Methionine Sulfoxide Reductase Protects Surface Adhesins on Streptococcus gordonii

The early oral biofilm colonizer Streptococcus gordonii produces hydrogen peroxide. To maintain functional proteins during oxidative stress, methionine sulfoxide reductase (MsrA) catalyzes the reduction of methionine sulfoxide to methionine. In the presence of H2O2, S. gordonii MsrA- showed reduced adhesion and biofilm formation compared to wild type.

Objective: Since the central divergent region of the adhesin SspA/B includes a Met-rich segment, MsrA was hypothesized to reduce H2O2-oxidized Met on adhesins and restore biofilm formation.

Methods: The MsrA- and msrA-FLAsH (fluorescent tag) complemented mutants were constructed in S. gordonii V288. Wild-type V288 and the MsrA- mutant were incubated ± H2O2 and biofilm formation was estimated on polystyrene. Cell wall proteins were analyzed by native PAGE and Western blot. MsrA and the Met-rich peptide segment of SspB were expressed and purified. Untreated, H2O2-oxidized and MsrA-reduced peptides were analyzed by Western blot and MASS-SPEC. MsrA enzyme activity was determined in cell fractions and FLAsH-tagged MsrA was detected by Western blot and localized in cells by confocal microscopy.

Results: In the presence of H2O2, the MsrA- mutant formed only 13% of the biofilm mass developed by the wild type and complemented mutant. The oxidized Met-rich peptide from SspB showed a PAGE band-shift, like SspA/B, which was restored by MsrA. Met residues in the peptide were oxidized by H2O2 and reduced by MsrA as shown with MASS-SPEC. In V288, MsrA enzyme activity was detected in spent media, cell washes, and lysates. Fractionated cell wall and cytosol contained FLAsH-tagged MsrA, and localization was confirmed by confocal microscopy.

Conclusions: MsrA reduces oxidized Met in the surface adhesin, SspA/B, thus facilitating biofilm formation of S. gordonii under oxidative stress. MsrA localizes to the cell wall of S. gordonii, and may function intracellularly and extracellularly. Supported by NIH R01 DE08590.