A family of phase- and size-variant membrane surface lipoprotein antigens (Vsps) of Mycoplasma bovis

Abstract

A set of strain-and size-variant highly immunogenic membrane surface protein antigens of Mycoplasma bovis, which has been identified by a monoclonal antibody, is shown in this report to make up a family of antigenically and structurally related lipid-modified proteins, designated Vsps (variable surface proteins). By systematic analysis of several isogenic clonal lineages of the type strain PG45, three members of this family have been identified, VspA, VspB, and VspC, each of which was shown to undergo independent high-frequency changes in size as well as noncoordinate phase variation between ON and OFF expression states. The monoclonal antibody-defined epitope common to VspA, VspB, and VspC was accessible on the cell surface in most, but not all, of the clonal populations analyzed and was present on a C-terminal limit tryptic fragment of each Vsp variant that was released from the membrane surface. VspA and VspC were distinguished from VspB by their selective detection with colloidal gold and by their distinctive reaction with a polyclonal antibody against M. bovis D490. VspA, VspB, and VspC were further distinguishable from one another by their characteristic patterns of degradation at carboxypeptidase Y pause sites. While these Vsp-specific structural fingerprints with an irregular periodic spacing were constant for similarly sized variants of a defined Vsp product, they showed distinct differences among variants differing in size. This variability included gain or loss of individual bands within distinct subsets of bands, as well as shifts of the entire banding patterns upor downwards, indicating that insertions or deletions underlying Vsp size variation can occur at various locations either within the C-terminal domain or within other regions of these proteins. This was similarly confirmed by comparative epitope mapping analysis of tryptic cleavage products generated from different Vsp size variants. The Vsp family of M. bovis described in this study represents a newly discovered system of surface antigenic variation in mycoplasmas displaying features which closely resemble but are also different from the characteristics reported for the Vlp (variable lipoprotein) system of M. hyorhinis. The isogenic lineages established here provide key populations for subsequent analysis of corresponding genes to further elucidate Vsp structure and variation, which may have important relevance for a better understanding of the pathogenicity of this agent.

Adaptive surface antigen variation in Mycoplasma bovis to the host immune response

“…No growth inhibition oc-curred and significant modifications appeared only for inocula of I-100 cells. The presence of such 'escape variants' within an initial mycoplasma population of only 1llOO cells is statistically unlikely since the frequency of Vsps was reported to range between I OF" and 1O-5 per cell per generation [16]. We therefore concluded that the observed changes in Vsp surface antigen expression were indeed induced by the respective antibody as sensory signal.…”

“…M bovis strain 1067 was isolated in 1983 from a case of bovine mastitis [24] and successfully used to experimentally induce reproducible arthritis in calves [20]. Clonal variants of M. bovis type strain PG45 expressing a single size variant of VspA (65 and 63 kDa), VspB (46,44.5,43 or 42 kDa) or Vsp C (79 or 75 kDa), or lacking all three Vsps (designated ABC-OK) have been previously described [16]. Each organism was propagated at 37°C in a standard mycoplasma medium [23].…”

“…Our previous investigations have shown that M. bovis employs two surface antigenic variation systems which could act independently to facilitate immune evasion by this organism [14-171. The first represents a family of membrane surface lipoproteins designated Vsps (variable surface proteins) [16], with as yet three defined members (VspA, VspB, VspC) which undergo a high rate of phase and size variation in vitro. A second, unrelated membrane surface protein designated pMB67 [17] proved to be as highly variable as the Vsps.…”

A 41-kDa variable surface protein of Mycoplasma gallisepticum has a counterpart in Mycoplasma imitans and Mycoplasma iowae

“…Nevertheless, pMG41 and pMI41 are the first reported examples of variable surface proteins in mycoplasmas which are not described as integral membrane proteins. All other described phase-variable mycoplasma antigens so far are integral membrane proteins that are membrane-anchored either via lipid [12, 14,17] or via hydrophobic transmembrane domains (191; D. Yogev, D. Menaker and R. Rosengarten, submitted).…”

“…for Triton X-l 14 (TX-l 14; Boehringer Mannheim) phase fractionation, SDS-PAGE and Western immunoblotting of mycoplasma proteins are described elsewhere [9,13,14]. For SDS-PAGE, samples (if not specifically indicated, each corresponding to 400 ~1 of a mid-log-phase culture) were boiled for 5 min under reducing conditions and loaded onto 9% polyacrylamide gels containing 3% (w/v) urea.…”