli (44, 56, 83). In E. coli, ZupT is a low-affinity Zn transporter (46, 90).Thus, bioinformatics predicts that Y. pestis KIM10+ has just one high-affinity ABC transporter for Zn and the low-affinity PitA transporter that has been implicated in Zn uptake at higher concentrations in E. coli (9).
Zur, a Fur-family member, is a transcriptional regulator that responds to Zn and represses transcription of genes for Zn uptake systems in a variety of bacteria (49, 66, 70, 77, 92, 96, 105, 106). As reported previously (66), an endemic/enzootic strain of Y. pestis has a functional Zur regulon; in Y. pestis KIM10+ locus tag y0573 encodes Zur (25) (Fig. (Fig.1B),1B), suggesting this epidemic strain has a functional Zn regulator. Bacterial Zn efflux systems include ZntA, ZntB, ZitB, and FieF (formerly YiiP, which also effluxes Fe2+) (73, 100, 105). The Y. pestis KIM10+ genome (25) encodes homologs of all four known Zn effluxers - ZntA (Y0410), ZntB (Y1994), ZitB (Y3050), and FieF (Y0060) (Fig. (Fig.1C).1C). Thus, Y. pestis KIM10+ appears to regulate Zn homeostasis by a combination of regulatory and efflux mechanisms.
Since the ZnuABC transporter is the only apparent high-affinity Zn uptake system in Y. pestis, we constructed a ΔznuBC2077 mutation to assess its role in Zn homeostasis (Fig. (Fig.1A).1A). Compared to the ZnuABC+ parent strain (KIM6+), the ΔznuBC2077 mutant (KIM6-2077+) had a significant growth defect in Chelex-100-treated PMH2 that was alleviated by complementation with the cloned znuACB locus on pZnu2 (Fig. (Fig.2A).2A). Zn supplementation did not significantly increase the growth of KIM6+, suggesting that the remaining Zn (0.52 ± 0.24 μM) in Chelex-100-treated PMH2 is sufficient to fully support growth of a ZnuABC+ Y. pestis strain (Fig. (Fig.2B).2B). Addition of the Zn chelator TPEN [N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine] to 0.5 μM was required to inhibit growth of KIM6+ to an extent similar to that of the ΔznuBC2077 mutant without TPEN (Fig. (Fig.2A),2A), indicating that Zn uptake by the parental strain is highly efficient.
Given that Chelex-100-treated PMH2 also is Fe deficient, it is possible that the growth defect of KIM6-2077+ was caused by a defect in Fe acquisition. Supplementation of Chelex-100-treated PMH2 with 1 or 10 μM FeCl3 increased the growth rate and yield of the ΔznuBC2077 mutant and KIM6+. However, at both Fe concentrations, KIM6-2077+ had a growth defect compared to its ZnuABC+ parent KIM6+ (Fig. (Fig.2B;2B; data not shown). In contrast, supplementation of PMH2 with 1 or 10 μM ZnCl2 restored growth of KIM6-2077+ to that of KIM6+ (Fig. (Fig.2B).2B). Collectively, these results indicate that the growth defect of the ΔznuBC2077 mutant is due to a defect in Zn uptake not Fe acquisition. They also point to the existence of a second transport system capable of accumulating Zn in the presence of relatively low concentrations of the metal.
To confirm that Zur mediates Zn-dependent repression, we constructed a zur::kan mutant, as well as znuA::lacZ and znuC::lacZ transcriptional reporters. The Y. pestis zur::kan mutant does not exhibit a growth defect compared to its parent when growth with or without Zn supplementation. Transcription from the znuA promoter (Fig. (Fig.1)1) was modestly repressed (~1.7-fold) in a Zur-dependent manner during growth in Chelex-100-treated PMH2 with 10 μM Zn added, while the znuC promoter was unaffected. In a Znu− background; however, transcription from both the znuA and the znuC promoters was greatly increased, approximately 9- and 4-fold, respectively, compared to the parent strain (Fig. (Fig.3).3). These results support our growth curves indicating that the Znu+ strain KIM6+ is able to acquire sufficient Zn from submicromolar concentrations of the metal present in Chelex-100-treated PMH2 to allow full growth. Despite the znu deletion, the znuA and znuC promoters were still repressed by 10 μM Zn approximately 14- and 3-fold, respectively (Fig. (Fig.3).3). This result is in accord with our conclusion (above) that Znu is the primary Zn uptake system in Y. pestis during in vitro growth, as well as our conjecture that the bacterium also contains a second, relatively high-affinity Zn uptake system. If a second Zn uptake system were not operating, Zn would not be taken up by the cells at the low concentrations used, and transcriptional repression via Zur would not occur. In contrast, addition of 10 μM Fe did not repress expression from either promoter in either a znu+ or Δznu background (Fig. (Fig.3).3). Supplementation of PMH2 with TPEN severely reduced growth but increased transcription from the znuA and znuC promoters. In addition, a Znu− background, increased transcription from both promoters compared to the parent strain (Fig. (Fig.3).3). These results demonstrate Zur-dependent repression of both promoters.
In recent years, the virulence of a variety of bacterial pathogens has been shown to be dependent upon Zn acquisition from the host via Znu or related C9 ABC transporters (1, 15, 24, 42, 57, 64, 67, 74, 90, 104, 107). Experiments were undertaken, therefore, to determine whether the Znu system is required for the virulence of Y. pestis. The KIM6+ strain and its derivatives used to test Zn uptake and regulation during in vitro growth are completely avirulent because they lack the pCD1 virulence plasmid which encodes a type III secretion system and its effector proteins (80) (Table (Table1).1). To restore this plasmid to the znu mutant, we electroporated the recombinant pCD1Ap plasmid into KIM6-2077+ (31, 43), generating KIM5-2077(pCD1Ap)+. As described in Materials and Methods, we used s.c. and i.n. infection of Swiss-Webster mice as models of bubonic and pneumonic plague (31, 38, 43). Although the variance between the trials reported here is larger than in previous studies, statistical analysis confirmed the surprising result that the Δznu mutant manifested no significant loss of virulence by either route of infection, at least as determined by LD50 analysis. For s.c. infection, the mutant and its parent had LD50s (± the standard deviations) of 305 ± 316 and 57 ± 50 (P = 0.4), respectively. For i.n. infection, LD50s of 266 ± 149 (Znu+) and 1,113 ± 1,376 (Znu− mutant) were calculated (P = 0.3). We then extended these analyses by comparing survival curves for the parental and Znu− mutant strains. At the lowest doses for the s.c. infections (an average of 9 and 15 cells for the mutant and parental strains, respectively), all mice survived infection with the Znu− mutant, while half the mice died by 8 days postinfection with the parental strain (Fig. (Fig.4A).4A). At a higher dose (an average of 117 and 180 cells for the mutant and parental strains, respectively), there was no significant difference in survival between the two strains (Fig. (Fig.4B).4B). Comparable results were obtained by i.n. infection; only at the lowest dose (an average of 52 and 100 cells for the mutant and parental strains, respectively) was there a significant difference between the Znu− mutant and fully virulent controls (Fig. 4C and D). Over multiple trials, the low dose for the Znu− mutant was ~2-fold lower than that of the parent, which might have accounted for the survival differences we observed. However, some individual experiments had bacterial doses for both i.n. and s.c. infections that were nearly identically in both strains. In all of these instances, all mice infected with the znu mutant survived, while those infected with the parental strains had fatality rates of 50% (i.n.) and 27% (s.c.). Within the mammal, Zn is highly sequestered (35, 85, 88, 103) and, therefore, cannot be obtained by pathogens without efficient uptake mechanisms. The finding that loss of the Znu transporter results in only a modest increase in survival in low-dose infections strongly supports the existence of an alternative, high-affinity Zn acquisition system.
As noted earlier, there is experimental evidence that non-Znu C9 transporters can possess specificity for more than one metal ligand (10, 26, 50, 54, 55, 67, 75, 91). TroABCD of Treponema pallidum, which also encodes ZnuABC, and MntABC of Neisseria gonorrhoeae, which does not encode Znu, represent examples of non-Znu C9 importers capable of high-affinity uptake of Zn (26). Most importantly, Fe uptake via Yfe is inhibited by Mn and Zn, suggesting that both of these metals compete for uptake through this system (10-11). It seemed plausible, therefore, that Yfe could be the putative alternative high-affinity Zn transporter in Y. pestis. As one approach to examine this possibility, we used ITC to compare the Mn2+- and Zn2+-binding capabilities of YfeA, the SBP component of Yfe. Of note, under the near-physiological conditions used in these experiments, Fe2+ was not included because its propensity to rapidly oxidize to Fe3+ under aerobic conditions results in the production of insoluble Fe-hydroxide polymers (86, 93). Table Table22 summarizes the ITC data, while representative isotherms are shown in Fig. Fig.5.5. As indicated by the sharp transitions in the integrated heats (kcal/mol) occurring at metal/YfeA ratios of 1, YfeA has a single metal occupancy binding site with exceedingly high affinities for both metals (dissociation constants [KDs] of 17.8 ± 4.4 nM and 6.6 ± 1.2 nM for Mn2+ and Zn2+, respectively). These KDs are in-line with those determined by ITC for Znu and other non-Znu C9 SBPs (26, 102). Overall, the free energies of binding (ΔG) for the Mn2+- and Zn2+-YfeA complexes are favorable, and relatively similar, with −10.4 × 103 and −11.0 × 103 kcal/mol, respectively (Table (Table2).2). However, despite the similarities in ΔGs and KDs as well, the thermodynamic parameters defining the manner in which YfeA associates with the metals are dramatically different. The YfeA-Mn2+ complex forms as a result of markedly favorable entropic (−TΔS = −7.0 × 103 kcal/mol) and minor enthalpic (ΔH = −3.4× 103 kcal/mol) parameters, whereas the YfeA-Zn2+ complex forms as a result of minor entropic (−TΔS = −2.3 × 103 kcal/mol) and highly favorable enthalpic (ΔH = −8.7 × 103 kcal/mol) parameters. Interestingly, these results are highly similar to those reported for TroA in which the Mn2+- and Zn2+-TroA complexes were entropically and enthalpically driven, respectively (26).
To test whether Yfe transported Zn, we constructed a ΔznuBC ΔyfeAB double mutant (KIM6-2077.1+) and assessed growth in Chelex-100-treated PMH2. The double mutant grew, as well as the single ΔznuBC mutant (KIM6-2077) (Fig. (Fig.6).6). Thus, Yfe is not the second, high-affinity Zn transporter.
Given the apparent contradiction of in vitro Zn binding by YfeA and the inability of the Yfe transporter to aid in Zn acquisition in Y. pestis, we wanted to garner evidence for binding of Zn by YfeA in the bacterial cell. If such binding occurred, overexpression of YfeA in the bacterium might interfere with the growth response to Zn supplementation. Consequently, we added submicromolar concentrations of Zn to Chelex-100-treated PMH2 and tested the growth response of a ΔznuBC ΔmntH ΔyfeA triple mutant (KIM6-2077.3+; see below) without or with expression of the Yfe system from pYfe3. Figure Figure7A7A shows that pYfe3 causes overexpression of the YfeA SBP even in the presence of 10 μM Zn. Previously, we have shown that transcription from the yfeABCD promoter is repressed by Fe in a Fur-dependent manner but does not respond to Zn (11, 79). The immunoblot analysis shows that this regulation translates to the protein level: excess Fe but not Zn lowers YfeA protein levels. A comparison of Fig. 7B and C shows that all four tested Zn concentrations resulted in significantly less growth stimulation when the triple mutant carried pYfe3. The growth defect, due to overexpression of YfeA, was alleviated by supplementation with excess Zn (10 μM; data not shown). Thus, YfeA, at least when overexpressed, appears to bind Zn in the cell periplasm and prevents its uptake.
To corroborate these findings, we sought evidence that Zn binding by YfeA interfered with the ability of the Yfe system to acquire Fe. The Y. pestis Yfe and Feo systems are both required for normal growth under microaerophilic conditions in Chelex-100-treated PMH2 supplemented with 10 μM ferrozine (Fe2+ chelator). Mutation of either system causes a growth defect which is increased when both systems are mutated (10, 11, 81). The addition of 10 μM Zn to Chelex-100-treated PMH2-10 μM ferrozine further reduced the growth of a feoB mutant (Fig. (Fig.8A)8A) but not of the yfe or yfe feoB mutants (Fig. 8B and C). These results suggest that excess Zn interferes with the ability of the remaining Yfe system in the feoB mutant to accumulate Fe for growth under microaerophilic conditions. They also suggest that Zn does not interfere with ferrous uptake via the Feo system.
As noted earlier, in vitro growth and regulation studies, as well as in vivo virulence testing support the hypothesis that Y. pestis possesses two high-affinity Zn importers. Since bioinformatic analysis identified only the ZnuABC transporter in Y. pestis KIM10+, we have begun to search for the secondary Zn importer. Metal specificities cannot always be determined from sequence homologies (67). Some transporters import Zn as well as their primary divalent cation, at least in some organisms. The specificities of other divalent cation transporters are relatively untested. We selected some candidate loci for the above reasons and others because they encode transporters that are repressed by Zn in an endemic/enzootic strain of Y. pestis (66).
Yfe and MntH serve redundant Mn import functions in Y. pestis, and MntH is a member of the NRAMP1 family that transports several divalent cations. In some bacteria, MntH has been shown to have a higher specificity for Mn uptake, although there are studies showing competition by Fe, Zn, and some other divalent cations (2, 45, 71, 76). Consequently, there is a possibility that MntH is the second Zn import system. We constructed combinations of double mutants in these three systems (Znu, Yfe, and MntH), as well as a triple mutant, and tested their growth in Chelex-100-treated PMH2. The ΔyfeAB ΔmntH mutant (KIM6-2122.1+) grew as well as the parental KIM6+ strain. Only a znu mutation caused a significant growth defect under these conditions; mutations in yfe or mntH did not increase this growth defect (Fig. (Fig.6).6). Indeed, the ΔznuBC ΔmntH ΔyfeAB triple mutant (KIM6-2077.3+) had a growth rate and final yield similar to that of the single znu mutant (KIM6-2077+) (Fig. (Fig.6).6). Thus, it seems unlikely that MntH contributes significantly to high-affinity Zn acquisition in Y. pestis.
Next, we tested the role of two Zur-regulated genes encoding transport components (66). FiuA (Y2842) shows similarity to iron/siderophore SBPs and is repressed 228-fold by excess Zn in the endemic/enzootic Y. pestis strain 201. Y1245 shows similarity to NicO a nickel transporter and is repressed 2.9-fold by Zn in strain 201 (25, 66). However, Y. pestis Δy2842 (KIM6-2186+), Δy2842 ΔznuBC (KIM6-2077.5+), and Δy1245 ΔznuBC (KIM6-2077.6+) mutants all failed to show a growth defect in Chelex-100-treated PMH2 compared to either their Znu+ and/or Znu− parent strains (data not shown).
Finally, we also examined the Feo, EfeUOB, and Fet MP ferrous transporters as potential Zn uptake systems. Although the recently described Efe system did not appear to be affected by addition of Zn, the specificity of this Ftr1p-like system remains to be fully defined (16, 47). Although Feo is generally considered specific for Fe, Mn transport by an Feo-like system has been described in Porphyromonas gingivalis (21, 51). FetMP (Y2370/y2368), encoded within the pgm locus, are putative membrane and periplasmic proteins, respectively (37). In E. coli, homologues FetMP have been shown to accumulate ferrous iron (58). A similar genetic locus has been implicated in Fe uptake in a marine bacterium (28). However, the specificity of this new and understudied type of transporter has not been addressed. Recombinant clones of feoABC expressed from its native promoter, as well as efeUOB (y2450-y2452) or fetMP expressed from their native promoters or an arabinose-inducible promoter, failed to complement the growth defect of our Y. pestis znu mutant (data not shown). Finally, Y. pestis ΔfetMP and ΔfetMP ΔznuBC mutants had no growth defect compared to their Znu+ and Znu− parents (data not shown).
Bioinformatic analysis indicates that Y. pestis has one high-affinity Zn transporter, ZnuABC. Mutation of this system resulted in a growth defect in a Chelex-100-treated, defined medium, PMH2. Growth comparable to the Znu+ parent was restored by the cloned znuABC locus or by supplementation with 1 μM Zn but not 1 or 10 μM Fe (Fig. (Fig.2;2; data not shown). Analysis of Zur-dependent Zn transcriptional repression of the znuA and znuC promoters demonstrated that the znuC promoter is slightly less responsive to Zn levels than the znuA promoter (Fig. (Fig.3).3). The ~9-fold increase in transcription from znuA in the Znu+ strain compared to the Znu− mutant suggests that the ZnuABC system is a primary Znu uptake system from low Zn levels in vitro. The relatively low levels of transcription in the Znu+ parent strain also indicates that the Znu system is sufficient to provide most of the cellular requirements of Y. pestis even in Zn-depleted PMH2, which has a residual Zn contamination of 0.52 ± 0.24 μM.
A Zur regulon was defined in Y. pestis strain 201, a Microtus strain attenuated or avirulent in humans (66). Although only the promoter regions for znuA, znuCB, and ykgM-rpmJ2 (encoding ribosomal proteins) were shown to interact directly with Zur, a number of genes were found to be responsive to Zn supplementation via Zur. Genes encoding transport components (e.g., y1245 [ypo2673], y2842 [ypo1343], and ypo2675, as well as several uncharacterized ABC transporters [ypo2660, ypo1348, and ypo3908]) were repressed. In addition, genes of the defective urease locus (ypo2665-ypo2671), a variety of metabolic and putative exported proteins, as well as some regulators were also repressed. YPO designations are locus tag numbers for epidemic strain CO92 and were used for gene designations in the Microtus strain 201 by Li et al. (66). In other cases, Zn supplementation increased gene expression (e.g., genes encoding an oligopeptide transporter [ypo2182-ypo2186], a variety of metabolic and putative exported proteins, and some regulators). Although many of these genes do not appear to be directly regulated by Zur, inasmuch as they lack apparent Zur binding sequences, it is clear that Zn homeostasis has large effects on gene expression that go beyond controlling Zn transporters (66). Thus, the metabolic status of Y. pestis appears to be affected by Zn availability.
While ZnuABC is a critical high-affinity Zn uptake system, the Y. pestis znu mutant still has a growth response to the addition of 0.4 μM Zn to Chelex-100-treated PMH2 (Fig. (Fig.7).7). This and transcriptional repression of the znuA and znuC promoters in our znu mutant by micromolar Zn supplementation of PMH2 (Fig. (Fig.3)3) are strong indicators for a second high-affinity Zn uptake system in Y. pestis. Our in vivo findings that LD50 studies showed no significant loss of virulence by the znu mutant but that survival of mice infected with this mutant increased moderately at low inoculum doses also support this hypothesis. In fact, it suggests that this alternative transporter may play a greater role in Zn physiology during infection than is apparent from the in vitro studies. Our studies lead to the hypothesis that mutation of both znu and the second, unidentified high-affinity Zn transporter will be required to observe a drastic effect of Zn deprivation on the virulence of Y. pestis.
Recently, Znu in Yersinia ruckeri, a fish pathogen, has been characterized. Here, a znu mutant, compared to its Znu+ parent strain, showed only a modest in vitro growth defect but had an ~350-fold lower bacterial load in rainbow trout by day 9 of the infection. The authors of that study concluded that Y. ruckeri had a second Zn importer that allowed in vitro growth but was either ineffective or was not expressed during infection of fish (20).
In contrast to our findings with Y. pestis, mutation of znu ABC transporters in Brucella abortus, Campylobacter jejuni, Haemophilus ducreyi, Pasteurella multocida, Salmonella enterica serovar Typhimurium, and Streptococcus pyogenes causes a loss of virulence or host colonization (1, 15, 24, 42, 57, 64, 67, 104, 107). The large differences observed between parental and mutant strains suggest that these bacteria may differ from Y. pestis in that they lack a second high-affinity Zn transporter effective in host environments. It is intriguing to note that znu mutations in Proteus mirabilis and extraintestinal E. coli also have more limited affects on colonization and growth in models of urinary tract infections than do znu mutations in the pathogens noted above (74, 90). Cochallenge studies with the P. mirabilis parent and mutant were necessary to demonstrate decreased in vivo fitness of the mutant. For the uropathogenic E. coli (UPEC) strain CFT073, a znu mutation lowered bacterial loads ~100-fold in the kidneys of mice; in contrast, this mutation did not affect bacterial loads in the bladder. Both UPEC and P. mirabilis znu mutants responded to the addition of 5 μM Zn in vitro (74, 90). Thus, both of these organisms may also express a second high-affinity Zn uptake system that can function under in vivo metal-limiting conditions, although not with the same degree of efficiency as the putative second transporter in Y. pestis.
Our attempts to identify the second high-affinity Zn uptake system in Y. pestis have been unsuccessful to date. The Yfe transporter, a member of the C9 family was a logical candidate due to previous uptake studies showing inhibition of radiolabeled Fe uptake by Zn (10, 81). We also tested mutations in mntH, y1245 (a putative nickel transporter) and y2842 (encoding a putative iron/siderophore SBP); transcription of both y1245 and y2842 were repressed by Zn (66). None of these mutations caused an increased growth requirement for Zn. We also attempted to restore Zn-deficient growth by complementation of our znu mutant with recombinant clones of ferrous transporters FeoABC, FetMP, or EfeUOB. The metal specificities of these transporters, especially Fet and Efe, have not been fully elucidated. However, none of these systems expressed from native and/or artificial promoters on moderate-copy-number plasmids enhanced the growth of the znu mutant under Zn-depleted conditions. Thus, the second high-affinity Zn transporter of Y. pestis remains to be identified and is likely to represent a new family of Zn transporters or a cation transporter with a previously unrecognized ability to accumulate Zn from low concentrations.
In previous reports, we have shown that Yfe, a non-Znu C9 transporter, facilitates the uptake of Mn and Fe (10, 81). Our ITC studies not only confirmed the high-affinity nature of the Yfe-Mn2+ interaction (KD = 17.8 ± 4.4 nM), they also extended the aforementioned observations to include the capacity of YfeA to bind Zn2+ (KD = 6.6 ± 1.2 nM). To better understand the metal specificities of YfeA and ZnuA revealed by our binding studies, both proteins were modeled based on the solved structures of C9 SBPs (Fig. (Fig.9).9). The modeling predicts that YfeA and ZnuA share the three-dimensional topologies common to their C9 counterparts (7, 17, 60, 62, 63, 65, 68, 69, 84, 89, 102, 108), most notably a long α-helical backbone bridging the N- and C-terminal α/β-fold domains, which interface to form the metal-binding pocket. In addition, the His-rich tract of ZnuA, a distinctive structural feature of Zn-dedicated C9 SBPs, is predicted to form a flexible loop proximal to the metal-binding site similar to those observed in ZnuAs from other bacteria (7, 17, 65, 102, 108). With the amino acid composition of the binding pocket of YfeA comprising 2 His, 1 Glu, and 1 Asp (Fig. (Fig.9),9), the permissible coordinate bonds for liganding a metal range from 4 to 6. In contrast, the metal-binding pocket of ZnuA contains three His residues that provide three coordinate bonds, with a fourth possibly mediated by water, as observed in the structure of the Synechocystis ZnuA (Fig. (Fig.9)9) (7). Mn and Fe maintain strong preferences for coordination geometries consisting of either 5 or 6, whereas Zn2+ has adaptable coordination geometries (4, 5, or 6) with 4 being optimal (18, 39). ZnuA of Y. pestis, therefore, appears to allow for the sole binding of Zn2+ by restricting the number of coordinate bonds, whereas YfeA appears to allow for multiple transition metal (Zn2+, Mn2+, and Fe2+) specificities by maintaining a flexible number of coordinate bonds. Interestingly, sequence alignments reveal that the configuration of metal-binding residues of YfeA is common to non-Znu SBPs (19, 50), with one notable exception being TroA, which has 3 His and 1 Asp (62). Despite this difference with YfeA, TroA also binds Mn2+ and Zn2+ with equally high affinities (26). It is possible therefore, that Zn binding is a widespread property of non-Znu SBPs. The structure of PsaA bound to Zn (60), the interference of Zn with the uptake functions of the Mnt, Sca, Sit, and Yfe ABC transporters (10, 59, 76, 90), and the binding studies reported herein collectively support this conjecture.
Our ITC binding analyses (Fig. (Fig.5),5), combined with previous studies demonstrating the inhibitory effect of Zn on the uptake of Fe by Yfe (10) suggested that Yfe might serve as a secondary Zn transporter in Y. pestis. In the ΔznuABC ΔmntH ΔyfeA strain, however, the overexpression of Yfe from a plasmid not only failed to complement the function of Znu under metal-limiting conditions, it abrogated the positive influence of Zn supplementation on the growth of the mutant (Fig. (Fig.7)7) and inhibited Yfe's ability to acquire Fe2+ (Fig. (Fig.8).8). The simplest explanation for these results is that YfeA binds Zn in the bacterial periplasm, in accord with the in vitro ITC analyses (Fig. (Fig.5),5), but that the metal does not get transported into the cytoplasm. When an ABC transporter complex engages its substrate-loaded SBP, conformational changes in the SBP upon the hydrolysis of ATP lead to transfer of the substrate to the permease and its subsequent transport into the cytoplasm (23). In the case of C9 SBPs, the rigid α-helical backbone restricts the mobility of the ligand-binding subdomains such that the conformational change required to release the bound metal is small (60, 62); moreover, permease recognition and/or substrate release may be dependent upon the cation being liganded. Several possibilities can be envisioned, therefore, to explain why overexpression of Yfe in the presence of Zn supplementation disrupts the ability of Y. pestis to achieve transition metal homeostasis. Quite possibly, Zn-loaded YfeA fails to engage the permease or engages it but fails to induce the appropriate conformational changes required to release the metal. Alternatively, the Yfe permease engages the YfeA-Zn complex and induces the appropriate conformational changes in the SBP, releasing Zn to a transport-incompetent permease; the metal then would return to the periplasmic space where it would be tightly bound by overexpressed apo-YfeA. Regardless of which mechanism is correct, the net effect would be to “freeze” periplasmic Zn in a YfeA-bound state, rendering it inaccessible to an alternative, as-yet-unidentified, Zn transporter.
The above observations suggest that Yfe potentially comes into contact with Zn, in effect, creating a scenario in which Yfe, Znu, and the unidentified second Zn-transporter compete for the same metal. Therefore, given the inhibitory effect of Zn on the transport capability of Yfe, combined with the tendency of Zn to replace metal ligands and form stable complexes with Mn- and Fe-metalloproteins (39, 97), it is likely that Y. pestis must calibrate the binding and uptake capabilities of multiple metal transporters to maintain transition metal homeostasis. The capacity of the secondary transporter in Zn uptake with respect to the metal transport functions of Yfe and Znu is unknown. In the absence of Znu, however, the minor decrease in virulence indicates that the secondary transporter is sufficient for acquiring Zn, but these results also indicate that the transporter alone is not as efficient as Znu in siphoning Zn from Yfe, and possibly, other transporters with Zn-binding capacities. In this regard, an important feature of ZnuA may be the His-rich loop. Currently, the function of this loop is not clear, but evidence suggests that the His-rich tract serves as a Zn-chaperone that fosters the uptake of Zn by attracting and ferrying the metal into the binding pocket (26, 83, 102). Studies by Berducci et al. provided the first evidence for Zn sequestration by ZnuA (13). These authors showed in E. coli that under conditions of Zn limitation, expression of ZnuA prevented acquisition of the metal by Cu, Zn superoxide dismutase within the periplasmic space. In addition, providing evidence that the loop behaves as a mobile extension scavenging Zn, several studies demonstrated metal-binding sites outside of the binding pocket cleft (26, 102, 108). Collectively, these studies imply that the His-rich loop facilitates sequestration of Zn in the periplasmic space, thereby preventing the adventitious binding of the metal by other transporters. Clearly, in Y. pestis, there are aspects of Zn acquisition and metal transporter cooperativity that we have yet to completely understand, although it is conceivable that in order to circumvent a situation in which Zn competes against Mn and Fe2+ for acquisition by YfeA, Znu and the unidentified secondary Zn-transporter must be coordinately expressed.
This study was supported by Public Health Service grants AI-26756 (J.D.R.) and AI-33481 (R.D.P.) and by the Connecticut Children's Medical Center Arrison and Burr Curtis Research Funds (J.C.S.).
Editor: S. M. Payne
Published ahead of print on 20 September 2010.
‡Supplemental material for this article may be found at http://iai.asm.org/.