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

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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 virulentBrucella ovisPA 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 roughB. abortusRB51, 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 theB. ovisOM, and important differences between the OMs ofB. ovisPA andB. abortusRB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM ofB. ovisPA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Δomp22mutant 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 Δomp25dmutant were found, especially considering that the fully virulent Δomp25cmutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival ofB. ovisinside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both ofB. ovisin rams and of otherBrucellaspecies, should be thoroughly evaluated in future studies.