Coordinated Zinc Homeostasis Is Essential for the Wild-Type Virulence of Brucella abortus

ZntR controls the expression of zntA in a zinc-responsive manner. Quantitative RT-PCR was used to assess the amount of znuA and zntA mRNA produced by B. abortus 2308 or B. abortus ΔzntR incubated in zinc-replete medium, as well as when the same strains were incubated in medium containing the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) at a final concentration of 100 μM for 30 min. 16S rRNA levels were assessed as a control, and all mRNA levels were normalized to the level of the parental strain 2308 grown in the absence of TPEN. The graphic bars depict the average level of specific mRNA for each strain and condition listed from two biological replicate experiments, each analyzed in triplicate, and the error bars represent the standard deviations. (A) Relative expression levels of zntA in B. abortus 2308 or the B. abortus ΔzntR mutant strain grown in the absence (−) or presence (+) of TPEN. Statistical significance between the levels of zntA mRNA in a given strain grown with and without TPEN was determined using a t test (*, P < 0.05; NS, not significant). (B) Relative expression levels of znuA in B. abortus 2308 or the B. abortus ΔzntR mutant strain grown in the absence (−) or presence (+) of TPEN.

Zur binds directly to the znu (zinc uptake) promoter regions and requires zinc for interactions with DNA. (A) Recombinant Zur (rZur) protein was assessed for binding to the znuA-znuC intergenic region using electrophoretic mobility shift assays (EMSAs). Increasing concentrations of rZur were incubated with radiolabeled znuA-znuC intergenic region DNA, and in some binding reactions, unlabeled specific (znuA-znuC intergenic region DNA) or nonspecific (zntA-zntR intergenic region DNA) competitor DNA fragments were included as controls. The presence (+) and absence (−) of components in the binding reaction mixture are indicated. In addition to testing the binding of rZur to the znuA-znuC intergenic region, interactions between rZur and DNA corresponding to the virB promoter were also assessed as a negative control. (B) EMSAs performed with the znuA-znuC intergenic region DNA and rZur protein in the presence of EDTA and various divalent metal cations. All binding reaction mixtures contained 250 nM rZur and 50 μM ZnCl₂, and, where indicated, 250 μM EDTA was included in some samples. To assess the metal specificity of rZur binding to DNA, an additional divalent metal cation (Zn²⁺, Fe²⁺, Mn²⁺, Ni²⁺, or Cu²⁺) was added to the binding reaction mixtures at a final concentration of 25 μM in the presence of EDTA.

ZntR binds with high affinity to the Brucella zntR-zntA promoter region. (A) Recombinant ZntR (rZntR) protein was assessed for binding to the zntA-zntR intergenic region using electrophoretic mobility shift assays (EMSAs). Increasing concentrations of rZntR were incubated with radiolabeled zntA-zntR intergenic region DNA, and in some binding reactions, unlabeled specific (zntA-zntR intergenic region DNA) or nonspecific (cueO promoter region DNA) competitor DNA fragments were included as controls. The presence (+) and absence (−) of components in the binding reaction mixture are indicated. (B) Specificity of rZntR binding to the zntA-zntR promoter regions. EMSAs were performed as described in panel A, but here, increasing concentrations of rZntR were incubated with radiolabeled znuA-znuC intergenic region DNA or radiolabeled virB promoter (P_virB) region DNA.

Mutation of zntA, encoding a high-affinity zinc exporter, results in elevated sensitivity to zinc toxicity. (A) Isogenic zur, zntR, and zntA mutants were constructed in Brucella abortus 2308, and these strains were tested in a disk diffusion assay for their comparative susceptibilities to toxic levels of zinc. The results are plotted as the average diameter (±standard deviation) of the zone of inhibition around a disk containing ZnCl₂, and the results are from a representative experiment that was performed independently at least three times with triplicate samples for each individual experiment. Statistical significance between results for a given strain and those for the parental strain 2308 was determined by a t test (*, P < 0.05). (B) The zntA mutant is uniquely sensitive to zinc concentrations. A disk diffusion assay was performed to compare the susceptibilities of B. abortus 2308 and the zntA mutant strain to toxic levels of divalent cations, including Ni²⁺, Zn²⁺, and Cu²⁺. The results are shown as the average diameter (±standard deviation) of the zone of inhibition around a disk containing NiCl₂, ZnCl₂, or CuCl₂, and the results are from a single representative experiment that was performed three times independently using triplicate samples for each experiment. Statistical significance between results for the zntA mutant strain and those for the parental strain 2308 was determined by a t test (*, P < 0.05). (C) Genetic complementation of zntA expression restores resistance to zinc toxicity. The wild-type zntA locus was reconstructed on the chromosome of the zntA mutant strain, and this complemented strain (zntA::comp) was assessed for its sensitivity to zinc toxicity using a disk diffusion assay. Data are from a representative experiment performed independently three times using triplicate samples for each experiment. Statistical significance between results for the zntA mutant strain and those for the parental strain 2308 was determined by a t test (*, P < 0.05).

Virulence of B. abortus 2308 and the isogenic zur, zntR, and zntA mutants in murine peritoneal macrophages and experimentally infected mice. (A) Macrophage survival and replication experiments. Cultured resident peritoneal macrophages from BALB/c mice were infected with B. abortus 2308 and the isogenic zur, zntR, and zntA mutant strains. At the indicated times postinfection, the macrophages were lysed, and the number of intracellular brucellae present in these phagocytes was determined by serial dilution, plating, and bacteriologic culture. The results are from a representative experiment that was performed independently four times. (B) Mouse infection experiments. BALB/c mice were infected intraperitoneally with B. abortus 2308 and the isogenic zur, zntR, and zntA mutant strains. Mice were sacrificed at weeks 1, 4, and 8 postinfection, and the number of brucellae colonizing the spleens was determined. The data are presented as the average number of brucellae ± the standard deviation from 5 mice colonized with a specific Brucella strain at a specific time point. These data are from a single experiment using 5 mice per bacterial strain (i.e., n = 5). Statistical significance between results for the zntR mutant strain and those for the parental strain 2308 at 8 weeks postinfection was determined by a t test (*, P < 0.05).