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

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 expressedvariants, 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.