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 ⌬omp22 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 ⌬omp25d mutant were found, especially considering that the fully virulent ⌬omp25c 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.The genus Brucella comprises six classical species (Brucella melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae) that infect terrestrial mammals (41), although in recent years an increasing number of Brucella strains have been isolated from marine mammals and proposed to be included in two new species, B. cetaceae and B. pinnipediae (9).Brucella spp. may have smooth or rough lipopolysaccharide (S-LPS or R-LPS) depending on the presence or absence, respectively, of O-polysaccharide chains. Rough mutants derived from smooth Brucella strains show an important reduction in virulence (1,26,30,40,45). Accordingly, the O-polysaccharide chains of LPS are thought to be necessary for the pathogenicity of Brucella strains bearing S-LPS. However, B. ovis and B. canis lack O chains in the LPS but are pathogenic for rams and dogs, respectively, and induce long-lasting infections with high levels of splenic colonization in laboratory animals (34). Since O chains mask other components of the Brucella spp. outer membrane (OM), OM proteins (OMPs) are more exposed at the bacterial surface of rough B. ovis and B. canis (4, 7) and their involvement in virulence in rough Brucella strains may be more relevant than in smooth Brucella strains.The Brucella spp. Omp25/Omp31 family comprises seven homologous OMPs. Omp25 and Omp31...

ABSTRACTBrucella ovisis a rough bacterium—lacking O-polysaccharide chains in the lipopolysaccharide—that is virulent in its natural host and whose virulence mechanisms remain almost unexplored. In a search for additional traits that distinguishB. ovisfrom smoothBrucella, which require O-polysaccharide chains for virulence, we have analyzed the significance inB. ovisof the main virulence factors described for smoothBrucella. Attempts to obtain strains of virulentB. ovisstrain PA that are mutated in the BvrR/BvrS two-component regulatory system were unsuccessful, suggesting the requirement of that system forin vitrosurvival, while the inactivation ofbacA—in contrast to the results seen with smoothBrucella—did not affect splenic colonization in mice or behavior in J774.A1 murine macrophages. Defects in the synthesis of cyclic ß-1,2 glucans reduced the uptake ofB. ovisPA in macrophages and, although the intracellular multiplication rate was unaffected, led to attenuation in mice. Growth of strains with mutations in the type IV secretion system (encoded by thevirBoperon) and the quorum-sensing-related regulator VjbR was severely attenuated in the mouse model, and although the mutant strains internalized like the parental strain in J774.A1 murine macrophages, they were impaired for intracellular replication. As described forB. melitensis, VjbR regulates the transcription of thevirBoperon positively, and theN-dodecanoyl-dl-homoserine lactone (C12-HSL) autoinducer abrogates this effect. In contrast, no apparent VjbR-mediated regulation of thefliFflagellar gene was observed inB. ovis, probably due to the two deletions detected upstream offliF. These results, together with others reported in the text, point to similarities between rough virulentB. ovisand smoothBrucellaspecies as regards virulence but also reveal distinctive traits that could be related to the particular pathogenicity and host tropism characteristics ofB. ovis.

Analysis of the occurrence and distribution of the Omp25/Omp31 family of surface proteins in the six classical Brucella species. Veterinary Microbiology, Elsevier, 2009, 137 (1-2) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 30A c c e p t e d M a n u s c r i p t proteins of this family among species could be related to the difference in pathogenicity and 5 host preference they exhibit. Accordingly, in this work we have analyzed the expression of 6 the genes coding for the Omp25/Omp31 family in the six classical Brucella species and a set 7 of B. ovis mutant strains with each omp gene inactivated. Immunoblot of whole cell lysates 8 with antibodies raised against the purified recombinant outer membrane proteins (OMPs) did 9 not show the simultaneous presence of the seven OMPs in any of the Brucella strains studied, 10 but different Omp25/Omp31 profiles were detected, in our experimental conditions, between 11the Brucella strains representative of the six classical species. Transcripts for omp31, omp25 12 and omp25c were, in general, the most abundant of the family and some hits were found in B. 13 ovis for a posttranscriptional regulation mechanism and for a compensatory mechanism 14 increasing the synthesis of a protein to compensate for the absence of another one.

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