Phenotypic switching of variable surface lipoproteins in Mycoplasma bovis involves high-frequency chromosomal rearrangements

Abstract

Mycoplasma bovis, an important pathogen of cattle, was recently shown to possess a family of phase-and size-variable membrane surface lipoprotein antigens (Vsps). These proteins spontaneously undergo noncoordinate phase variation between ON and OFF expression states, generating surface antigenic variation. In the present study, we show that the spontaneously high rate of Vsp phenotypic switching involves DNA rearrangements that occur at high frequency in the M. bovis chromosome. A 1.5-kb HindIII genomic fragment carrying the vspA gene from M. bovis PG45 was cloned and sequenced. The deduced VspA amino acid sequence revealed that 80% of the VspA molecule is composed of reiterated intragenic coding sequences, creating a periodic polypeptide structure. Four distinct internal regions of repetitive sequences in the form of in-tandem blocks extending from the N-terminal to the C-terminal portion of the Vsp product were identified. Southern blot analysis of phenotypically switched isogenic lineages representing ON or OFF phase states of Vsp products suggested that changes in the Vsp expression profile were associated with detectable changes at the DNA level. By using a synthetic oligonucleotide representing a sequence complementary to the repetitive vspA gene region as a probe, we could identify the vspA-bearing restriction fragment undergoing high-frequency reversible rearrangements during oscillating phase transition of vspA. The 1.5-kb HindIII fragment carrying the vspA gene (on state) rearranged and produced a 2.3-kb HindIII fragment (OFF state) and vice versa. Two newly discovered vsp genes (vspE and vspF) were localized on two HindIII fragments flanking the vsp gene upstream and downstream. Southern blot hybridization with vspE-and vspF-specific oligonucleotides as probes against genomic DNA of VspA phase variants showed that the organization and size of the fragments adjacent to the vspA gene remained unchanged during VspA ON-OFF switching. The mechanisms regulating the vsp genes are yet unknown; our findings suggest that a recombinative mechanism possibly involving DNA inversions, DNA insertion, or mobile genetic elements may play a role in generating the observed high-frequency DNA rearrangements.

Molecular and Antigenic Characterization of aMycoplasma BovisStrain Causing an Outbreak of Infectious Keratoconjunctivitis

“…This result is not surprising because this primer set targets conserved motives flanking tandem repeat regions, which are known to vary in the number of repeats, at least between the M. bovis reference strain PG45 T and field strain 422. Moreover, different repertoires of Vsp genes have been observed in M. bovis, 23,29,37 and primer set B can potentially amplify at least Vsp 422-4 and Vsp 422-8 in strain 422. Sequencing of vsp A revealed that Sar1 is characterized by a number of R A tandem repeats in the initial portion of the Vsp A -like gene (10 repeats, 8 R A 1 and 2 R A 2) that is different from the number observed in the strains so far described (Table 4).…”

“…This number is lower than the number of repeats of the type strain PG45 T , but higher than the number reported for M. bovis strain 422. 23,29 When vsp A PCR (primer set B) was used to compare Sar1 to M. bovis field strains isolated in northern and central Italy, it was observed that the Sar1 PCR pattern (Fig. 1B, lane 3, 1 band of about 340 bp) is different from the pattern formed by the northern Italy strains (Fig.…”

“…The presence of M. agalactiae, M. bovoculi, and M. conjunctivae was excluded on the basis of the negative result obtained with primer sets C, D, and E. (Table 3), common to the 39 mycoplasma Sar1 samples investigated, was checked against the GenBank database and produced a significant alignment with M. bovis strains 120/81 and MC3386 (100% identity, E score 0). The analysis of the vsp A -like sequence of M. bovis Sar1 revealed the presence of an R A repeat pattern 23,29 typical for this isolate (Table 4). In fact, Sar1 possesses a number of 10 R A tandem repeats in the initial portion of the vsp Alike gene (8 R A 1 and 2 R A 2).…”

Molecular Biology and Pathogenicity of Mycoplasmas

“…However, only one vsa gene is expressed, while the others are transcriptionally silent (45). Structurally, the expressed vsa gene displays features similar to the vlp and the vsp genes (267,485). The expressed vsa gene is divided into three regions: region I, encoding a putative lipoprotein signal sequence; region II, the region encoding the conserved N terminus of the mature Vsa protein; and region III, a variable region containing the 3Ј repetitive elements (45,421).…”

“…Mycoplasma bovis, an important pathogen of cattle, presents another interesting example. A family of proteins undergoing noncoordinate high-frequency phase variation, as well as size variation (designated Vsps), is used to achieve extensive antigenic variation (34,267,268,387). Thirteen Vsp ORFs, not all similarly oriented, were identified within a 16-kb genomic fragment carrying the vsp locus (268).…”