A greater effect on clarithromycin resistance of mef(A)-associated msr(D) than mef(E)-associated msr(D) in Streptococcus pyogenes.

The mef(A)- and its subclass mef(E) systems had long been considered to constitute one of the primary macrolide-resistant mechanisms in Streptococcus pyogenes. However, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A)/mef(E) genes plays a predominant role in these systems. In previous studies, furthermore, mef(A)-associated msr(D)10-85 of an S. pyogenes strain (10-85) exhibited a greater increase in clarithromycin minimum inhibitory concentration (MIC) than mef(E)-associated msr(D)13-O-10 of another strain (13-O-10). Both msr(D) genes encode 487 amino acid residues, 13 amino acid residues of which are different from each other. In this study, we performed mutational analysis of the msr(D) genes and showed that a single-nucleotide polymorphism to cause a substitution of Asp238 with Gly is mainly associated with the greater increase in clarithromycin MIC by the msr(D)10-85 than by the msr(D)13-O-10 allele. In addition, another substitution of Ser with Arg at codon 194 is partially associated with the greater increase by the msr(D)10-85 than by the msr(D)13-O-10 allele.

Streptococcus pyogenes TrxSR Two-Component System Regulates Biofilm Production in Acidic Environments.

Bacteria form biofilms for their protection against environmental stress and produce virulence factors within the biofilm. Biofilm formation in acidified environments is regulated by a two-component system, as shown by studies on isogenic mutants of the sensor protein of the two-component regulatory system in Streptococcus pyogenes. In this study, we found that the LiaS histidine kinase sensor mediates biofilm production and pilus expression in an acidified environment through glucose fermentation. The liaS isogenic mutant produced biofilms in a culture acidified by hydrochloric acid but not glucose, suggesting that the acidified environment is sensed by another protein. In addition, the trxS isogenic mutant could not produce biofilms or activate the mga promoter in an acidified environment. Mass spectrometry analysis showed that TrxS regulates M protein, consistent with the transcriptional regulation of emm, which encodes M protein. Our results demonstrate that biofilm production during environmental acidification is directly under the control of TrxS.

Induction of antibody responses in mice immunized intranasally with Type I interferon as adjuvant and synergistic effect of chitosan.

Type I IFNs are a range of host-derived molecules with adjuvant potential; they have been used for many years in the treatment of cancer and viral hepatitis. Therefore, the safety of IFNs for human use has been established. In this study, we evaluated the mucosal adjuvanticity of IFN-ß administered intranasally to mice with diphtheria toxoid, and suggested a method to improve its adjuvanticity. When IFN-ß alone was used as a mucosal adjuvant, no clear results were obtained. However, simultaneous administration of IFN-ß and chitosan resulted in an enhancement of the specific serum immunoglobulin G (IgG) and IgA antibody responses, the mucosal IgA antibody response, and antitoxin titers. Furthermore, the intranasal administration of IFN-α alone resulted in a greater increase in antibody titer than IFN-ß, and a synergistic effect with chitosan was also observed. These findings suggest that intranasal administration of chitosan and Type I IFNs may display an effective synergistic mucosal adjuvant activity.

Prevalence of emm1 Streptococcus pyogenes having a novel type of genomic composition.

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). The complete genome sequence of a S. pyogenes strain 10-85 isolated from a STSS patient was recently announced. In this study, the genome sequence was dissected and it was found that the genomic region around 200 kbp (region A) and the genomic region around 1600 kbp (region B) were replaced by each other in strain 10-85, when compared with those in reference strains SF370 and A20. In order to address whether this replacement is unique to 10-85, we further analyzed 163 emm1-type strains. The results indicated that none of the strains isolated before 1990 had the replacement. In contrast, most of the strains isolated at least after 2000 appeared to have the 10-85-type replacement.

Predominant role of msr(D) over mef(A) in macrolide resistance in Streptococcus pyogenes.

In Japan, the number of patients with streptococcal toxic shock syndrome is reported to be increasing. mef(A) gene-positive macrolide-resistant emm1 strains are thought to possibly contribute to the rise in the frequency of STSS. Although analyses of macrolide-resistant mechanisms, including mef(A) resistance, have been performed mainly in Streptococcus pneumoniae, the role of this gene in Streptococcus pyogenes has not been completely investigated. Therefore, to the best of our knowledge, we established the first mef(A)-knockout strain using an emm1-type S. pyogenes strain, and tested its susceptibility to erythromycin, clarithromycin and azithromycin. We found that the antimicrobial susceptibilities were almost identical to those of the parental strain. Hence, we established a knockout strain for another gene, msr(D), that is located immediately downstream of mef(A). The macrolide resistances of the resulting strain significantly decreased, and were further altered when both mef(A) and msr(D) were knocked out. The introduction of the msr(D) gene into a macrolide-sensitive strain conferred more resistance than the introduction of the mef(A) gene. The erythromycin susceptibilities of knockout strains were further dissected using two additional emm4- and emm75-type S. pyogenes strains. We found almost identical results for both strains except for the mef(A) knockout emm4 type, whose susceptibility was altered, although the change was less than that for the msr(D) knockout. These results suggest that both mef(A) and msr(D) are involved in macrolide resistance in S. pyogenes, and that the msr(D) gene plays a more predominant role in macrolide resistance than mef(A).

Association of CovRS Two-Component Regulatory System with NADase Induction by Clindamycin Treatment in Streptococcus pyogenes.

Administration of high-dose clindamycin (CLI) and penicillin is recommended for the treatment of streptococcal toxic shock syndrome (STSS). However, CLI-resistant strains have been identified worldwide. In this study, some CLI-resistant strains demonstrated increased extracellular activity of the NAD-glycohydrolase (NADase) exotoxin following CLI treatment. These results support our previous conclusion that CLI-susceptible and CLI-resistant Streptococcus pyogenes strains exhibit CLI-dependent NADase induction. Furthermore, we investigated the mechanism of this phenomenon using 13 types of two-component sensor knockout strains derived from the CLI-susceptible strain 1529 that has a CLI-dependent NADase induction phenotype. Among the knockout strains, only 1529ΔcovS lost the phenotype. Additionally, 1529ΔspeB, 1529Δmga, and 1529Δrgg retained the CLI-dependent NADase induction phenotype. These findings indicate that CovS is related to this phenotype in a SpeB-independent manner.

General Phenotype of NADase Induction by CLI Treatment in Streptococcus pyogenes.

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic-shock syndrome (STSS). However, we previously reported that a "subinhibitory dose" of CLI induced the expression of the NAD-glycohydrolase (NADase) exotoxin in an emm1-type Streptococcus pyogenes 1529 strain isolated from an STSS patient. In this study, we examine NADase induction by CLI treatment using an extracellular NADase activity assay instead of the previous two-dimensional gel electrophoresis assay. The examination revealed that CLI administration can induce NADase expression in a dose-dependent manner. We analyzed 23 CLI-susceptible strains (5 emm1 strains, 6 emm3 strains, 3 emm4 strains, 1 emm6 strain, 3 emm12 strains, 1 emm28 strain, and 4 emm89 strains), and 19 of the 23 strains showed similar NADase induction phenotypes to that shown in strain 1529. These results indicate that NADase induction by CLI treatment is not restricted to specific strains and it could be a standard phenotype among CLI-susceptible S. pyogenes strains. We also analyzed four CLI-resistant strains. All four strains showed increased extracellular NADase activities at high concentrations of CLI that did not inhibit bacterial growth. These results indicated that the subinhibitory dose of CLI was not the critical factor for NADase induction.

Analysis of two-component sensor proteins involved in the response to acid stimuli in Streptococcus pyogenes.

The virulence of Streptococcus pyogenes depends on proteins that are produced by this bacterium. The production of virulence proteins depends on environmental factors, and two-component 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 two-component 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.

Clindamycin-induced CovS-mediated regulation of the production of virulent exoproteins streptolysin O, NAD glycohydrolase, and streptokinase in Streptococcus pyogenes.

The administration of high-dose clindamycin (CLI) along with penicillin is recommended for the treatment of streptococcal toxic shock syndrome. However, the prevalence of CLI-resistant Streptococcus pyogenes strains is increasing worldwide, and the effect of CLI on CLI-resistant S. pyogenes strains remains unknown. We aimed to evaluate the effect of CLI on the in vitro production of three major virulent exoproteins, namely, streptolysin O (Slo), NAD glycohydrolase (Nga), and streptokinase (Ska), by CLI-resistant S. pyogenes strains. After the incubation of M1 serotype CLI-resistant S. pyogenes D2TY in the presence of 1 microg/ml CLI, the amounts of Slo, Nga, and Ska and the levels of slo, nga, and ska mRNA in the supernatant were analyzed by Northern blotting and Western blotting, respectively. The results of both assays showed that the production of Slo, Nga, and Ska was higher with CLI treatment than without CLI treatment. We evaluated the role of the sensor kinase CovS, which is involved in the two-component system of S. pyogenes, in the CLI-induced production of these three exoproteins. Northern blotting analysis revealed that CLI induced the expression of covS mRNA in wild-type strain D2TY. Furthermore, both Northern blotting and Western blotting analyses showed that CLI decreased the levels of expression of Slo, Nga, and Ska in isogenic covS mutant D2TYcovS. These results suggest that CLI increases the production of three virulent exoproteins in CLI-resistant S. pyogenes strains via the action of CovS.

Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes.

We investigated culture supernatant proteins from the M1 serotype of Streptococcus pyogenes by two-dimensional gel electrophoresis and peptide mass mapping analysis, and characterized the single protein spots. Among them, we analysed the Spy0747 protein. This protein is homologous to the SsnA protein, a cell-wall-located DNase expressed in Streptococcus suis serotype 2. We designated the Spy0747 protein as SpnA. SpnA protein was also detected in the insoluble fraction of whole-cell lysates using shotgun proteomic analysis, suggesting that SpnA is also located in the cell wall. SpnA was expressed as a glutathione S-transferase-fusion protein in Escherichia coli. We confirmed that the recombinant protein had DNase activity that was dependent on Ca(2+) and Mg(2+), like SsnA. Blood bactericidal assays and mouse infection model experiments showed that the spnA knockout strain was less virulent than the parental strain, thus suggesting that SpnA could play an important role in virulence. Using PCR, we found that the spnA gene was present in all clinical S. pyogenes strains we examined. Our results, together with a previous report identifying Spy0747 as a surface-associated protein, suggest that SpnA is an important cell-wall-located DNase that is generally produced in S. pyogenes and is involved in virulence.

NADase as a target molecule of in vivo suppression of the toxicity in the invasive M-1 group A Streptococcal isolates.

BACKGROUND: NAD-glycohydrolase (NADase) secreted by M-1 group A streptococcal (GAS) isolates are suspected as one of the virulence factors to cause severe invasive disease including streptococcal toxic shock-like syndrome (STSS). M-1 GAS strains were divided into three groups based on NADase activity: high activity, low activity and no activity in our previous report. RESULTS: The representative high activity isolates taken from STSS patients showed higher virulence compared with isolates from the low activity group, when used to infect mice. The knockout mutant of the nga gene, which encodes NADase also showed reduced virulence in a mouse infection study. The cloned nga gene was able to significantly complement the lost virulence. In addition, the solution containing purified recombinant IFS, which is an inhibitor of NADase, partially rescued mice infected with S. pyogenes. CONCLUSIONS: These results indicate that NADase is important for the virulence of S. pyogenes in vivo and is the potential target to suppress the virulence.

Characterization of Streptococcus pyogenes isolated from balanoposthitis patients presumably transmitted by penile-oral sexual intercourse.

Streptococcus pyogenes is indigenous to the human pharynx and causes acute pharyngitis. Balanoposthitis is an inflammatory disease of the glans and the foreskin. However, balanoposthitis caused by S. pyogenes is not widely recognized as a sexually transmitted disease. In addition, bacteriological features of the isolates causing balanoposthitis are unclear. The four S. pyogenes strains isolated from adult balanoposthitis were examined. We performed emm typing, T antigen typing, RAPD assay, PCR assay for the streptococcal pyrogenic exotoxin-related genes and antibiotic-resistant genes, and antibiotic susceptibility assay. All four strains were suspected to be transmitted by penile-oral sexual intercourse, were found to be different by genetic analysis, and also harbored some antibiotic-resistant factors. We propose that S. pyogenes should be considered as a causative agent of sexually transmitted disease. The drug resistant S. pyogenes must be taken into account when balanoposthitis patients are treated with antibiotic.

Complete Genome Sequence of emm1 Streptococcus pyogenes 10-85, a Strain Isolated from a Patient with Streptococcal Toxic Shock Syndrome in Japan.

Here, we announce the complete genome sequence of Streptococcus pyogenes strain 10-85 (type emm1), isolated from a patient with streptococcal toxic shock syndrome (STSS). The strain lacks the genomic regions encoding SalR-SalK, a two-component regulatory system, and the adjacent type I restriction modification system.

Homologous role of CovRS two-component regulatory system in NAD+ -glycohydrolase activity in Streptococcus dysgalactiae subsp. equisimilis as in Streptococcus pyogenes.

Streptococcal toxic shock syndrome (STSS) is primarily caused by Streptococcus pyogenes, but it may also be caused by Streptococcus dysgalactiae subsp. equisimilis (SDSE). The analyses of S. pyogenes have revealed the important roles of NAD+ -glycohydrolase (Nga) and CovR/CovS, a two-component regulatory system. We examined these factors in SDSE by analyzing mainly two isogenic SDSE strains (12-10-1 and 12-10-3) from the blood of a patient with STSS. The Nga activities were measured and the nucleotide sequences of covR and covS genes were determined. We detected one nucleotide difference between the covR gene of 12-10-1 and that of 12-10-3, and the Nga activity of 12-10-1 was approximately 6.8-fold more than that of 12-10-3. The introduction of covR of 12-10-3 into 12-10-1 significantly reduced the Nga activity, but the introduction of 12-10-1 covR into itself had only a little effect. In addition, the knockout of covR or covS of 12-10-3 remarkably increased the Nga activity. We are the first to report that strains with wild-type and mutated covR were isolated simultaneously from an SDSE STSS patient and that the CovR/CovS two-component regulatory system is involved in the Nga activity in SDSE as well as in S. pyogenes.

Functional Predominance of msr(D), Which Is More Effective as mef(A)-Associated Than mef(E)-Associated, Over mef(A)/mef(E) in Macrolide Resistance in Streptococcus pyogenes.

Although mef(A) and its subclass mef(E) genes have long been considered to play a central role in macrolide efflux-based resistance, we have previously demonstrated that the msr(D) gene located immediately downstream of the mef(A) gene plays a predominant role in Streptococcus pyogenes macrolide resistance. The mef(A) and mef(E) genes are carried by different genetic elements and the resistance associated with mef(A) was reported to be higher than that associated with mef(E); therefore, we further investigated the functional relevance of mef(A)/mef(E) and its associated msr(D). We established additional mef(A)-, mef(E)-, and their associated msr(D)-knockout strains and confirmed the predominance of msr(D) over mef(A)/mef(E). In addition, we performed experiments introducing mef(A), mef(E), and their associated msr(D) genes into mef(A)/mef(E)-msr(D) double-knockout and mef(A)/mef(E) negative strains. Neither mef(A) nor mef(E) genes had effects on erythromycin resistance. However, both associated msr(D) showed significant effects, and the mef(A)-associated msr(D) exhibited more effect than the mef(E)-associated one. These results suggest that an overall functional predominance of msr(D) over mef(A)/mef(E) is conceivable in efflux-based macrolide resistance in at least some S. pyogenes strains. Furthermore, the higher resistance of mef(A) system over mef(E) system could be derived at least in part from functional differences of mef(A)- and mef(E)-associated msr(D).

Characterisation of clinically isolated Streptococcus pyogenes from balanoposthitis patients, with special emphasis on emm89 isolates.

PURPOSE: Streptococcus pyogenes causes a variety of diseases, such as pharyngitis and toxic shock syndrome. In addition, this bacterium is a causative agent of balanoposthitis. To reveal the bacteriological characteristics of the isolates from balanoposthitis patients, we analysed 47 isolates. In addition, novel clade genotype emm89 S. pyogenes isolates have been reported to be spreading worldwide recently. Hence, we further analysed eight emm89 isolates. METHODOLOGY: A drug susceptibility experiment was performed and emm types were determined. More detailed experiments, such as PCR analysis for the presence of virulence-associated genes and MLST analysis, were performed especially using emm89 isolates. RESULTS: All isolates were sensitive to ampicillin, but 34 % of the isolates were resistant to at least one antibiotic. The emm types of the isolates varied, with emm89 and emm11 being the most prevalent, but the emm1 type was not detected. The analysis of emm89 isolates revealed that drug susceptibilities varied. All isolates were negative for the hasABC gene and produced active NADase that are characteristics of novel clade genotype emm89 S. pyogenes. MLST analysis demonstrated that six isolates were of the ST101 type, the most predominant type reported thus far, but two isolates were of the ST646 type. According to the PCR analysis used to determine the presence of streptococcal pyrogenic exotoxin-related genes, the six ST101 isolates were further classified into four groups. CONCLUSION: These results suggest that balanoposthitis is caused by a variety of types of S. pyogenes, with novel clade genotype emm89 isolates playing a role in balanoposthitis infections in Japan.

Female GADD34 mice develop age-related inflammation and hepatocellular carcinoma.

AIM: To analyze the impact of sex on GADD34 function, we studied the aging of female GADD34-deficient mice and compared them with male GADD34-deficient mice. METHODS: We used GADD34-deficient mice on a C57BL/6 background. These mice were fed a normal diet throughout their life. Alternatively, they were fed a high-fat diet at 3 months-of-age. Liver tissues taken from mice were analyzed by hematoxylin-eosin staining and immunohistochemical methods. Fresh liver cells were analyzed by flow cytometry. RESULTS: We found that female GADD34-deficient mice did not develop obesity or fatty livers. However, female GADD34-deficient mice had infiltrations of myeloid cells in the liver, followed by liver atrophy. Many female GADD34-deficient mice developed hepatocellular carcinoma, whereas female wild-type (WT) mice did not show hepatocellular carcinoma during aging. Female GADD34-deficient mice and female WT mice developed the same percentages of lymphoma. Although a high-fat diet induced a higher level of steatosis in young male GADD34-deficient mice compared with WT mice, a high-fat diet induced the same level of steatosis in young female GADD34-deficient mice compared with WT mice. However, GADD34-deficient female young mice had a higher level of infiltration of myeloid cells and myofibroblasts than WT mice. CONCLUSIONS: In contrast to male GADD34-deficient mice, female GADD34-deficient mice did not show obesity as they aged. However, similar to the males, they developed inflammation followed by hepatocellular carcinoma. Geriatr Gerontol Int 2017; 17: 2593-2601.

Relevance of spontaneous fabT mutations to a streptococcal toxic shock syndrome to non-streptococcal toxic shock syndrome transition in the novel-type Streptococcus pyogenes isolates that lost a salRK.

Streptococcus pyogenes is a causative agent of streptococcal toxic shock syndrome (STSS). Mutations in covR/S or rgg, negative regulators, can reportedly modulate the severity of infection in this pathogen. Recently, we showed that the regions encoding the SalR-SalK, a two-component regulatory system, were deleted in some emm 1-type isolates (named as 'novel-type'). In this study, the two novel 'STSS' isolates 10-85stss and 11-171stss were more virulent than the two novel 'non-STSS' isolates 11O-2non and 11T-3non when examined using a mouse model of invasive infection. Genome-sequencing experiments using the three strains 10-85stss , 11-171stss , and 11O-2non detected only one single nucleotide polymorphism that causes a non-synonymous mutation in fabT encoding a transcriptional regulator in strain 11O-2non . Loss of fabT reduced the high level of virulence observed in the STSS isolates to that in the non-STSS isolates, and introduction of an intact fabT compensated the lower virulence of 11O-2non , suggesting that the mutation in fabT, but not in covR/S or rgg, is involved in the differential virulence among the novel-type clinical isolates. This type of non-synonymous fabT mutation was also identified in 12 non-STSS isolates (including 11O-2non and 11T-3non ), and most of those 12 isolates showed impaired FabT function.

The YvqE two-component system controls biofilm formation and acid production in Streptococcus pyogenes.

In Streptococcus pyogenes, proteins involved in determining virulence are controlled by stand-alone response regulators and by two-component regulatory systems. Previous studies reported that, compared to the parental strain, the yvqE sensor knockout strain showed significantly reduced growth and lower virulence. To determine the function of YvqE, we performed biofilm analysis and pH assays on yvqE mutants, and site-directed mutagenesis of YvqE. The yvqE deletion mutant showed a slower acid production rate, indicating that YvqE regulates acid production from sugar fermentation. The mutant strain, in which the Asp(26) residue in YvqE was replaced with Asn, affected biofilm formation, suggesting that this amino acid senses hydrogen ions produced by fermentative sugar metabolism. Signals received by YvqE were directly or indirectly responsible for inducing pilus expression. This study shows that at low environmental pH, biofilm formation in S. pyogenes is mediated by YvqE and suggests that regulation of pilus expression by environmental acidification could be directly under the control of YvqE.

Analysis of the roles of NrdR and DnaB from Streptococcus pyogenes in response to host defense.

Toxic shock syndrome caused by Streptococcus pyogenes (S. pyogenes) is a re-emerging infectious disease. Many virulence-associated proteins play important roles in its pathogenesis and the production of these proteins is controlled by many regulatory factors. CovS is one of the most important two-component sensor proteins in S. pyogenes, and it has been analyzed extensively. Our recent analyses revealed the existence of a transposon between covS and nrdR in several strains, and we speculated that this insertion has some importance. Hence, we examined the significances of the NrdR stand-alone regulator and DnaB, which is encoded by the gene located immediately downstream of nrdR in S. pyogenes infection. We established an nrdR-only knockout strain, and both nrdR and partial dnaB knockout strain. These established knockout strains exhibited a deteriorated response to H2 O2 exposure. nrdR and partial dnaB knockout strain was more easily killed by human polynuclear blood cells, but the nrdR-only knockout strain had no significant difference compared to wild type in contrast to the combined knockout strain. In addition, the mouse infection model experiment illustrated that nrdR and partial dnaB knockout strain, but not the nrdR-only knockout strain, was less virulent compared with the parental strain. These results suggest that DnaB is involved in response to host defense.