Characterization of a Multigene Family Undergoing High-Frequency DNA Rearrangements and Coding for Abundant Variable Surface Proteins in Mycoplasma agalactiae

Abstract

A family of abundant surface proteins (Vpmas [variable proteins of Mycoplasma agalactiae]) undergoing phase variation in M. agalactiae has been characterized using monoclonal antibodies and specific polyclonal sera. Two expressed members of 39 kDa (Vpma39) and 34 kDa (Vpma34), which varied in expression between clones of a lineage, shared a common amino-terminal sequence but were immunologically distinct. An aminoterminal oligonucleotide probe identified multiple vpma genes which were clustered within a 14-kb ClaI genomic fragment. Rearrangements were found to have occurred within the vpma locus between clones which correlated with changes in their Vpma phenotype. Two neighboring vpma genes were cloned and sequenced from one M. agalactiae clonal variant expressing Vpma39. The two genes, vpmaX and vpmaY, were orientated divergently and shared highly homologous 5 untranslated regions, 25-amino-acid (aa) lipoprotein leader sequences, and amino-terminal sequences. The vpmaY gene coded for 346 aa and 84% of the open reading frame, comprised of 1.5 units of a large repeat of 186 aa. Although the sequence for an entire second vpmaY repeat was present, it was prematurely terminated by insertion of two nucleotides. The vpmaX gene encoded 221 aa and possessed 102 aa of the 186-aa repeat of vpmaY. Many of the features in common between the vpma genes were also found to be shared by the vsp genes of M. bovis, which also undergo DNA rearrangements concomitant with phenotypic changes. Since M. bovis is the closest phylogenetic relative to M. agalactiae, the vpma and vsp gene families most probably represent homologous systems.

Validation of the suppressive subtractive hybridization method in Mycoplasma agalactiae species by the comparison of a field strain with the type strain PG2

“…2A). Sequence analysis of 5632-specific fragments from the "A" library by BlastX homology searches revealed that the insert carried by the A36 clone partially matched with the aminoterminal portion of the vpmaY gene product [13] and other variable surface proteins from M. agalactiae PG2 and Mycoplasma bovis. The nucleotides 2-100 of the 310-bp A36 insert were potentially coding for a 33-a.a. peptide having 81% identity with the a.a. 18-50 of VpmaY and possessing the consensus acylation/peptidase II cleavage motif AAKC found in the Vpma family.…”

“…Detection of hybridized probes was performed with the alkaline phosphataseconjugated anti-Dig Fab antibodies and CDPstar reagents (Roche). The A3F oligonucleotide probe and the PG2-vpmaU-Z specific probes were used as described elsewhere [13][14][15].…”

“…The 5632 strain has also been shown to differ from PG2 by the lack of surface antigen(s) recognized by the 1D4 monoclonal antibody in western or colony immunoblots [4], the presence of an insertion sequence, ISMag1 [19], the Dam-like methylation of its DNA and the composition of vpma gene repertoires [14]. The M. agalactiae type strain PG2 harbors a family of genes coding for abundant surface lipoproteins (Vpmas) that undergo high frequency phase variation; the vpma locus of the clone 55-5 of PG2 contains 6 vpma genes [13,15]. The degenerate oligonucleotide probe A3F can specifically hybridize with a conserved sequence that is present in the vpma genes.…”

Genetic immunization with the immunodominant antigen P48 of Mycoplasma agalactiae stimulates a mixed adaptive immune response in BALBc mice

“…Mycoplasma agalactiae, and mycoplasmas in general, trick the immune system either by phase and size variation of their surface antigens Glew et al, 2000) or by modulating the host immune responses through the expression of up-and downregulating cytokines inducing suppression or aspecific polyclonal stimulation of B and T lymphocytes (Razin et al, 1998). Membrane surface proteins can also play a basic role in pathogenesis of mycoplasmas especially by acting as cytoadhesins in the attachment of mycoplasmas to host cells (Zhang et al, 1995;Santona et al, 2002).…”

Role of the GapA and CrmA Cytadhesins of Mycoplasma gallisepticum in Promoting Virulence and Host Colonization

“…These mainly include gene families encoding related surface proteins and single-copy genes encoding molecules with adhesive properties. However, the significance and consequences of this phenomenon have been addressed in vivo only in the case of complex gene families (21,22).…”