Molecular, Antigenic, and Functional Analyses of Omp2b Porin Size Variants of Brucella spp

Abstract

Omp2a and Omp2b are highly homologous porins present in the outer membrane of the bacteria from the genus Brucella, a facultative intracellular pathogen. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. In this work, we present the cloning, purification, and characterization of four Omp2b size variants found in various Brucella species, and we compare their antigenic and functional properties to the Omp2a and Omp2b porins of Brucella melitensis reference strain 16M. The variation of the Omp2a and Omp2b porin sequences among the various strains of the genus Brucella seems to result mostly from multiple gene conversions between the two highly homologous genes. As shown in this study, this phenomenon has led to the creation of natural Omp2a and Omp2b chimeric proteins in Omp2b porin size variants. The comparison by liposome swelling assay of the porins sugar permeability suggested a possible functional differences between Omp2a and Omp2b, with Omp2a showing a more efficient pore in sugar diffusion. The sequence variability in the Omp2b size variants was located in the predicted external loops of the porin. Several epitopes recognized by anti-Omp2b monoclonal antibodies were mapped by comparison of the Omp2b size variants antigenicity, and two of them were located in the most exposed surface loops. However, since variations are mostly driven by simple exchanges of conserved motifs between the two genes (except for an Omp2b version from an atypical strain of Brucella suis biovar 3), the porin variability does not result in major antigenic variability of the Brucella surface that could help the bacteria during the reinfection of a host. Porin variation in Brucella seems to result mainly in porin conductivity modifications.Porins are abundant proteins in the outer membrane of gram-negative bacteria. They create a controlled permeability of the outer membrane toward small hydrophilic solutes such as sugars that are otherwise not allowed to diffuse through the outer membrane (for a review, see references 21, 22, and 24). In a sense, porins are the Achilles' heel of this protective barrier, since they can act as receptors for bacteriophage and colicins (13), surface-exposed antigens (37), complement-binding sites (29), and the gate of entry for some antibiotics (27,28). In some pathogenic bacteria, such as Neisseria gonorrhoeae, porin sequence variations between serotypes are well described and probably allow successive infection by different serotypes presenting different surface-exposed epitopes (20). Neisseria porin genes are subject to frequent horizontal transfer movements of genetic material, allowing for numerous changes in sequence that can hamper vaccine development (18).Brucellae are gram-negative pathogens infecting numerous mammalian species and causing economic losses in domestic cattle, sheep, and goats, as well as human health problems in zones where they are endemic. The mechanisms of pathogenicity of Brucella spp. are still poorly understoo...

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…However, in a planar lipid bilayer study, Omp2a behaved in a rather anomalous manner, in contrast to Omp2b, which produced conductance steps expected for stable, trimeric porins (424). In any case, the juxtaposition of two very homologous genes apparently produces constant variation in gene sequences via gene conversion mechanism (482), and the effect of this variation on the physiology and evolution of Brucella species remains an interesting topic. OM protein IIIa of Rhizobium leguminosarum is homologous to Brucella porins (128), but no further study of the Rhizobium porin seems to exist.…”

“…The proposal made by Ferenci was to look at the alignment of related porin sequences and assume that the less variant regions correspond to transmembrane strands. This works very nicely when many sequences from isolates of the same species, for example, exist (362,482). However, in other cases one has to start from an alignment of sequences of distantly related porins, and this requires the prior identification of transmembrane strands.…”

Polypyrrole-Supported Membrane Proteins for Bio-Inspired Ion Channels

“…Indeed, pores are larger in Omp2a than in its homologue Omp2b (85% sequence identity and both encoded in the same genetic locus). 27 Although the function of Omp2a is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel MPs is not known yet, very recent refolding studies suggested that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. 28,29 On the other hand, poly(N-methylpyrrole) (PNMPy) is the CP chosen to act as the supporting matrix for Omp2a.…”

Characterization of Novel Brucella Strains Originating from Wild Native Rodent Species in North Queensland, Australia

“…T demonstrated close clustering to an atypical B. suis strain (strain 83-210) isolated from a rodent in Australia (30,44). Further genetic analysis of the 16S rRNA genes from strains BO1…”