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Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation.

Chopra-Dewasthaly R, Citti C, Glew MD, Zimmermann M, Rosengarten R, Jechlinger W - Mol. Microbiol. (2008)

Bottom Line: Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector.Inactivation of xer1 abolished further Vpma switching and the 'phase-locked' mutants (PLMs) continued to steadily express only a single Vpma product.Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria. Rohini.Chopra-Dewasthaly@vu-wien.ac.at

ABSTRACT
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host-pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector. Inactivation of xer1 abolished further Vpma switching and the 'phase-locked' mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other 'difficult-to-manipulate' mycoplasmas.

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Related in: MedlinePlus

Comparative Western blot analysis of whole-cell extracts of M. agalactiae type strain PG2 (WT-PG2), two PLMs (PLMY and PLMU) and xer1-complemented PLMU (PLMU-CP) using six different Vpma-specific pAbs as described in Table 1. Designations of individual pAbs used for each Western blot are indicated in the right margins of each panel whereas relevant protein size standards are shown on the left margins.
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fig04: Comparative Western blot analysis of whole-cell extracts of M. agalactiae type strain PG2 (WT-PG2), two PLMs (PLMY and PLMU) and xer1-complemented PLMU (PLMU-CP) using six different Vpma-specific pAbs as described in Table 1. Designations of individual pAbs used for each Western blot are indicated in the right margins of each panel whereas relevant protein size standards are shown on the left margins.

Mentions: Western blot analysis using whole-cell extracts of PLMU confirmed the results of colony immunoblot analysis (Fig. 4). PLMU only reacted with pAb α-U, corresponding to the VpmaU protein, and was not recognized by any of the other five pAbs. Consistent expression of only VpmaU upon 15 successive passages of PLMU, both in the absence and presence of tetracycline selection, and the complete lack of any spontaneous reversions or vpma-specific rearrangements proved the ‘phase-locked’ VpmaU phenotype in this xer1 mutant (data not shown). This was in contrast to the wt parental strain PG2 which reacted with all six pAbs, and whose clonal variants expressing VpmaU led to a mixed colony Vpma-staining phenotype (positive, negative and sectorial) with almost all pAbs within two to five in vitro passages, and as well showed vpma-specific rearrangements observed by Southern blot analysis (data not shown) indicating Vpma phase variation.


Phase-locked mutants of Mycoplasma agalactiae: defining the molecular switch of high-frequency Vpma antigenic variation.

Chopra-Dewasthaly R, Citti C, Glew MD, Zimmermann M, Rosengarten R, Jechlinger W - Mol. Microbiol. (2008)

Comparative Western blot analysis of whole-cell extracts of M. agalactiae type strain PG2 (WT-PG2), two PLMs (PLMY and PLMU) and xer1-complemented PLMU (PLMU-CP) using six different Vpma-specific pAbs as described in Table 1. Designations of individual pAbs used for each Western blot are indicated in the right margins of each panel whereas relevant protein size standards are shown on the left margins.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2268961&req=5

fig04: Comparative Western blot analysis of whole-cell extracts of M. agalactiae type strain PG2 (WT-PG2), two PLMs (PLMY and PLMU) and xer1-complemented PLMU (PLMU-CP) using six different Vpma-specific pAbs as described in Table 1. Designations of individual pAbs used for each Western blot are indicated in the right margins of each panel whereas relevant protein size standards are shown on the left margins.
Mentions: Western blot analysis using whole-cell extracts of PLMU confirmed the results of colony immunoblot analysis (Fig. 4). PLMU only reacted with pAb α-U, corresponding to the VpmaU protein, and was not recognized by any of the other five pAbs. Consistent expression of only VpmaU upon 15 successive passages of PLMU, both in the absence and presence of tetracycline selection, and the complete lack of any spontaneous reversions or vpma-specific rearrangements proved the ‘phase-locked’ VpmaU phenotype in this xer1 mutant (data not shown). This was in contrast to the wt parental strain PG2 which reacted with all six pAbs, and whose clonal variants expressing VpmaU led to a mixed colony Vpma-staining phenotype (positive, negative and sectorial) with almost all pAbs within two to five in vitro passages, and as well showed vpma-specific rearrangements observed by Southern blot analysis (data not shown) indicating Vpma phase variation.

Bottom Line: Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector.Inactivation of xer1 abolished further Vpma switching and the 'phase-locked' mutants (PLMs) continued to steadily express only a single Vpma product.Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bacteriology, Mycology and Hygiene, Department of Pathobiology, University of Veterinary Medicine Vienna, Veterinärplatz 1, A-1210 Vienna, Austria. Rohini.Chopra-Dewasthaly@vu-wien.ac.at

ABSTRACT
Mycoplasma agalactiae, an important pathogen of small ruminants, exhibits antigenic diversity by switching the expression of multiple surface lipoproteins called Vpmas (Variable proteins of M. agalactiae). Although phase variation has been shown to play important roles in many host-pathogen interactions, the biological significance and the mechanism of Vpma oscillations remain largely unclear. Here, we demonstrate that all six Vpma proteins are expressed in the type strain PG2 and all undergo phase variation at an unusually high frequency. Furthermore, targeted gene disruption of the xer1 gene encoding a putative site-specific recombinase adjacent to the vpma locus was accomplished via homologous recombination using a replicon-based vector. Inactivation of xer1 abolished further Vpma switching and the 'phase-locked' mutants (PLMs) continued to steadily express only a single Vpma product. Complementation of the wild-type xer1 gene in PLMs restored Vpma phase variation thereby proving that Xer1 is essential for vpma inversions. The study is not only instrumental in enhancing our ability to understand the role of Vpmas in M. agalactiae infections but also provides useful molecular approaches to study potential disease factors in other 'difficult-to-manipulate' mycoplasmas.

Show MeSH
Related in: MedlinePlus