Characterization of New Members of the Group 3 Outer Membrane Protein Family of Brucella spp

Abstract

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.Brucellae are small, nonmotile, gram-negative coccobacilli that are able to infect a broad range of wildlife and domestic mammals. They remain a major zoonotic disease source affecting humans worldwide and are also a focus of concern as potential biological warfare agents (20). Although BrucelIa spp. are not particularly host specific, three major (B. abortus, B. melitensis, and B. suis) and minor (B. canis, B. ovis, and B. neotomae) species each have distinct host preferences and pathogenicities for humans (29). Malta fever (also known as Mediterranean, Gibraltar, or undulant fever) and porcine brucellosis-caused by B. melitensis and B. suis infection of humans, respectively-are usually by far more clinically apparent than Bang's disease (B. abortus infection), whereas among the remaining species only B. canis causes anecdotal mild infections in humans (10).The Brucella outer membrane was investigated to seek immunogenic and protective antigens for potential diagnostic and vaccine applications. The major outer membrane proteins (Omps) of Brucella spp. were thoroughly studied in this regard.

Quorum-Sensing and BvrR/BvrS Regulation, the Type IV Secretion System, Cyclic Glucans, and BacA in the Virulence of Brucella ovis: Similarities to and Differences from Smooth Brucellae

“…(37,56). They are also in accord with the findings that bvrR and bvrS mutants have been obtained only by transposon mutagenesis in B. abortus 2308 and B. suis 1330 (28,52) and that other attempts of deletion by homologous recombination have failed (49).…”

Release of Periplasmic Proteins of Brucella suis upon Acidic Shock Involves the Outer Membrane Protein Omp25

“…which is also present in the outer membrane Omp25/Omp31 protein family (35). However, for two released proteins, the identity of the cleavage site is restricted to AXA, while two others exhibited a very different APXXA sequence, further illustrating the difficulty of predicting the cleavage sites of signal peptides in Brucella proteins.…”

“…1B). The immunostaining detected in lanes 2 and 4, with an apparent molecular mass of Ͼ31 kDa, most probably corresponded to the Omp25 paralogue previously identified and named Omp25b (35).…”

“…strain C58, which triggers nopaline production in plants, utilizes the nopaline binding protein for its cellular uptake. As for Brucella, Rocha et al suggested that a ribose binding protein linked to the outer membrane might be involved in virulence, since it participates in the attachment of Brucella to sialic acid residues of eukaryotic cells (35). The question of whether the various Brucella species possess one or several homologues of these specific Agrobacterium nutrient receptors has not yet been specifically addressed.…”

Stability of Poly(ε-caprolactone) Microparticles Containing Brucella ovis Antigens as a Vaccine Delivery System Against Brucellosis

“…This extract is especially rich in Omp25 (25 to 27 kDa), Omp31 (31 to 34 kDa) (13), and Omp22 (Omp3b; 22 kDa) (14). Other proteins such as group 2 (Omp2a and Omp2b [36 to 38 kDa]) and three lipoproteins (L-Omp10, L-Omp16, and L-Omp19) (13,15), which are expressed in all Brucella spp.…”