RESUMO
The survival and replication of Brucella in macrophages is initially triggered by a low intraphagosomal pH. In order to identify proteins released by Brucella during this early acidification step, we analyzed Brucella suis conditioned medium at various pH levels. No significant proteins were released at pH 4.0 in minimal medium or citrate buffer, whereas in acetate buffer, B. suis released a substantial amount of soluble proteins. Comparison of 13 N-terminal amino acid sequences determined by Edman degradation with their corresponding genomic sequences revealed that all of these proteins possessed a signal peptide indicative of their periplasmic location. Ten proteins are putative substrate binding proteins, including a homologue of the nopaline binding protein of Agrobacterium tumefaciens. The absence of this homologue in Brucella melitensis was due to the deletion of a 7.7-kb DNA fragment in its genome. We also characterized for the first time a hypothetical 9.8-kDa basic protein composed of five amino acid repeats. In B. suis, this protein contained 9 repeats, while 12 were present in the B. melitensis orthologue. B. suis in acetate buffer depended on neither the virB type IV secretory system nor the omp31 gene product. However, the integrity of the omp25 gene was required for release at acidic pH, while the absence of omp25b or omp25c displayed smaller effects. Together, these results suggest that Omp25 is involved in the membrane permeability of Brucella in acidic medium.
Assuntos
Ácidos/farmacologia , Arginina/análogos & derivados , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella suis/efeitos dos fármacos , Brucella suis/metabolismo , Proteínas Periplásmicas/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Arginina/metabolismo , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Brucella suis/genética , Soluções Tampão , Extensões da Superfície Celular/efeitos dos fármacos , Genes Bacterianos/genética , Genômica , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Dados de Sequência Molecular , Mutação/genética , Proteínas Periplásmicas/química , Permeabilidade , Fagossomos/microbiologia , Ligação Proteica , Ribose/metabolismo , Homologia de Sequência , Solubilidade , Fatores de VirulênciaRESUMO
Impairment of the omp25 gene in Brucella spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in B. melitensis or B. abortus. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in B. suis. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type B. suis with mutants systematically having B. suis omp25-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in B. suis, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of Bartonella quintana. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in B. suis or in other Brucella species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future Brucella interspecies study results.
