Omp2b Porin Alteration in the Course of Evolution of Brucella spp

Abstract

The genus Brucella comprises major pathogenic species causing disease in livestock and humans, e.g. B. melitensis. In the past few years, the genus has been significantly expanded by the discovery of phylogenetically more distant lineages comprising strains from diverse wildlife animal species, including amphibians and fish. The strains represent several potential new species, with B. inopinata as solely named representative. Being genetically more distant between each other, relative to the "classical" Brucella species, they present distinct atypical phenotypes and surface antigens. Among surface protein antigens, the Omp2a and Omp2b porins display the highest diversity in the classical Brucella species. The genes coding for these proteins are closely linked in the Brucella genome and oriented in opposite directions. They share between 85 and 100% sequence identity depending on the Brucella species, biovar, or genotype. Only the omp2b gene copy has been shown to be expressed and genetic variation is extensively generated by gene conversion between the two copies. In this study, we analyzed the omp2 loci of the non-classical Brucella spp. Starting from two distinct ancestral genes, represented by Australian rodent strains and B. inopinata, a stepwise nucleotide reduction was observed in the omp2b gene copy. It consisted of a first reduction affecting the region encoding the surface L5 loop of the porin, previously shown to be critical in sugar permeability, followed by a nucleotide reduction in the surface L8 loop-encoding region. It resulted in a final omp2b gene size shared between two distinct clades of non-classical Brucella spp. (African bullfrog isolates) and the group of classical Brucella species. Further evolution led to complete homogenization of both omp2 gene copies in some Brucella species such as B. vulpis or B. papionis. The stepwise omp2b deletions seemed to be generated through recombination with the respective omp2a gene copy, presenting a conserved size among Brucella spp., and may involve short direct DNA repeats. Successive Omp2b porin alteration correlated with increasing porin permeability in the course of evolution of Brucella spp. They possibly have adapted their porin to survive environmental conditions encountered and to reach their final status as intracellular pathogen.

Keywords: Brucella; Omp2 porin; diversity; evolution; gene conversion; loop.

FIGURE 1

Brucella spp. omp2 clustering analysis, constructed using the omp2a and omp2b sequences of the non-classical and classical Brucella species of this study (GenBank accession numbers are in Supplementary Figure S1). The strains are represented by circles, increasing in size for strains with identical sequences. Empty circles represent the omp2b sequences while yellow-colored circles represent the omp2a sequences of the respective strains. Two half-colored circles are present in the tree for strains carrying identical omp2a and omp2b sequences, i.e. B. papionis F8/08/60 and B. ceti B14/94. The gene conversion events for each strain or groups of strains are schematized by sliced boxes representing omp2b (upper part) and omp2a (lower part), colored in yellow when omp2a-specific sequences are present. Relevant omp2 size variations are indicated in blue, with the Greek I and D letters meaning insertion and deletion, respectively. The size in bp of each event, as well as the encoded segment concerned (L3, L5, and L8) are indicated.

FIGURE 2

Schematic multiple nucleotide sequence alignment of the omp2a and omp2b genes of Brucella strains. This schematic representation was done according to the detailed alignment show in the Supplementary Figure S1. Nucleotide sequences are represented by rectangles divided into boxes of 30 nucleotides. The Brucella sp. 83–211 omp2b gene sequence was used as a reference (white boxes). The boxes containing omp2a-specific nucleotides are colored in yellow. The numbers in the corresponding boxes indicate the number of omp2a-specific nucleotides present in the sequence considered. Numbers in parentheses represent insertions and deletions. Numbers in red indicate nucleotide differences that are not due to gene conversion.

FIGURE 3

Clustering of Brucella spp. and representative classical strains of this study by whole genome SNP calling and Bio-NJ analysis. The color code reflects animal or human host assignment. Strain names are indicated (strain 83/13 is an alias of 83–211). Blue letters A to Q indicate the corresponding omp2 locus type according to Figure 2 and Supplementary Figure S1.

FIGURE 4

Multiple nucleotide sequence alignment of the omp2 segments encoding the L3, L5, and L8 loops (upper, middle, and lower panel, respectively), from representative strains containing insertions or deletions. Strains and omp2 genes used are indicated on the left of each segment, omp2a genes are indicated as 2a in yellow. omp2a-specific nucleotides are highlighted in yellow, according to the omp2a and omp2b reference sequences used from Brucella sp. 83–211. Nucleotides colored in red indicate differences that are not due to gene conversion, according to the same reference sequences. Direct DNA repeats are underlined. *Indicates identical nucleotides.

FIGURE 5

Amino acid sequence alignment of the Omp2b and Omp2a L5 loops from representative strains of this study, showing the progressive loss of amino acids in accordance with the respective nucleotide sequence data shown in Figures 1–3. The strains are indicated on the left, when Omp2a is used the corresponding strain number is highlighted in yellow. The predicted L5 loop, according to Paquet et al. (2001), is highlighted in blue. Underlined B9 and B10 sequences indicate the predicted flanking transmembrane β-strands (Paquet et al., 2001). Omp2a-specific amino acids are highlighted in yellow, in accordance with the Omp2a and Omp2b reference sequence used from Brucella sp. 83–211. Omp2 amino acids colored in red are independent from gene conversion according to the same reference sequences. The middle panel of the figure highlights the negatively charged amino acids of the segment. The right panel indicate the available sugar permeability data from previously published strains (Paquet et al., 2001). *Indicates identical amino acids.