Streptococcus pyogenes does not produce catalase, but it can grow in aerobic environments and survive in the presence of peroxide. One of the stress proteins of this organism, peroxide resistance protein (Dpr), has been studied to examine its role in resistance to hydrogen peroxide, but the protective mechanism of Dpr is not clear. The aim of this study was to characterize the dpr gene and its role in dealing with different stresses. A dpr deletion mutant was constructed by double-crossover mutagenesis. The dpr mutant was more sensitive to H 2 O 2 , and complementation could partially restore the defect in the mutant. Pretreatment with the iron chelator deferoxamine mesylate rescued the survival activity of the mutant under oxidative stress conditions. The dpr mutant also showed a low survival rate in the long-term stationary phase, when it was treated with extreme acids, and under alkaline pH conditions compared to the wild-type strain. The growth of the dpr mutant was slower than that of the wild-type strain in iron-limiting conditions. The dpr mutant showed high sensitivity to iron and zinc but not to manganese, copper, nickel, and calcium. Recombinant Dpr protein was purified and showed iron-binding activity, whereas no DNA-binding activity was found. These data indicate that an ironbinding protein, Dpr, provides protection from hydrogen peroxide stress by preventing the Fenton reaction, and Dpr was identified as a novel stress protein that protects against several stresses in group A streptococci.

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Streptococcus pyogenes is a human pathogen that causes various diseases. Numerous virulence factors secreted by S. pyogenes are involved in pathogenesis. The peroxide regulator (PerR) is associated with the peroxide resistance response and pathogenesis, but little is known about the regulation of the secretome involved in virulence. To investigate how PerR regulates the expression of the S. pyogenes secretome involved in virulence, a perR deficient mutant was used for comparative secretomic analysis with a wild-type strain. The conditioned medium containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for two-dimensional gel electrophoresis analysis, where protease inhibitors were applied to avoid the degradation of extracellular proteins. Differentially expressed protein spots were identified by liquid chromatography electrospray ionization tandem MS. More than 330 protein spots were detected on each gel. We identified 25 unique up-regulated proteins and 13 unique downregulated proteins that were directly or indirectly controlled by the PerR regulator. Among these identified proteins, mitogen factor 3 (MF3), was selected to verify virulence and the expression of gene products. The data showed that MF3 protein levels in conditioned medium, as measured by immunoblot analysis, correlated well with protein levels determined by two-dimensional gel electrophoresis analysis. We also demonstrated that PerR bound to the promoter region of the mf3 gene. The result of an infection model showed that virulence was attenuated in the mf3 deficient mutant. Additional growth data of the wild-type strain and the mf3 deficient mutant suggested that MF3 played a role in digestion of exogenous DNA for promoting growth. To summarize, we conclude that PerR can positively regulate the expression of the secreted protein MF3 that contributes to the virulence in S. pyogenes. The analysis of the PerR-regulated secretome provided key information for the elucidation of the host-pathogen interactions and might assist in the

The peroxide regulator (PerR) is a ferric uptake repressor-like protein, which is involved in adaptation to oxidative stress and iron homeostasis in group A streptococcus. A perR mutant is attenuated in surviving in human blood, colonization of the pharynx, and resistance to phagocytic clearance, indicating that the PerR regulon affects both host environment adaptation and immune escape. Sda1 is a phage-associated DNase which promotes M1T1 group A streptococcus escaping from phagocytic cells by degrading DNA-based neutrophil extracellular traps. In the present study, we found that the expression of sda1 is up-regulated under oxidative conditions in the wild-type strain but not in the perR mutant. A gel mobility shift assay showed that the recombinant PerR protein binds the sda1 promoter. In addition, mutation of the conserved histidine residue in the metal binding site of PerR abolished sda1 expression under hydrogen peroxide treatment conditions, suggesting that PerR is directly responsible for the sda1 expression under oxidative stress. Our results reveal PerR-dependent sda1 expression under oxidative stress, which may aid innate immune escape of group A streptococcus.