Role of the Omp25/Omp31 family in outer membrane properties and virulence of Brucella ovis

Abstract

The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Deltaomp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the Deltaomp25d mutant were found, especially considering that the fully virulent Deltaomp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies.

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.

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.

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.

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.