The virulence of Streptococcus pyogenes depends on proteins that are produced by this bacterium. The production of virulence proteins depends on environmental factors, and twocomponent regulatory systems are considered to be involved in sensing these factors. One of the environmental factors is acid stimuli. We established knockout strains in all speculated twocomponent regulatory sensor proteins of the M1 clinical strain of S. pyogenes and examined their relevance to acid stimuli. The parental strain and its derived knockout strains were cultured in a medium adjusted to pH 7.6 or 6.0, and their growth in broth was compared. The spy1622 sensor knockout strain showed significant growth reduction compared with the parental strain in broth at pH 6.0, suggesting that the Spy1622 two-component sensor protein is involved in sensing acid stimuli. To further examine the role of the Spy1622 two-component sensor protein in virulence, blood bactericidal assays and mouse infection model experiments were performed. We found that the spy1622 knockout strain was less virulent than the parental strain, which suggests that the Spy1622 two-component sensor protein could play an important role in virulence. INTRODUCTIONStreptococcus pyogenes is a Gram-positive bacterium that infects the upper respiratory tract, for example the tonsils and pharynx, and causes serious post-infectious diseases such as rheumatic fever and glomerulonephritis. Furthermore, S. pyogenes causes even more serious human diseases such as streptococcal toxic shock syndrome (STSS), whose mortality rate is considerable (Cone et al., 1987;Cunningham, 2000). Because of the worldwide prevalence of STSS, a number of studies have been undertaken to identify relevant virulence factors (Hauser et al., 1991;Reichardt et al., 1992). These factors include M protein, streptococcal inhibitor of complement, streptococcal pyrogenic toxins, haemolysins, and several DNases (Cunningham, 2000).The production of virulence proteins depends on environmental factors such as pH. Bacterial growth and the host environment, such as that in phagocytic cells, can cause local and temporal pH changes, and bacteria can subsequently sense the pH change as a signal to modify their growth mode. In addition, bacteria can modulate their gene expression in response to changes in pH (Loughman & Caparon, 2006). Hence, pH is considered to play an important role in host colonization and infection by S. pyogenes.The regulation of virulence protein expression in S. pyogenes is mainly controlled by stand-alone response regulators and two-component regulatory systems (TCSs). Stand-alone transcriptional regulators have no identified sensory domain (Kreikemeyer et al., 2003). The three characterized stand-alone regulators include Mga, Rgg and RofA-like family members (McIver et al., 1995;Beckert et al., 2001;Chaussee et al., 1999;Lyon et al., 1998;Molinari et al., 2001;Podbielski et al., 1999). A TCS consists of a sensor protein and a regulatory protein.Thirteen TCSs have been identified in the available S. ...

Background Streptococcus pyogenes (S. pyogenes) causes various serious diseases including necrotizing fasciitis and streptococcal toxic shock syndrome. One serious problem observed recently with S. pyogenes therapy is attenuation of the antibiotic effect, especially penicillin treatment failure and macrolide resistance. Hainosankyuto, a traditional Japanese medicine based on ancient Chinese medicine, has been used for treatment of infectious purulent diseases in Japan. In this study, we investigated the protective and therapeutic efficacy of Hainosankyuto against S. pyogenes-skin infection.Methodology/Principal FindingsA broth microdilution method revealed that Hainosankyuto did not show a direct anti-bacterial effect against S. pyogenes. Force-feeding Hainosankyuto to infected mice for 4 consecutive days increased the survival rate and reduced the size of local skin lesions compared with mice fed PBS. Although we did not find the significant recovery of survival rate in Hainosankyuto administration only after S. pyogenes infection, the sizes of ulcer lesion were significant smaller after Hainosankyuto administration compared with mice fed PBS. No difference was observed in the anti-bacterial effect of Hainosankyuto between macrolide-susceptible and -resistant strains. Blood bactericidal assay showed that the survival rate of S. pyogenes using the blood from Hainosankyuto -treated mice was lower than that using the blood from untreated mice. We also found increased levels of IL-12, IFN-γ and a decreased level of TNF-α in the serum of S. pyogenes-infected mice treated with Hainosankyuto. Mouse peritoneal macrophage from Hainosankyuto-treated mice had significant phagocytic activity and increased mRNA levels of IL-12, IFN-γ and decreased mRNA level of TNF-α compared with control macrophage.Conclusions/SignificanceHainosankyuto increased survival rate after S. pyogenes infection and up-regulated both blood bactericidal activity and macrophage phagocytic activity through modulation of inflammatory cytokines. Our data also suggest Hainosankyuto may be useful for the treatment of S. pyogenes infection more prophylactically than therapeutically.

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.

The LytR family of cell envelope-associated transcriptional attenuators in bacteria has been brought into focus of scientific interest on the expression of various virulence factors, as well as bacterial cell envelope maintenance. However, this protein of Streptococcus pyogenes has been only described as cell surface-associated protein, and its function is completely unknown. We created lytR mutant strains from two independent S. pyogenes strains to analyze the function of LytR. The protease assay in culture supernatant showed that lytR mutant had the higher cysteine protease activity than wild-type. Two-dimensional gel electrophoresis and western blotting analysis revealed that the amount of cysteine protease, SpeB in lytR mutant was more compared with that in wild-type. The level of speB mRNA in lytR mutant also increased compared with that of wild-type. The membrane integrity and potential in lytR mutant also were decreased compared with that of wild-type. Murine infection model showed that less survival was detected in mice inoculated with lytR mutant than that with wild-type, and the size of wound lesion of mice with lytR mutant was larger than that with wild-type. Our data suggest that the lytR regulates the expression of SpeB in S. pyogenes with relation to membrane integrity.