The Cation Diffusion Facilitator Family Protein EmfA Confers Resistance to Manganese Toxicity in Brucella abortus 2308 and Is an Essential Virulence Determinant in Mice

Abstract

The gene designated bab_rs23470 in the Brucella abortus 2308 genome encodes an ortholog of the cation diffusion facilitator family protein EmfA which has been linked to resistance to Mn toxicity in Rhizobium etli A B. abortusemfA null mutant derived from strain 2308 displays increased sensitivity to elevated levels of Mn in the growth medium compared to that of the parent strain but wild-type resistance to Fe, Mg, Zn, Cu, Co, and Ni. Inductively coupled plasma mass spectroscopy also indicates that the B. abortusemfA mutant retains significantly higher levels of cellular Mn after exposure to this metal than the parent strain, which is consistent with the proposed role of EmfA as a Mn exporter. Phenotypic analysis of mutants indicates that EmfA plays a much more important role in maintaining Mn homeostasis and preventing the toxicity of this metal in Brucella than does the Mn-responsive transcriptional regulator Mur. EmfA is also an essential virulence determinant for B. abortus 2308 in C57BL/6 and C57BL/6^Nramp1+/+ mice, which suggests that avoiding Mn toxicity plays a critical role in Brucella pathogenesis.IMPORTANCE Mn nutrition is essential for the basic physiology and virulence of Brucella strains. The results of the study presented here demonstrate that the cation diffusion facilitator (CDF)-type metal exporter EmfA plays critical roles in maintaining Mn homeostasis and preventing Mn toxicity in Brucella and is an essential virulence determinant for these bacteria. EmfA and other cellular components involved in Mn homeostasis represent attractive targets for the development of improved vaccines and chemotherapeutic strategies for preventing and treating brucellosis in humans and animals.

Keywords: Brucella; EmfA; Mur; cation diffusion facilitator; manganese; manganese homeostasis; manganese toxicity.

FIG 1

B. abortus 2308 does not require Mur for growth in the presence of excess exogenous Mn. Twenty-five-microliter suspensions of B. abortus 2308 and the isogenic mur mutant EAM001 were placed on Schaedler agar plates supplemented with MnCl₂, and growth was observed following 72 h of incubation at 37°C with 5% CO₂. The pictures shown represent growth in a single representative experiment. One plate containing each level of metal was examined in each individual experiment, and the experiment was repeated 3 times with the same results.

FIG 2

The B. abortus mur mutant contains similar cellular Mn levels as the wild type. The cellular Mn levels of B. abortus 2308 and the mur mutant EAM001 were measured following growth to mid-log phase in brucella broth (A) and a subsequent 2-h exposure to 50 μM MnCl₂ in this medium (B). Cellular Mn content was determined by ICP-MS, and cellular protein levels were determined using the Bradford assay. The data were evaluated using one-way analysis of variance (ANOVA) and Tukey’s multiple-comparison test, and no statistically significant differences were detected between the parent strain and emfA mutant.

FIG 3

EmfA protects B. abortus 2308 from Mn toxicity. Twenty-five-microliter suspensions of B. abortus 2308 and derivative strains were placed on Schaedler agar plates supplemented with MnCl₂, and growth was observed following 72 h of incubation at 37°C with 5% CO₂. The pictures shown represent growth in a single representative experiment. One plate containing each level of metal was examined in each individual experiment, and the experiment was repeated 4 times with the same results.

FIG 4

The B. abortus emfA mutant is more sensitive to growth restriction by exogenous Mn than the parental 2308 strain during cultivation in brucella broth. The results presented are the OD₆₀₀ values obtained for 3 cultures of each B. abortus strain under each experimental condition after 40 h of growth in brucella broth supplemented with the indicated concentration of Mn in a single experiment. The experiment was repeated 3 times with the same results.

FIG 5

EmfA does not protect B. abortus 2308 from Fe, Mg, Zn, Ni, Co, or Cu toxicity. Twenty-five-microliter suspensions of B. abortus 2308 and the isogenic emfA mutant JEP61 were placed on Schaedler agar plates supplemented with FeCl₃, MgCl₂, ZnSO₄, NiCl₂, CoCl₂, or CuCl₂, and growth was observed following 72 h of incubation at 37°C with 5% CO₂. The pictures shown represent growth in a single representative experiment. One plate containing each level of metal was examined in each individual experiment, and the experiment was repeated 3 times with the same results.

FIG 6

The B. abortus emfA mutant has increased cellular Mn content following Mn challenge compared to that of B. abortus 2308. Cellular Mn levels were measured in B. abortus 2308, the emfA mutant JEP61, and the complemented emfA mutant MJJ012 (ΔemfA^C) following growth to the mid-log phase in brucella broth (A) and following a subsequent 2-h exposure to 50 μM MnCl₂ in this medium (B). ICP-MS was used to measure cellular Mn levels, and the Bradford assay used to determine cellular protein levels. *, P < 0.05; ***, P < 0.005 for comparisons of 2308 versus the emfA mutant or the complemented emfA mutant using one-way ANOVA and Tukey’s multiple-comparison test.

FIG 7

The B. abortus emfA mutant displays attenuation in mice. Spleen colonization profiles of Brucella abortus 2308, the emfA mutant, and the complemented emfA mutant (ΔemfA^C) in C57BL/6 and C57BL/6^Nramp1+/+ mice are shown. Ten mice were individually infected with 5 × 10⁴ of each bacterial strain via the intraperitoneal route, and five mice from each experimental group were evaluated at 2 weeks and 5 weeks postinfection. *, P < 0.05; ***, P < 0.005 for comparisons of 2308 versus the emfA mutant or complemented emfA mutant using one-way ANOVA and Tukey’s multiple-comparison test.