Dataset Information

Characterization of a variant vlhA gene of Mycoplasma synoviae, strain WVU 1853, with a highly divergent haemagglutinin region.

ABSTRACT: BACKGROUND: In Mycoplasma synoviae, type strain WVU 1853, a single member of the haemaglutinin vlhA gene family has been previously shown to be expressed. Variants of vlhA are expressed from the same unique vlhA promoter by recruiting pseudogene sequences via site-specific recombination events, thus generating antigenic variability. Using a bacterial stock of M. synoviae WVU 1853 that had been colony purified thrice and maintained in our laboratory at low passage level, we previously identified a vlhA gene-related partial coding sequence, referred to as MS2/28.1. The E. coli-expressed product of this partial coding sequence was found to be immunodominant, suggesting that it might be expressed. RESULTS: Reverse transcription-PCR amplification (RT-PCR), using a sense primer located at the 5'-end region of the expected vlhA transcript and a reverse primer located at the 3' end of MS2/28.1 coding sequence, yielded a consistent amplification product showing that MS2/28.1 was indeed transcribed. Nucleotide sequence analysis of the RT-PCR product identified an 1815-nucleotide full-length open reading frame (ORF), immediately preceded by a nucleotide sequence identical to that previously reported for expressed vlhA genes. PCR amplifications using genomic DNA isolated from single colonies further confirmed that the full-length ORF of MS2/28.1 was located downstream of the unique vlhA promoter sequence. The deduced 604-amino acid (aa) sequence showed a perfect sequence identity to the previously reported vlhA expressed genes along the first 224 residues, then highly diverged with only 37.6% aa identity. Despite the fact that this M. synoviae clone expressed a highly divergent and considerably shorter C-terminal haemagglutinin product, it was found to be expressed at the surface of the bacterium and was able to haemagglutinate chicken erythrocytes. Importantly, the E. coli-expressed C-terminal highly divergent 60 residues of MS2/28.1 proved haemagglutination competent. CONCLUSIONS: In contrast to the previously characterized vlhA expressed variants, MS2/28.1 displayed a highly divergent sequence, while still able to haemagglutinate erythrocytes. Overall, the data provide an indication as to which extent the M. synoviae vlhA gene could vary its antigenic repertoire.

SUBMITTER: Khiari AB

PROVIDER: S-EPMC2825196 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Characterization of a variant vlhA gene of Mycoplasma synoviae, strain WVU 1853, with a highly divergent haemagglutinin region.

Khiari Awatef Béjaoui AB Guériri Ibtissem I Mohammed Radhia Ben RB Mardassi Boutheina Ben Abdelmoumen BB

BMC microbiology 20100112

<h4>Background</h4>In Mycoplasma synoviae, type strain WVU 1853, a single member of the haemaglutinin vlhA gene family has been previously shown to be expressed. Variants of vlhA are expressed from the same unique vlhA promoter by recruiting pseudogene sequences via site-specific recombination events, thus generating antigenic variability. Using a bacterial stock of M. synoviae WVU 1853 that had been colony purified thrice and maintained in our laboratory at low passage level, we previously iden ...[more]

PMID: 20067616

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Project description:Mycoplasma synoviae depends on its adhesin VlhA to mediate cytadherence to sialylated host cell receptors. Allelic variants of VlhA arise through recombination between an assemblage of promoterless vlhA pseudogenes and a single transcription promoter site, creating lineages of M. synoviae that each express a different vlhA allele. The predicted full-length VlhA sequences adjacent to the promoter of nine lineages of M. synoviae varying in avidity of cytadherence were aligned with that of the reference strain MS53 and with a 60-a.a. hemagglutinating VlhA C-terminal fragment from a Tunisian lineage of strain WVU1853(T). Seven different sequence variants of an imperfectly conserved, single-copy, 12-a.a. candidate cytadherence motif were evident amid the flanking variable residues of the 11 total sequences examined. The motif was predicted to adopt a short hairpin structure in a low-complexity region near the C-terminus of VlhA. Biotinylated synthetic oligopeptides representing four selected variants of the 12-a.a. motif, with the whole synthesized 60-a.a. fragment as a positive control, differed (P<0.01) in the extent they bound to chicken erythrocyte membranes. All bound to a greater extent (P<0.01) than scrambled or irrelevant VlhA domain negative control peptides did. Experimentally introduced branched-chain amino acid (BCAA) substitutions Val3Ile and Leu7Ile did not significantly alter binding, whereas fold-destabilizing substitutions Thr4Gly and Ala9Gly tended to reduce it (P<0.05). Binding was also reduced to background levels (P<0.01) when the peptides were exposed to desialylated membranes, or were pre-saturated with free sialic acid before exposure to untreated membranes. From this evidence we conclude that the motif P-X-(BCAA)-X-F-X-(BCAA)-X-A-K-X-G binds sialic acid and likely mediates VlhA-dependent M. synoviae attachment to host cells. This conserved mechanism retains the potential for fine-scale rheostasis in binding avidity, which could be a general characteristic of pathogens that depend on analogous systems of antigenically variable adhesins. The motif may be useful to identify previously unrecognized adhesins.

Dataset Information

Characterization of a variant vlhA gene of Mycoplasma synoviae, strain WVU 1853, with a highly divergent haemagglutinin region.

Publications

Characterization of a variant vlhA gene of Mycoplasma synoviae, strain WVU 1853, with a highly divergent haemagglutinin region.

Similar Datasets

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