Redox-Regulated Adaptation of Streptococcus oligofermentans to Hydrogen Peroxide Stress

Preexposure to a low concentration of H₂O₂ significantly increases the survivability of catalase-negative streptococci in the presence of a higher concentration of H₂O₂ However, the mechanisms of this adaptation remain unknown. Here, using a redox proteomics assay, we identified 57 and 35 cysteine-oxidized proteins in Streptococcus oligofermentans bacteria that were anaerobically cultured and then pulsed with 40 μM H₂O₂ and that were statically grown in a 40-ml culture, respectively. The oxidized proteins included the peroxide-responsive repressor PerR, the manganese uptake repressor MntR, thioredoxin system proteins Trx and Tpx, and most glycolytic proteins. Cysteine oxidations of these proteins were verified through redox Western blotting, immunoprecipitation, and liquid chromatography-tandem mass spectrometry assays. In particular, Zn²⁺-coordinated Cys139 and Cys142 mutations eliminated the H₂O₂ oxidation of PerR, and inductively coupled plasma mass spectrometry detected significantly decreased amounts of Zn²⁺ in H₂O₂-treated PerR, demonstrating that cysteine oxidation results in Zn²⁺ loss. An electrophoretic mobility shift assay (EMSA) determined that the DNA binding of Mn²⁺-bound PerR protein (PerR:Zn,Mn) was abolished by H₂O₂ treatment but was restored by dithiothreitol reduction, verifying that H₂O₂ inactivates streptococcal PerR:Zn,Mn through cysteine oxidation, analogous to the findings for MntR. Quantitative PCR and EMSA demonstrated that tpx, mntA, mntR, and dpr belonged to the PerR regulons but that only dpr was directly regulated by PerR; mntA was also controlled by MntR. Deletion of mntR significantly reduced the low-H₂O₂-concentration-induced adaptation of S. oligofermentans to a higher H₂O₂ concentration, while the absence of PerR completely abolished the self-protection. Therefore, a low H₂O₂ concentration resulted in the cysteine-reversible oxidations of PerR and MntR to derepress their regulons, which function in cellular metal and redox homeostasis and which endow streptococci with the antioxidative capability. This work reveals a novel Cys redox-based H₂O₂ defense strategy employed by catalase-negative streptococci in Mn²⁺-rich cellular environments.IMPORTANCE The catalase-negative streptococci produce as well as tolerate high levels of H₂O₂ This work reports the molecular mechanisms of low-H₂O₂-concentration-induced adaptation to higher H₂O₂ stress in a Streptococcus species, in which the peroxide-responsive repressor PerR and its redox regulons play the major role. Distinct from the Bacillus subtilis PerR, which is inactivated by H₂O₂ through histidine oxidation by the Fe²⁺-triggered Fenton reaction, the streptococcal PerR is inactivated by H₂O₂ oxidation of the structural Zn²⁺ binding cysteine residues and thus derepresses the expression of genes defending against oxidative stress. The reversible cysteine oxidation could provide flexibility for PerR regulation in streptococci, and the mechanism might be widely used by lactic acid bacteria, including pathogenic streptococci, containing high levels of cellular manganese, in coping with oxidative stress. The adaptation mechanism could also be applied in oral hygiene by facilitating the fitness and adaptability of the oral commensal streptococci to suppress the pathogens.