Znu is the predominant zinc importer in Yersinia pestis during in vitro growth but is not essential for virulence

Abstract

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.

FIG. 1.

Genetic organization of the znuACB (A), zur (B), and Zn efflux genes (C) of Y. pestis KIM10+. ORF arrows indicate the direction of transcription and are drawn to scale. The space between znuA and znuC is a divergent promoter region. The deletion in znuCB and the kanamycin cassette (kan) insertion into zur are shown. Protein characteristics are also listed. *, ZnuA protein characteristics are for the processed form of the protein.

FIG. 2.

Growth of the Y. pestis znu mutant is reduced in Chelex-100-treated PMH2 at 37°C due to Zn deprivation. Where indicated, FeCl₃, ZnCl₂, or TPEN were added at the concentrations shown. NA, no additions. For simplicity, growth results with 1 μM FeCl₃ are not shown. Strains: KIM6+, Pgm⁺ Znu⁺; KIM6-2077+, Pgm⁺ Znu⁻; KIM6-2077(pZnu2)+, Pgm⁺ Znu⁻/Znu⁺. pZnu2 encodes the znuACB locus expressed from native promoters. The growth curves shown are from one of two independent experiments; both yielded similar results.

FIG. 3.

Zn- and Zur-dependent regulation of the znuA and znuCB promoters. Y. pestis KIM6+ (Znu⁺ Zur⁺; black bars), KIM6-2078 (Znu⁺ Zur⁻; white bars), and KIM6-2077+ (Znu⁻ Zur⁺; gray bars) carrying either the znuA::lacZ (A) or znuC::lacZ (B) reporters were grown in Chelex-100-treated PMH2 at 37°C with no additions (NA), 10 μM FeCl₃, 10 μM ZnCl₂, or 1 μM TPEN. The values are averages of replicate samples from two or more independent experiments. Error bars indicate the standard deviations.

FIG. 4.

Survival analysis of mice following s.c. or i.n. infection with Znu⁺ and Znu⁻ Y. pestis strains. s.c. and i.n. infections with low- and mid-range (Mid) infectious doses by KIM5-2077(pCD1Ap)+ (ΔznuBC2077 mutant) or control Znu⁺ strains are shown. Numbers in parentheses are the average dose from two or more infections; P values are shown in each panel. For s.c. infections, 8 mice were used for each dose and bacterial strain; for i.n. infections, 12 mice were used for each dose and bacterial strain. For i.n. infections only, the Znu⁺ strains include KIM5-2088.7(pCD1Ap)+, as well as KIM5(pCD1Ap)+. KIM5-2088.7(pCD1Ap)+ has mutations in yfe feo and mntH but has wild-type virulence properties in i.n. infections (data not shown).

FIG. 5.

ITC analyses of YfeA titrated with Mn²⁺ (A) and Zn²⁺ (B). Rates of heat release are shown above the corresponding integrated heats. Note the differences in the scales of the y axes. In the bottom panels, lines represent curves which were fitted to a single binding site model. Binding stoichiometries represent the averages ± the standard deviations of three independent experiments.

FIG. 6.

The Y. pestis Yfe and MntH transporters do not assist in Zn acquisition. Y. pestis strains with mutations in yfeAB, mntH, and/or znuCB were grown in Chelex-100-treated PMH2 at 37°C. All strains are Pgm⁺. Strains: KIM6+, Znu⁺ Yfe⁺ MntH⁺; KIM6-2077+, ΔznuBC; KIM6-2122.1+, ΔyfeA ΔmntH; KIM6-2077.1+, ΔznuBC ΔyfeA; KIM6-2077.2+, ΔznuBC ΔmntH; and KIM6-2077.3+, ΔznuBC ΔmntH ΔyfeA. The growth curves shown are from one of two independent experiments; both yielded similar results.

FIG. 7.

The Y. pestis znu yfe mntH mutant (KIM6-2077.3+) growth response to submicromolar concentrations of Zn is impaired by overexpression of the Yfe transporter. Cells were grown in Chelex-100-treated PMH2 at 37°C with no additions (NA), increasing concentrations of Zn as indicated (all panels), or 10 μM Fe (panel A). pYfe3 encodes the yfeABCDE locus expressed from its native promoters. (A) Western blot with antiserum against YfeA. YfeA, 5 μg of the purified protein; Yfe⁺, KIM6+; Yfe⁺⁺, KIM6-2077.3(pYfe3)+. The growth curves (B and C) and Western blot shown are from one of two independent experiments that yielded similar results. The expression levels of YfeA were not affected by mutations in mntH and znuABC (data not shown).

FIG. 8.

Excess Zn impairs Fe acquisition via the Yfe transporter during microaerophilic growth. Y. pestis KIM6-2088 (Yfe⁺ Feo⁻; panel A), KIM6-2088.1 (Yfe⁻ Feo⁻; panel B), and KIM6-2031.1 (Yfe⁻ Feo⁺; panel C) were grown at 37°C in Chelex-100-treated PMH2 containing10 μM ferrozine with no other additions (NA), 10 μM FeCl₃, or 10 μM ZnCl₂. All strains are Δpgm mutants that lack the Ybt siderophore-dependent Fe transport system. The growth curves shown are from one of two or more independent experiments; all yielded similar results.

FIG. 9.

Predicted three-dimensional structures of Y. pestis YfeA and ZnuA. (A) Front and side profiles of YfeA (left; pale green) and ZnuA (right; pale blue) show the modeled topologies. The rigid α-helix spanning the length of the proteins is colored wheat, while the histidines the His-rich loop of ZnuA are pale orange. Arrows point to the metal-binding pockets. (B) Basic and acidic metal-binding pocket residues, colored blue and red, respectively, are shown with the metals corresponding to the templates from which they were modeled (see Materials and Methods). Mn²⁺ is magenta, whereas Zn²⁺ is orange.