A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviae

Abstract

High‐frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5′ to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3′ end of the gene, but conservation of the 5′ end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5′ coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single‐copy 5′ end of vlhA, but extending over one of four distinct overlapping regions of the 3′ coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5′ end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families.

Classification of Mycoplasma synoviae strains using single-strand conformation polymorphism and high-resolution melting-curve analysis of the vlhA gene single-copy region

“…It is also very unlikely that PCR products consisted of two variant sequences, since all M. synoviae cultures used in this study were grown from single colonies and all produced only one (and not more than one) shoulder peak. More importantly, within a single strain of M. synoviae, the vlhA single-copy gene region targeted for PCR in this study is known to be conserved (Bencina et al, 2001;Hong et al, 2004;Noormohammadi et al, 2000Noormohammadi et al, , 2002. Examination of the nucleotide sequence of the targeted vlhA gene region revealed an uneven distribution of G/C nucleotides through the length of the region.…”

“…An arbitrary-primed PCR has previously been described for detection of M. synoviae strain variations (Fan et al, 1995), but the reproducibility of the method is still under question and the results are difficult to interpret. In addition, the approach does not determine whether the profile variation detected relates to genomic rearrangements that commonly occur within single isolates (Noormohammadi et al, 2000). PCR followed by sequencing of the amplified product has also been described (Hong et al, 2004) for detection of M. synoviae strain variations; however, this approach is considerably more time consuming and the results often require interpretation.…”

“…RFLP has been described elsewhere (Morrow et al, 1990b) for classification of M. synoviae strains and is currently in use in our laboratory for classification of new field M. synoviae strains. However, RFLP is time consuming, since it requires isolation and culture of the organism and extraction of genomic DNA, and can be unreliable, particularly because of genomic rearrangements that may occur in progenies of a single M. synoviae isolate (Noormohammadi et al, 2000). Also, sequence variation in an individual isolate may go undetected, because only a relatively small number of restriction enzymes scans a subset of a putatively variable site.…”

“…Also, sequence variation in an individual isolate may go undetected, because only a relatively small number of restriction enzymes scans a subset of a putatively variable site. An arbitrarily primed PCR has also been described (Fan et al, 1995) (Noormohammadi et al, 2000) has been used for classification of M. synoviae strains and epidemiological investigations (Bencina et al, 2001;Hong et al, 2004). These studies have suggested that strain identification based on the vlhA PCR should be regarded as a preliminary typing method for M. synoviae.…”

Genetic variation in sialidase and linkage to N-acetylneuraminate catabolism in Mycoplasma synoviae

“…The vlhA locus in the M. synoviae strain 53 genome is flanked by homologs of gapDH adjacent to the sialic acid degradation locus. Hypervariability in the VlhA hemagglutinins expressed within and among strains is generated by site-specific recombinations among a large assemblage of vlhA pseudogenes constituting the 69 kb locus [44,45]. In contrast, there are many potentially independently-transcribed vlhA genes dispersed throughout the M. gallisepticum genome [37,46], supporting the hypothesis that, like nanI, vlhA also may have been exchanged between these species by horizontal transfer [11,47].…”