Oxidative Stress of Ozone Down-regulates Streptococcus mutans Extra-cellular Protein Expression

Objectives: In several reports including our previous presentation in IADR-SEA 2005 Meeting, it has been demonstrated that ozone gas can inactivate cariogenic bacteria. To determine the effect of the oxidative stress generated by ozone on bacterial cell associated proteins, two-dimensional gel electrophoresis (2-DE) and protein mass analysis were performed. Methods: Aliquots were prepared from freshly cultured Streptococcus mutans MT8148 (S. mutans) in brain heart infusion broth (Becton Dickinson, Sparks, MD) and resuspended in PBS at an OD₅₀₀=2. Ozone gas was sparged for 0 (no ozone-stress), 30, 60 and 240 s, from the HealOzone device (Kavo, Germany). Solubilized extracellular proteins were separated by 2-DE on 24 cm SDS-PAGE gels over the pH ranges 3.0–10.0. Protein spots on the gel-images were analyzed by using Progenesis PG220 protein gel-spot analyzing software (Shimadzu Biotech, Kyoto, Japan). Selected protein spots were digested with trypsin and analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI/ToF-ToF). Peptide mass fingerprinting (PMF) analyses of proteins was performed. The peak list generation made use from the Mass and Mass/Mass spectrometry to query the MASCOT and SWISS-PROT. Results: Image analysis revealed highly reproducible 2-DE gels displaying approximately 160 spots of S. mutans surface-associated proteins from the no ozone-stressed aliquot. Compared to that almost no differences could be recognized on 30 s ozone-stressed gel-image. However, when the ozone was sparged for more than 60 s the spot number dramatically reduced and less than 20 spots could be detected on 240 s ozone-stressed gel-image. Most remarkable change was recognized in glyceraldehydes-3-phosphate dehydrogenase (GAPDH). Conclusions: Expression of surface-associated proteins was down-regulated on ozone induced oxidative stress when ozone was sparged for more than 60 s. Possibly, high concentration of ozone causes an irreversible damage to the S. mutans cell surface-associated proteins. This study was supported by Center of Excellence (COE) Program for Frontier Research on Molecular Destruction and Reconstruction of Tooth and Bone at Tokyo Medical and Dental University.