Characterization of three predicted zinc exporters in Brucella ovis identifies ZntR-ZntA as a powerful zinc and cadmium efflux system not required for virulence and unveils pathogenic Brucellae heterogeneity in zinc homeostasis

Abstract

Brucella ovis causes non-zoonotic ovine brucellosis of worldwide distribution and is responsible for important economic losses mainly derived from male genital lesions and reproductive fails. Studies about the virulence mechanisms of this rough species (lacking lipopolysaccharide O-chains) are underrepresented when compared to the main zoonotic Brucella species that are smooth (with O-chains). Zinc intoxication constitutes a defense mechanism of the host against bacterial pathogens, which have developed efflux systems to counterbalance toxicity. In this study, we have characterized three potential B. ovis zinc exporters, including the ZntA ortholog previously studied in B. abortus. Despite an in-frame deletion removing 100 amino acids from B. ovis ZntA, the protein retained strong zinc efflux properties. Only indirect evidence suggested a higher exporter activity for B. abortus ZntA, which, together with differences in ZntR-mediated regulation of zntA expression between B. ovis and B. abortus, could contribute to explaining why the ΔzntR mutant of B. abortus is attenuated while that of B. ovis is virulent. Additionally, B. ovis ZntA was revealed as a powerful cadmium exporter contributing to cobalt, copper, and nickel detoxification, properties not previously described for the B. abortus ortholog. Deletion mutants for BOV_0501 and BOV_A1100, also identified as potential zinc exporters and pseudogenes in B. abortus, behaved as the B. ovis parental strain in all tests performed. However, their overexpression in the ΔzntA mutant allowed the detection of discrete zinc and cobalt efflux activity for BOV_0501 and BOV_A1100, respectively. Nevertheless, considering their low expression levels and the stronger activity of ZntA as a zinc and cobalt exporter, the biological role of BOV_0501 and BOV_A1100 is questionable. Results presented in this study evidence heterogeneity among pathogenic Brucellae regarding zinc export and, considering the virulence of B. ovis ΔzntA, suggest that host-mediated zinc intoxication is not a relevant mechanism to control B. ovis infection.

Keywords: Brucella ovis; ZntA; ZntR; ZnuA; cadmium; cobalt; virulence; zinc.

Figure 1

Schematic representation of BOV_0501 (A) and BOV_A1100 (B) inner membrane proteins of Brucella ovis PA and the ZntA Zn²⁺ exporter of B. abortus 2308 (C). N- and C-terminal domains (NTD and CTD) are shown, and the predicted transmembrane domains (TM) are numbered. In BOV_A1100 and ZntA, the 6 TM domains that are found in all P_1B-ATPases are numbered 1 to 6, while the N-terminal TM regions that can be present or not depending on the subgroup of P_1B-ATPases are identified as A or B (30). In panel (A) the transmembrane motifs, the Gly residue adjacent to the cytoplasmic side of TM3, and the His residues of the NTD and CTD of BOV_0501, usually found in metal exporters of the cation diffusion facilitator (CDF) family (31–33), are shown. In panels (B,C) the characteristic motifs conserved in the actuator and nucleotide-binding domains of P_1B-ATPases are shown in the long cytoplasmic domains connecting TM2 with TM3 and TM4 with TM5 (30, 34–36). Motifs located within TM4-TM6 and that are related to the metal specificity-selectivity of the transporter are also represented (30, 37–39). In panel (C) the two GXXCXXC motifs and the His-rich region, defined as metal binding motifs (40–44), are marked in the cytoplasmic NTD of B. abortus 2308 ZntA, and the region of the NTD that is absent in the B. ovis ZntA is drawn in blue (see also Figure 2).

Figure 2

Genetic organization of the ZntR-ZntA zinc export system in B. ovis PA and B. abortus 2308 (A); the nucleotide sequence of B. abortus 2308 zntA and flanking regions (B); and zntA downstream region of B. ovis PA (C). In panel (A) the predicted DNA binding site of the ZntR transcriptional regulator is marked, the 300 nt region of B. abortus 2308 zntA absent in B. ovis PA is represented in blue, the zntA downstream region differing between B. ovis PA and B. abortus 2308 is represented in red, and location of the Bru-RS1 and Bru-RS2 Brucella repetitive elements (45) is represented with boxes. In panel (B) the direct repeats probably involved in the deletion process of the 300 nt absent in zntA of B. ovis PA (blue region) are underlined. The GXXCXXC and His-rich motifs of ZntA, which could be involved in Zn binding (40–44), are framed in green and blue, respectively. Characteristic motifs located within the cytoplasmic domains of P_1B-type ATPases (30, 34–36) are framed in black, and those identified in the transmembrane domains of P_1B-2 ATPases are framed in purple (30, 37–39). The inverted repeats that could be involved in ZntR binding (46) and the two nt differences (C → T) between B. abortus 2308 and B. ovis PA located in the zntR-zntA intergenic region are marked with orange arrows and blue circles, respectively. Bru-RS1 and Bru-RS2 elements (45) downstream zntA in B. abortus 2308 are framed in red. Black arrows mark the inverted repeats detected downstream zntA in B. abortus 2308 (10 nt) and B. ovis PA (48 nt), which are located inside regions differing in sequence between both strains (red sequences). In panel (C) the 84 nt of the zntA downstream region identical to the 3′-end of IS711 (47, 48) is framed.

Figure 3

Growth curves of parental B. ovis PA transformed with empty pBBR1MCS4 or with pNVzntA_PAcom5 or pNVzntA₂₃₀₈com5 in the presence of 5 mM ZnCl₂ (A), proteins of the same strains separated by SDS-PAGE and stained with Coomassie blue (B), and gene expression in the three strains grown in normal medium (C), and of B. ovis PA cultured in the presence or absence of 1 mM ZnCl₂ (D). In panel (A) all strains exhibited the same behavior in the normal medium as that shown in red for B. ovis PA-pBBR1MCS4, which was also same as that of parental B. ovis PA (data not shown). In panel (C) statistically significant differences (p ≤ 0.05) when compared to B. ovis PA-pBBR1MCS4 strain are marked with asterisks, and differences between B. ovis PA-pNVzntA_PAcom5 and B. ovis PA-pNVzntA₂₃₀₈com5 are marked with asterisks in brackets. In panel (D) statistically significant differences (p ≤ 0.05) when compared to B. ovis PA cultured in the absence of 1 mM ZnCl₂ are marked with asterisks. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.

Figure 4

Growth curves of parental B. ovis PA and the ΔzntR and ΔzntA isogenic mutants transformed with empty pBBR1MCS4 or with pNVzntA_PAcom5 or pNVzntA₂₃₀₈com5 in the presence of 5 mM ZnCl₂ (A), proteins of the same strains separated by SDS-PAGE and stained with Coomassie blue (B), growth curves of the ΔzntA mutant in the presence of several ZnCl₂ concentrations (C), and gene expression in the ΔzntR mutant grown in normal medium (D). In panel (A) all strains exhibited the same behavior in the normal medium as that shown in red for B. ovis PA (data not shown). In panel (C) blue curves correspond to ZnCl₂ concentrations, giving an equivalent growth pattern in parental B. ovis PA and its isogenic ΔzntA mutant. In panel (D) statistically significant differences (p ≤ 0.05) when compared to B. ovis PA are marked with asterisks. ***p ≤ 0.001.

Figure 5

Growth curves of parental B. ovis PA, the ΔzntR mutant, and the ΔzntA mutant transformed with empty pBBR1MCS4 or with pNVzntA_PAcom5 or pNVzntA₂₃₀₈com5 in the presence of 0.2 mM CdCl₂ (A), growth curves of the ΔzntA mutant in the presence of several CdCl₂ concentrations (B), and gene expression in parental B. ovis PA cultured in the presence or absence of 0.05 mM CdCl₂ (C). In panel (A) all strains exhibited the same behavior in the normal medium as that shown in red for B. ovis PA (data not shown). In panel (B) blue curves correspond to CdCl₂ concentrations, giving an equivalent growth pattern in parental B. ovis PA and its isogenic ΔzntA mutant. In panel (C) statistically significant differences (p ≤ 0.05) when compared to B. ovis PA cultured in the absence of 0.05 mM CdCl₂ are marked with asterisks. **p ≤ 0.01.

Figure 6

Role of the ZntR-ZntA export system in the survival of B. ovis PA exposed to high concentrations of CoCl₂ (A), CuCl₂ (B), or NiCl₂ (C). The growth curves of ΔzntA-pNVzntA_PAcom5 and ΔzntA-pNVzntA₂₃₀₈com5 in each panel are similar. All strains gave the same growth profile in normal medium and are not represented in the figure (see growth of B. ovis PA in red lanes as representative curve). Similarly, B. ovis PA, B. ovis ΔzntR, and B. ovis ΔzntA transformed with non-recombinant pBBR1MCS4 behaved as the respective parental strains and are not shown in the figure. In panel (A) parental B. ovis PA transformed with pNVzntA_PAcom5 or pNVzntA₂₃₀₈com5 also exhibited a growth pattern in the presence of 0.5 mM CoCl₂ close to that of the parental strain in normal medium (data not shown).

Figure 7

The ability of overexpression of BOV_0501 to restore the growth defects of the B. ovis PA ΔzntA mutant in the presence of ZnCl₂ 0.2 mM (A) or of overexpression of BOV_A1100 to restore the growth defects of the B. ovis PA ΔzntA mutant in the presence of 0.5 mM CoCl₂ (B). All strains gave the same growth profile in normal medium, including strains transformed with empty pBBR1MCS4, and are not represented in the figure (see growth of B. ovis PA in red lanes as representative curve).

Figure 8

Virulence of the B. ovis PA deletion mutants in J774A.1 murine macrophages (A) and mice intraperitoneally infected with 10⁶ CFU/mice (B). Intracellular bacteria in murine macrophages were enumerated at three time points after infection, and results for each strain and time point are expressed as means ± SD (n = 3) of the log₁₀ CFU/well. Strain colonization in mice was evaluated by CFU accounts in spleen at weeks 3 and 7 post-infection, and results are expressed as means ± SD (n = 5) of the log₁₀ CFUs/spleen. No statistically significant differences were detected in any strain compared to the parental strain.