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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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Colony immunoblot analysis of M. agalactiae type strain PG2 and its derivative mutants using the six Vpma-specific pAbs recognizing specific surface exposed epitopes. PLMU and PLMY represent the two xer1-disrupted PLMs expressing exclusively VpmaU and VpmaY respectively. WT-PG2 and PLMU-CP representing the xer1-complemented PLMU show sectorial staining phenotype with all Vpma-specific pAbs reflecting high frequency Vpma phase variations. Designations of Vpma-specific pAbs as used for each column, and as described in Table 1, are indicated at the bottom.
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fig02: Colony immunoblot analysis of M. agalactiae type strain PG2 and its derivative mutants using the six Vpma-specific pAbs recognizing specific surface exposed epitopes. PLMU and PLMY represent the two xer1-disrupted PLMs expressing exclusively VpmaU and VpmaY respectively. WT-PG2 and PLMU-CP representing the xer1-complemented PLMU show sectorial staining phenotype with all Vpma-specific pAbs reflecting high frequency Vpma phase variations. Designations of Vpma-specific pAbs as used for each column, and as described in Table 1, are indicated at the bottom.

Mentions: Colony immunoblotting revealed positive, negative and sectorial staining with all six anti-Vpma pAbs reflecting the surface exposure, as well as the hypervariability of all target epitopes in the PG2 strain (Fig. 2, row 1).


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)

Colony immunoblot analysis of M. agalactiae type strain PG2 and its derivative mutants using the six Vpma-specific pAbs recognizing specific surface exposed epitopes. PLMU and PLMY represent the two xer1-disrupted PLMs expressing exclusively VpmaU and VpmaY respectively. WT-PG2 and PLMU-CP representing the xer1-complemented PLMU show sectorial staining phenotype with all Vpma-specific pAbs reflecting high frequency Vpma phase variations. Designations of Vpma-specific pAbs as used for each column, and as described in Table 1, are indicated at the bottom.
© Copyright Policy
Related In: Results  -  Collection

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

fig02: Colony immunoblot analysis of M. agalactiae type strain PG2 and its derivative mutants using the six Vpma-specific pAbs recognizing specific surface exposed epitopes. PLMU and PLMY represent the two xer1-disrupted PLMs expressing exclusively VpmaU and VpmaY respectively. WT-PG2 and PLMU-CP representing the xer1-complemented PLMU show sectorial staining phenotype with all Vpma-specific pAbs reflecting high frequency Vpma phase variations. Designations of Vpma-specific pAbs as used for each column, and as described in Table 1, are indicated at the bottom.
Mentions: Colony immunoblotting revealed positive, negative and sectorial staining with all six anti-Vpma pAbs reflecting the surface exposure, as well as the hypervariability of all target epitopes in the PG2 strain (Fig. 2, row 1).

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