BvrR/BvrS-controlled outer membrane proteins Omp3a and Omp3b are not essential for Brucella abortus virulence

Abstract

The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps.

FIG. 1.

Construction of B. abortus omp3a and omp3b mutants. (A) RT-PCR using specific primers for omp3a (top) and omp3b (bottom). Total RNA was extracted and retrotranscribed, and cDNA was amplified by PCR with primers Omp25U1/Omp25L1 and 3bZ-1/3bZ-2. Lanes: M, molecular size markers; WT, B. abortus 2308; omp3a, B. abortus omp3a mutant; omp3b, B. abortus omp3b mutant; C-, PCR-negative control with water; C+, PCR-positive control with Brucella genomic DNA. (B) Detection of Omp3a by Western blotting in cell envelope Sarkosyl-resistant fractions using anti-Omp3a MAbs. Lanes: WT, B. abortus 2308; bvrR, B. abortus bvrR::Tn5 mutant; bvrR⁺, reconstituted B. abortus bvrR⁺; omp3a, B. abortus omp3a deletion mutant; omp3b, B. abortus omp3b deletion mutant.

FIG. 2.

Sensitivity to polymixin B and human complement. (A) Bacterial survival in the presence of polymyxin B. The graph shows the percent survival after 1 h at 37°C with different peptide concentrations. (B) Bacterial survival after 30- or 90-min incubation with human nonimmune serum. Data represent the means ± standard deviations of percentages of viable bacteria in relation to a bacterial control without polymyxin B or a bacterial control with heat-inactivated human serum. Samples were compared using the Mann-Whitney U test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 (with respect to the WT strain).

FIG. 3.

Gentamicin survival assay by double immunofluorescence 30 min postinfection in HeLa cells. (A) Proportion of cells with associated (intra- and extracellular) bacteria. (B) Proportion of intracellular bacteria with respect to the total of intra- and extracellular bacteria. (C) Absolute number of intra- and extracellular bacteria per infected cell. Data represent means ± standard deviations. Samples were compared using the Student t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 (with respect to the WT strain).

FIG. 4.

Intracellular replication of B. abortus strains in epithelial HeLa cells, RAW 264.7 macrophages (Mφ), and PMN. Data represent means ± standard deviations of plate counts. Samples were compared using the Mann-Whitney U test. **, P < 0.005; ***, P < 0.0005 (with respect to the WT strain).

FIG. 5.

Induction of TNF-α in murine RAW 264.7 macrophages infected with different B. abortus strains. *, P < 0.05 (with respect to the WT strain).

FIG. 6.

Infection of the spleens of BALB/c mice with different B. abortus strains. Mice were infected intraperitoneally with 10⁵ CFU/mouse, except for the bvrS mutant, for which the dose was 10⁸ CFU/mouse. Values are means ± standard deviations (n = 5). The detection limit was 0.6 log CFU/spleen (3 to 4 CFU/spleen).