The manganese transporter MntH is a critical virulence determinant for Brucella abortus 2308 in experimentally infected mice

Abstract

The gene designated BAB1_1460 in the Brucella abortus 2308 genome sequence is predicted to encode the manganese transporter MntH. Phenotypic analysis of an isogenic mntH mutant indicates that MntH is the sole high-affinity manganese transporter in this bacterium but that MntH does not play a detectable role in the transport of Fe(2+), Zn(2+), Co(2+), or Ni(2+). Consistent with the apparent selectivity of the corresponding gene product, the expression of the mntH gene in B. abortus 2308 is repressed by Mn(2+), but not Fe(2+), and this Mn-responsive expression is mediated by a Mur-like repressor. The B. abortus mntH mutant MWV15 exhibits increased susceptibility to oxidative killing in vitro compared to strain 2308, and a comparative analysis of the superoxide dismutase activities present in these two strains indicates that the parental strain requires MntH in order to make wild-type levels of its manganese superoxide dismutase SodA. The B. abortus mntH mutant also exhibits extreme attenuation in both cultured murine macrophages and experimentally infected C57BL/6 mice. These experimental findings indicate that Mn(2+) transport mediated by MntH plays an important role in the physiology of B. abortus 2308, particularly during its intracellular survival and replication in the host.

FIG. 1.

Manganese-responsive repression of an mntH-lacZ transcriptional fusion in B. abortus 2308 is mediated by Mur. (A) Expression of an mntH-lacZ fusion in B. abortus 2308 following 24 h of growth in brucella broth or brucella broth supplemented with 50, 250, or 500 μM MnCl₂. (B) Activity of the mntH-lacZ fusion in B. abortus 2308 and the isogenic mur mutant (Fur2) following 24 h of growth in unsupplemented brucella broth (white bars), brucella broth supplemented with 500 μM MnCl₂ (hatched bars), or brucella broth supplemented with 500 μM FeCl₂ (black bars). β-Galactosidase activity is presented on the y axis in Miller units (53). The data presented are means and standard deviations for triplicate determinations from a single culture in a single experiment. The data presented are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained. *, P < 0.05; **, P < 0.005 (for comparisons of β-galactosidase activities in unsupplemented medium and medium supplemented with MnCl₂ or FeCl₃).

FIG. 2.

Supplemental MnCl₂ restores wild-type growth of the B. abortus mntH mutant in broth culture. (A) Growth of B. abortus 2308 (▴), MWV15 (2308 mntH) (□), and MWV15.C [MWV15(pEA31)] (▪) in brucella broth and MWV15 in brucella broth supplemented with 50 μM MnCl₂ (⧫) or 50 μM FeCl₂ (⋄). (B) Growth of B. abortus 2308 (▴), MWV15 (□), and MWV15.C (▪) in low-manganese minimal medium and MWV15 in low-manganese medium supplemented with 50 μM MnCl₂ (⧫). The data presented are the means and standard deviations for triplicate determinations from a single flask for each strain at each experimental time point in a single experiment. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained. *, P < 0.05; **, P < 0.005 (for comparisons of strain 2308 with MWV15 or MWV15.C).

FIG. 3.

Growth of the B. abortus mntH mutant in low-manganese medium supplemented with Mn²⁺, Fe²⁺, Zn²⁺, Co²⁺, Ni²⁺, and Cu²⁺. Bacterial cell cultures were inoculated into low-manganese medium at cell densities of approximately 10⁵ CFU/ml, and the number of bacteria present in these cultures following 48 h of incubation was enumerated by serial dilution and plating. Shown are the B. abortus parental strain (2308), the mntH mutant (MWV15), and the mntH mutant grown in low-manganese minimal medium supplemented with MnCl₂ at concentrations of 1, 5, and 10 μM (designated Mn1, Mn5, and Mn10, respectively). MWV15 cultures supplemented with 100 μM Fe(NH₄)₂(SO₄)₂ (Fe 100), 100 μM ZnCl₂ (Zn 100), 5 μM CoCl₂·6H₂O (Co 5), and 20 μM NiCl₂·6H₂O (Ni 20) are shown as well. Growth of the B. abortus mntH mutant MWV15 carrying a plasmid-borne copy of mntH (MWV15.C) in low-manganese minimal medium is also shown. The data presented are the means and standard deviations for triplicate determinations from single flasks for each strain and experimental condition in a single experiment. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained. *, P < 0.05; **, P < 0.005 (for comparisons of strain 2308 with MWV15 or MVW15.C).

FIG. 4.

Resistance of B. abortus 2308, MWV15 (2308 mntH), and MWV15.C [MWV15(pEA31)] to H₂O₂ (A), paraquat (B), and menadione (C) in disk sensitivity assays. The data presented are zones of inhibition around disks containing H₂O₂ (A), paraquat (B), or menadione (C). The values are the means ± standard deviations of the zone sizes obtained from three separate experiments, and three separate determinations of growth were obtained for each strain in each experiment. **, P ≤ 0.005; ***, P ≤ 0.001 (for comparisons of MWV15 with strain 2308 or MWV15.C).

FIG. 5.

SOD activity in B. abortus 2308, MWV15, and MWV15C. Cu/Zn SOD (SodC) (cyanide-sensitive) and Mn SOD (SodA) (cyanide- and H₂O₂-resistant) activities, detected in the parental 2308 strain (lane A), the mntH mutant MWV15 (lane B), and a derivative of MWV15 carrying a plasmid-borne copy of mntH (lane C). The B. abortus sodC mutant MEK2 (26) was included as a control (lane D). The gel presented is representative of multiple experiments (≥3) in which equivalent results were observed.

FIG. 6.

Survival and replication of B. abortus 2308 (▴), MWV15 (2308 mntH) (□), and MWV15C [MWV15(pEA31)] (▪) in cultured resident peritoneal macrophages from C57BL/6 mice. The data are means and standard deviations of the number of intracellular brucellae recovered for each strain from three separate wells of cultured macrophages at each experimental time point in a single experiment. The data are representative of multiple (≥3) experiments from which equivalent results and statistical trends were obtained. *, P < 0.05; **, P < 0.005 (for comparisons of MWV15 with 2308 and MWV15.C).

FIG. 7.

Spleen colonization profiles of B. abortus 2308 (▴), MWV15 (2308 mntH) (□), and MWV15.C [MWV15(pEA31)] (▪) in C57BL/6 mice. The data presented are means and standard deviations of the number of brucellae detected in the spleens of five mice infected with each strain at each experimental time point in a single experiment. **, P < 0.005 (for comparisons of MWV15 with 2308 and MWV15.C).