Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium

Abstract

Mycoplasma genitalium is the smallest self-replicating microorganism and is implicated in human diseases, including urogenital and respiratory infections and arthritides. M. genitalium colonizes host cells primarily through adherence mechanisms mediated by a network of surface-associated membrane proteins, including adhesins and cytadherence-related proteins. In this paper, we show that cytadherence in M. genitalium is affected by an unrelated protein known as peptide methionine sulfoxide reductase (MsrA), an antioxidant repair enzyme that catalyzes the reduction of methionine sulfoxide [Met(O)] residues in proteins to methionine. An msrA disruption mutant of M. genitalium, constructed through homologous recombination, displayed markedly reduced adherence to sheep erythrocytes. In addition, the msrA mutant was incapable of growing in hamsters and exhibited hypersensitivity to hydrogen peroxide when compared to wild-type virulent M. genitalium. These results indicate that MsrA plays an important role in M. genitalium pathogenicity, possibly by protecting mycoplasma protein structures from oxidative damage or through alternate virulence-related pathways.

FIG. 1

SDS-PAGE and Western blot profiles. (A) Overexpression and purification of M. pneumoniae MsrA. Lane 1, molecular size markers; lane 2, E. coli BL21 cells harboring M. pneumoniae msrA overexpression construct pMPMSRA before the addition of IPTG; lane 3, E. coli BL21 cells harboring the M. pneumoniae msrA overexpression construct pMPMSRA 2 h after the addition of 1 mM IPTG; lane 4, E. coli cells overexpressed M. pneumoniae MsrA after purification with a nickel affinity column (Ni-NTA agarose). MsrA∗ indicates the overexpressed and purified His₁₀ tag MsrA of M. pneumoniae. (B) Western blotting showing reactivity of anti-M. pneumoniae MsrA antibodies. Lane 1, wild-type M. genitalium strain G37; lane 2, msrA mutant M. genitalium strain MS5; lane 3, overexpressed and purified His₁₀ tag MsrA protein of M. pneumoniae. MsrA∗ indicates the overexpressed and purified MsrA of M. pneumoniae, and MsrA indicates the native MsrA protein of M. genitalium.

FIG. 2

Southern hybridization profiles of DNA from M. genitalium strains. G37, wild-type M. genitalium; MS5, M. genitalium msrA mutant. Genomic DNA from G37 and MS5 were digested with restriction enzymes EcoRV (E), StyI (S), and EcoRV and StyI (E+S) together. Southern blots were probed with a 1.5-kb M. genitalium msrA gene fragment (A) and a 2.5-kb gentamicin resistance gene fragment (B).

FIG. 3

Schematic representation of msrA locus in M. genitalium strains G37 (wild type) and MS5 (msrA mutant). Stippled boxes represent msrA region, a lightly hatched box represents the gentamicin resistance gene, and open boxes represent flanking regions of mg218 locus. The different sizes of EcoRV and StyI fragments observed in Southern hybridization are represented below the msrA locus of each strain. EI, EcoRI, EV, EcoRV; H, HindIII; N, NdeI; St, StyI; X, XbaI.

FIG. 4

Mycoplasma HA assay using sheep erythrocytes. (A) M. genitalium wild-type strain G37; (B) M. genitalium msrA mutant strain MS5.