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Molecular Pathogenesis

Role of the Omp25/Omp31 Family in Outer Membrane Properties and Virulence of Brucella ovis

Paola Caro-Hernández, Luis Fernández-Lago, María-Jesús de Miguel, Ana I. Martín-Martín, Axel Cloeckaert, María-Jesús Grilló, Nieves Vizcaíno
Paola Caro-Hernández
1Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
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Luis Fernández-Lago
1Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
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María-Jesús de Miguel
2Centro de Investigación y Tecnología Agroalimentaria del Gobierno de Aragón, Unidad de Sanidad Animal, Carretera de Montañana 930, 50059 Zaragoza, Spain
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Ana I. Martín-Martín
1Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
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Axel Cloeckaert
3INRA, UR1282, Infectiologie Animale et Santé Publique, IASP, Nouzilly F-37380, France
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María-Jesús Grilló
2Centro de Investigación y Tecnología Agroalimentaria del Gobierno de Aragón, Unidad de Sanidad Animal, Carretera de Montañana 930, 50059 Zaragoza, Spain
4Instituto de Agrobiotecnología y Recursos Naturales, CSIC-UPNA, Ctra. Mutilva Baja, 31192 Pamplona, Spain
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Nieves Vizcaíno
1Departamento de Microbiología y Genética, Edificio Departamental, Universidad de Salamanca, Plaza Doctores de la Reina s/n, 37007 Salamanca, Spain
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  • For correspondence: vizcaino@usal.es
DOI: 10.1128/IAI.00486-07
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  • FIG. 1.
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    FIG. 1.

    Growth curves in liquid TSB-YE-HS, as determined by the evolution of OD600 with time, of parental B. ovis PA (A and B) and the mutant strains with the genes omp31, omp25, omp25c, omp25d (A), and omp22 (B) inactivated. CFU/ml counts are shown at time points 25 and 46 h for parental and Δomp22 mutant strains (B). Cultures were started at an OD600 of 0.05 and were incubated at 37°C with shaking under a 10% CO2 atmosphere.

  • FIG. 2.
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    FIG. 2.

    Western blot (A and B) and iELISA (C and D) analysis of the B. ovis strains obtained in this work. Western blotting was performed with MAb G02, specific for the B. ovis Omp31 protein, diluted 1/5 (A) or with MAb C09, specific for the Brucella spp. Omp25 protein, diluted 1/6,000 (B). The positions of the molecular mass protein standards are shown by arrows on the left. For iELISA, MAbs specific for Omp31 (G02 and G10, diluted 1/50) (C) or specific for Omp25 (C09, diluted 1/6,400, and F04, diluted 1/800) (D) were used. Black, white, and shaded columns correspond to parental B. ovis PA, mutant strains, and complemented mutant strains, respectively.

  • FIG. 3.
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    FIG. 3.

    Autoagglutination capacity of parental B. ovis PA, its derived strains obtained in this work, and B. abortus RB51. The OD600 values for bacterial suspensions adjusted to an initial OD600 of 0.8 were scored at several time points of static incubation, and the percent OD600 was determined with respect to the OD600 of the initial suspension (100% of OD600). B. ovis PA mutants PNV25dA and PNV22A and the corresponding strains complemented with wild-type omp25d and omp22, respectively, behaved as parental B. ovis PA and are not represented. The SD, which was always lower than 5% of the mean, is not shown.

  • FIG. 4.
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    FIG. 4.

    Kinetics of splenic infection of parental B. ovis PA (A and B), and the mutant strains with the genes omp25, omp25c, omp25d, and omp22 (A) and omp31 (B) inactivated. Mice were inoculated intraperitoneally with 5 × 106 CFU. Results are expressed as the mean ± SD (n = 5) of the log CFU/spleen.

Tables

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  • TABLE 1.

    Brucella strains used in this work

    Brucella strainRelevant genotypeRelevant characteristic(s)Origin
    Wild-type strainsa
        B. abortus RB51Rough strain obtained under laboratory conditionsBCCNb
        B. ovis PAParental strainNaturally rough virulent Brucella strainBCCN
    Mutant strains
        B. ovis PNV31AΔomp31 Kanr B. ovis PA with a Kan resistance cassette replacing the XcmI-SalI fragment of omp31This work
        B. ovis PNV25A omp25::Kanr B. ovis PA with a Kan resistance cassette inserted into the AflII site of omp25This work
        B. ovis PNV25cAΔomp25c Kanr B. ovis PA with a Kan resistance cassette replacing the AspI-HinCII fragment of omp25cThis work
        B. ovis PNV25dAΔomp25d Kanr B. ovis PA with a Kan resistance cassette replacing the NheI-ClaI fragment of omp25dThis work
        B. ovis PNV22AΔomp22 Kanr B. ovis PA with a Kan resistance cassette replacing the RsrII-StuI fragment of omp22This work
    Complemented mutant strains
        B. ovis PNV31A-comΔomp31 omp31 Kanr Ampr B. ovis PNV31A complemented with omp31 cloned in pNV31300cThis work
        B. ovis PNV25A-com omp25::Kanromp25 Kanr Ampr B. ovis PNV25A complemented with omp25 cloned in pNV25comcThis work
        B. ovis PNV25cA-comΔomp25c omp25c Kanr Ampr B. ovis PNV25cA complemented with omp25c cloned in pNV25c4cThis work
        B. ovis PNV25dA-comΔomp25d omp25d Kanr Ampr B. ovis PNV25dA complemented with omp25d cloned in pNV25d4cThis work
        B. ovis PNV22A-comΔomp22 omp22 Kanr Ampr B. ovis PNV22A complemented with omp22 cloned in pNV22DcThis work
    • ↵ a Reference strains of the Brucella species (46) were also used for the bacteriological typing of the B. ovis strains obtained in this work.

    • ↵ b BCCN, Brucella Culture Collection of Nouzilly, INRA Centre de Tours, France. B. abortus RB51 and B. ovis PA are strains BCCN V5 and BCCN 76-250, respectively.

    • ↵ c Plasmids were constructed by cloning the corresponding B. ovis PA wild-type omp gene in pBBR1MCS-4.

  • TABLE 2.

    DNA primers used in this study

    Primer use and nameNucleotide sequence
    Construction of mutant B. ovis PA strains
        31MUT-F5′-AGA ATA AAA CAC ATG CCC-3′
        31MUT-R5′-GCT GAA TGC GGA GAT GGT-3′
        25MUTZ-F5′-CGA CCT TAT CCT CCT GAA-3′
        25MUTZ-R5′-CCA GCA AAA CGT CGC AAA-3′
        25cdMUT-F5′-TGC GTG GTT CAG ATT TCG-3′
        25cdMUT-R5′-TTG CCG CTT CCA TCA GGT-3′
        25cMUT-R5′-AGC CGT AAC CAA CCT GAC-3′
        22MUT-F5′-GGC AAA GAA GAA GAA TAC-3′
        22MUT-R5′-CTG CTG GAA TGC CCT GAA-3′
    Complementation of mutant B. ovis PA strains
        31sd5′-TGA CAG ACT TTT TCG CCG AA-3′
        31ter5′-CAT TCA GGA CAA TTC CCG CC-3′
        25A-F5′-GGA CCG CGC AAA ACG TAA-3′
        25-R5′-ACC GGA TGC CTG AAA TCC-3′
        25C-F5′-CTG TGT CCT GTT TGC TAC-3′
        25C-R5′-TAT TGG GTG AGG ATT GAC-3′
        25D-F5′-CTA CAT ACA TTC GAC CAG-3′
        25D-R5′-CTG AAG GGT AAA TGC GGC-3′
        22-F5′-TCA AGC ATG TTT CCC GCC-3′
        22-R5′-GTT TGA ATC CCG GCT GTT-3′
  • TABLE 3.

    Susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum of B. abortus RB51, parental B. ovis PA, and its derived strains obtained in this worka

    Brucella strain% Survival after 1-h exposure to:Inhibition zone diam (cm) with hydrogen peroxide% Survival after 4-h exposure to nonimmune ram serum
    Polymyxin B (1 mg/ml)Na deoxycholate (0.1 mg/ml)
    B. abortus RB510.00 ± 0.00B88.69 ± 5.454.28 ± 0.11C25.73 ± 4.84B
    B. ovis PA (parental)69.59 ± 5.2689.34 ± 9.305.04 ± 0.0585.52 ± 5.20
    B. ovis PNV31A70.07 ± 13.8375.51 ± 20.976.23 ± 0.11B64.43 ± 6.97C
    B. ovis PNV31A-com69.83 ± 8.6362.99 ± 22.90D6.64 ± 0.37B(D)69.44 ± 3.62D
    B. ovis PNV25A75.73 ± 22.1363.81 ± 3.98D5.88 ± 0.02B71.67 ± 2.89D
    B. ovis PNV25A-com75.42 ± 4.4164.74 ± 15.67D6.08 ± 0.43B87.21 ± 2.27(D)
    B. ovis PNV25cA27.70 ± 11.65B39.21 ± 7.92B5.88 ± 0.15B55.68 ± 7.32B
    B. ovis PNV25cA-com7.88 ± 5.18B(D)32.54 ± 18.15B7.08 ± 0.04B(B)17.51 ± 14.17B(B)
    B. ovis PNV25dA61.16 ± 8.9355.44 ± 21.69C6.05 ± 0.07B54.05 ± 7.42B
    B. ovis PNV25dA-com58.02 ± 12.8748.70 ± 15.19C6.29 ± 0.27B56.13 ± 2.98B
    B. ovis PNV22A52.74 ± 3.9730.68 ± 3.60B5.90 ± 0.25B25.38 ± 3.98B
    B. ovis PNV22A-com38.52 ± 1.91C23.31 ± 8.23B6.09 ± 0.02B17.41 ± 5.47B
    • ↵ a Assays were performed as detailed in Materials and Methods. Values are means ± SD (n = 3). Statistical comparisons were performed with Fisher's protected least significant differences test. Significant differences between the mutant or complemented strains and parental B. ovis PA or between the complemented strains and the corresponding mutant strain (in parenthesis) are as follows: B, P < 0.0005; C, P < 0.005; D, P < 0.05.

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Role of the Omp25/Omp31 Family in Outer Membrane Properties and Virulence of Brucella ovis
Paola Caro-Hernández, Luis Fernández-Lago, María-Jesús de Miguel, Ana I. Martín-Martín, Axel Cloeckaert, María-Jesús Grilló, Nieves Vizcaíno
Infection and Immunity Jul 2007, 75 (8) 4050-4061; DOI: 10.1128/IAI.00486-07

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Role of the Omp25/Omp31 Family in Outer Membrane Properties and Virulence of Brucella ovis
Paola Caro-Hernández, Luis Fernández-Lago, María-Jesús de Miguel, Ana I. Martín-Martín, Axel Cloeckaert, María-Jesús Grilló, Nieves Vizcaíno
Infection and Immunity Jul 2007, 75 (8) 4050-4061; DOI: 10.1128/IAI.00486-07
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KEYWORDS

Bacterial Outer Membrane Proteins
Brucella ovis
virulence factors

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