Group A Streptococcus Strains Research Articles

Serological Evidence of Immune Priming by Group A Streptococci in Patients with Acute Rheumatic Fever.

Acute rheumatic fever (ARF) is an autoimmune response to Group A Streptococcus (GAS) infection. Repeated GAS exposures are proposed to ‘prime’ the immune system for autoimmunity. This notion of immune-priming by multiple GAS infections was first postulated in the 1960s, but direct experimental evidence to support the hypothesis has been lacking. Here, we present novel methodology, based on antibody responses to GAS T-antigens, that enables previous GAS exposures to be mapped in patient sera. T-antigens are surface expressed, type specific antigens and GAS strains fall into 18 major clades or T-types. A panel of recombinant T-antigens was generated and immunoassays were performed in parallel with serum depletion experiments allowing type-specific T-antigen antibodies to be distinguished from cross-reactive antibodies. At least two distinct GAS exposures were detected in each of the ARF sera tested. Furthermore, no two sera had the same T-antigen reactivity profile suggesting that each patient was exposed to a unique series of GAS T-types prior to developing ARF. The methods have provided much-needed experimental evidence to substantiate the immune-priming hypothesis, and will facilitate further serological profiling studies that explore the multifaceted interactions between GAS and the host.

Novel genomic rearrangements mediated by multiple genetic elements in Streptococcus pyogenes M23ND confer potential for evolutionary persistence.

Symmetric genomic rearrangements around replication axes in genomes are commonly observed in prokaryotic genomes, including Group A Streptococcus (GAS). However, asymmetric rearrangements are rare. Our previous studies showed that the hypervirulent invasive GAS strain, M23ND, containing an inactivated transcriptional regulator system, covRS, exhibits unique extensive asymmetric rearrangements, which reconstructed a genomic structure distinct from other GAS genomes. In the current investigation, we identified the rearrangement events and examined the genetic consequences and evolutionary implications underlying the rearrangements. By comparison with a close phylogenetic relative, M18-MGAS8232, we propose a molecular model wherein a series of asymmetric rearrangements have occurred in M23ND, involving translocations, inversions and integrations mediated by multiple factors, viz., rRNA-comX (factor for late competence), transposons and phage-encoded gene segments. Assessments of the cumulative gene orientations and GC skews reveal that the asymmetric genomic rearrangements did not affect the general genomic integrity of the organism. However, functional distributions reveal re-clustering of a broad set of CovRS-regulated actively transcribed genes, including virulence factors and metabolic genes, to the same leading strand, with high confidence (p-value ~10-10). The re-clustering of the genes suggests a potential selection advantage for the spatial proximity to the transcription complexes, which may contain the global transcriptional regulator, CovRS, and other RNA polymerases. Their proximities allow for efficient transcription of the genes required for growth, virulence and persistence. A new paradigm of survival strategies of GAS strains is provided through multiple genomic rearrangements, while, at the same time, maintaining genomic integrity.

Combinatorial Synthetic Peptide Vaccine Strategy Protects against Hypervirulent CovR/S Mutant Streptococci.

Cluster of virulence responder/sensor (CovR/S) mutant group A streptococci (GAS) are serious human pathogens of multiple M protein strains that upregulate expression of virulence factors, including the IL-8 proteaseStreptococcus pyogenescell envelope proteinase (SpyCEP), thus blunting neutrophil-mediated killing and enabling ingress of bacteria from a superficial wound to deep tissue. We previously showed that a combination vaccine incorporating J8-DT (conserved peptide vaccine from the M protein) and a recombinant SpyCEP fragment protects against CovR/S mutants. To enhance the vaccine's safety profile, we identified a minimal epitope (S2) that was the target for anti-SpyCEP Abs that could protect IL-8 from SpyCEP-mediated proteolysis. Abs from healthy humans and from mice experimentally infected with GAS also recognized S2, albeit at low titers. Native SpyCEP may be poorly immunogenic (cryptic or subdominant), and it would be to the organism's advantage if the host did not induce a strong Ab response against it. However, S2 conjugated to diphtheria toxoid is highly immunogenic and induces Abs that recognize and neutralize SpyCEP. Hence, we describe a two-component peptide vaccine that induces Abs (anti-S2) that protect IL-8 from proteolysis and other Abs (anti-J8) that cause strain-independent killing in the presence of neutrophils. We show that either component alone is ineffectual in preventing skin infection and bacteremia due to CovR/S mutants but that the combination induces complete protection. This protection correlated with a significant influx of neutrophils to the infection site. The data strongly suggest that the lack of natural immunity to hypervirulent GAS strains in humans could be rectified by this combination vaccine.

Group A Streptococcus Persists in the Lysosomes of Phagocytic Cells

Group A Streptococcus (GAS) is a gram‐positive bacterium that is responsible for invasive diseases such as necrotizing fasciitis and streptococcal toxic shock‐like syndrome. Normally, phagocytic cells of the innate immune system such as macrophages rid the body of pathogens by ingestion and digestion of pathogenic microbes. Pathogens are internalized into structures called phagosomes, which then fuse with lysosomes, facilitating the degradation of the pathogen. Acidification of the phagolysosome/lysosome compartment is an important part of the process to activate the proteolytic enzymes of the lysosome. To visualize GAS as it is trafficked through this pathway, we transformed bacteria with a pH‐sensitive green fluorescent protein, mWASABI. Continued mWASABI fluorescence and persistence of GAS within the lysosomal compartment suggests that GAS survives phagolysosomal degradation by preventing acidification of the compartment. We verified that the mWASABI protein expressed by GAS is appropriately quenched in low pH environments and that GAS is not acid‐tolerant. We hypothesize that streptolysin O (SLO), a GAS secreted protein capable of creating pores, contributes to GAS prevention of acidification. To study this, a mutant strain of GAS lacking the SLO gene (ΔSLO) was transformed to express mWASABI. Surprisingly, ΔSLO mutants also persisted in the lysosomal compartment and were equally fluorescent to wild type bacteria. We are currently exploring whether overexpression of mWASABI may be masking the quenching of the pH‐sensitive probe. We are also exploring whether other pore‐forming toxins contribute to preventing lysosomal acidification. Determining the mechanism for how GAS prevents lysosomal acidification will be important in the development of therapeutics aimed at enhancing the ability of phagocytic cells to destroy GAS.

Flesh-eating Streptococcus pyogenes triggers the expression of receptor activator of nuclear factor-κB ligand.

Human CD46 is a receptor for the M protein of group A streptococcus (GAS). The emm1 GAS strain GAS472 was isolated from a patient suffering from streptococcal toxic shock-like syndrome. Human CD46-expressing transgenic (Tg) mice developed necrotizing fasciitis associated with osteoclast-mediated progressive and severe bone destruction in the hind paws 3 days after subcutaneous infection with 5 × 10(5) colony-forming units of GAS472. GAS472 infection induced expression of the receptor activator of nuclear factor-κB ligand (RANKL) while concomitantly reducing osteoprotegerin expression in the hind limb bones of CD46 Tg mice. Micro-computed tomography analysis of the bones suggested that GAS472 infection induced local bone erosion and systemic bone loss in CD46 Tg mice. Because treatment with monoclonal antibodies (mAbs) against mouse CD4(+) and CD8(+) T lymphocytes did not inhibit osteoclastogenesis, T lymphocyte-derived RANKL was not considered a major contributor to massive bone loss during GAS472 infection. However, immunohistochemical analysis of the hind limb bones showed that GAS472 infection stimulated RANKL production in various bone marrow cells, including fibroblast-like cells. Treatment with a mAb against mouse RANKL significantly inhibited osteoclast formation and bone resorption. These data suggest that increased expression of RANKL in heterogeneous bone marrow cells provoked bone destruction during GAS infection.

Virulence factors of Streptococcus pyogenes strains from women in peri-labor with invasive infections.

Invasive group A streptococcal (GAS) infections constitute an important epidemiological problem. Many cases occur in women during the postnatal period. The objective of this study was to evaluate the presence of the genes responsible for production of iron-chelating protein (perR) and superantigens (speA, speB, speC, speF, speG, speH, speI, speJ, speK, speL, speM, smeZ, and ssa) in S. pyogenes strains isolated from invasive infections in women after delivery. Furthermore, this study sought to verify whether S. pyogenes strains show special phenotypic and genotypic (sla, spy1325) characteristics that may play a decisive role in adherence to the genital tract epithelium. Moreover, the emm-types and antibiotic susceptibility were determined. We tested 30 invasive S. pyogenes strains isolated from postpartum invasive infection and 37 GAS control strains isolated from the genital tracts of asymptomatic multiparous women. The majority of the tested strains were shown to express two types of emm genes (1 and 28), though emm −12, −28, −75 and −89 were uniquely expressed in the group of strains isolated from invasive infections. A significantly higher prevalence of perR in the strains from puerperal fever was shown. Significant differences were also found between the two groups with respect to the incidence of the genes related to adherence; GAS strains originating from women with sepsis/puerperal fever showed presence of these genes less frequently than those of the control group. Although differences in frequencies of the gene coding for various superantigens were noted between the compared groups of GAS strains, they were not significant.

Validation of an automated colony counting system for group A Streptococcus.

BackgroundThe practice of counting bacterial colony forming units on agar plates has long been used as a method to estimate the concentration of live bacteria in culture. However, due to the laborious and potentially error prone nature of this measurement technique, an alternative method is desirable. Recent technologic advancements have facilitated the development of automated colony counting systems, which reduce errors introduced during the manual counting process and recording of information. An additional benefit is the significant reduction in time taken to analyse colony counting data. Whilst automated counting procedures have been validated for a number of microorganisms, the process has not been successful for all bacteria due to the requirement for a relatively high contrast between bacterial colonies and growth medium. The purpose of this study was to validate an automated counting system for use with group A Streptococcus (GAS).ResultsMethods: Twenty-one different GAS strains, representative of major emm-types, were selected for assessment. In order to introduce the required contrast for automated counting, 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) dye was added to Todd–Hewitt broth with yeast extract (THY) agar. Growth on THY agar with TTC was compared with growth on blood agar and THY agar to ensure the dye was not detrimental to bacterial growth. Automated colony counts using a ProtoCOL 3 instrument were compared with manual counting to confirm accuracy over the stages of the growth cycle (latent, mid-log and stationary phases) and in a number of different assays. The average percentage differences between plating and counting methods were analysed using the Bland–Altman method.ConclusionsResults: A percentage difference of ±10 % was determined as the cut-off for a critical difference between plating and counting methods. All strains measured had an average difference of less than 10 % when plated on THY agar with TTC. This consistency was also observed over all phases of the growth cycle and when plated in blood following bactericidal assays. Agreement between these methods suggest the use of an automated colony counting technique for GAS will significantly reduce time spent counting bacteria to enable a more efficient and accurate measurement of bacteria concentration in culture.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-1875-z) contains supplementary material, which is available to authorized users.

Accelerating the development of a group A Streptococcus vaccine: an urgent public health need.

Group A Streptococcus (GAS) infections cause substantial worldwide morbidity and mortality, mostly associated with suppurative complications such as pharyngitis, impetigo, and non-suppurative immune syndromes such as acute rheumatic fever, rheumatic heart disease, and acute post-streptococcal glomerulonephritis. Deaths occur mostly in children, adolescents, and young adults in particular pregnant women in low- and middle-income countries. GAS strains are highly variable, and a GAS vaccine would need to overcome the issue of multiple strains. Several approaches have been used multivalent vaccines using N-terminal polypeptides of different M protein; conserved M protein vaccines with antigens from the conserved C-repeat portion of the M protein; incorporation selected T- and B-cell epitopes from the C-repeat region in a synthetic polypeptide or shorter single minimal B-cell epitopes from this same region; and non-M protein approaches utilizing highly conserved motives of streptococcal C5a peptidase, GAS carbohydrate and streptococcal fibronectin-binding proteins. A GAS vaccine represents urgent need for this neglected disease and should therefore deserve the greatest attention of international organizations, donors, and vaccine manufacturers.

The distribution of the emm types and antimicrobial susceptibility to group A streptococcus in children of Beijing, 2014

Objective To investigate the distribution of the emm types and antimicrobial susceptibility to group A streptococcus(GAS) in Beijing. Methods A total of 371 strains of GAS were isolated from the oropharyngeal swabs which were collected from 36 hospitals in Beijing from May to July in 2014.The emm types were performed by PCR and sequencing.The minimum inhibitory concentrations(MIC) in 10 kinds of antibiotics(Penicillin, Ampicillin, Erythromycin, Clinamycin, Tetracycline, Levofloxacin, Tigecycline, Vancomycin, Linezolid and Streptogramin ) were detected by VITEK-2 compact with GPS-67 test kit.Antimicrobial susceptibility was evaluated based on M100-S22 criteria and susceptible rate, intermediary rate, and drug resistance rate was analyzed. Results Five emm types and 15 subtypes were identified, and emm1 type was 54.7%(203/371 strains), emm 12 type was 44.2%(164/371 strains) and other emm types were 1.1%(4/37 strains). And 207 strains of GAS were selected for antimicrobial susceptibility test.All of 207 isolates were sensitive to Penicillin, Ampicillin, Levofloxacin, Tigecycline, Vancomycin, Linezolid and Streptogramin.The percentages of the strains that were of resistance to Clindamycin, Erythromycin and Tetracycline were 98.5%(204/207 strains), 98.1%(203/207 strains) and 92.8%(192/207 strains), respectively.There were no statistically significant difference in the rates of the strains that were resistant to Erythromycin, Lindamycin and Tetracycline among different districts, ages and clinical diagnosis(P>0.05). Conclusions The emm types of all strains were distributed intensively in Beijing in 2014, and emm1, emm12 constituted the main types.The strains of GAS are sensitive to β-lactams and quinolones antibiotics and highly resistant to the macrolide antibiotics and resistant higher to Clindamycin and Erythromycin, which suggests a reasonably use of antibiotics in hospitals nowadays. Key words: Group A streptococcus; emm gene; Antimicrobial susceptibility; Child

Molecular Epidemiology of Group A Streptococcus Infections in Cambodian Children, 2007-2012.

To the Editors: Group A Streptococcus (GAS) is responsible for significant morbidity and mortality globally. Two strong vaccine candidates are currently under evaluation: a 30-valent type–specific M protein–based vaccine and a vaccine targeting the conserved J8 region of M protein.1 The 30-valent vaccine covers the most frequent serotypes circulating in high-income countries, but coverage may be suboptimal in low-income settings with greater GAS emm-type diversity.2 Sixty-eight allelic variants have been described for J8, and the relation between allelic diversity and vaccine efficacy is unclear.1 Limited epidemiologic data are available from many regions making vaccine coverage estimates imprecise. There are no previous data for low-income countries in South East Asia.2 The clinical microbiology database at Angkor Hospital for Children, a nongovernmental pediatric hospital serving the population of northern Cambodia, was searched to identify clinical GAS isolates cultured between January 1, 2007 and December 31, 2012. These isolates underwent molecular emm-typing. emm-clusters, and the J8 vaccine antigen content, were deduced from the emm-typing result as previously described.3 The 30-valent vaccine coverage was estimated using currently available cross-opsonization data.3,4 Strain diversity was assessed by Simpson Reciprocal Index. One hundred fifty GAS isolates from 149 patients were characterized. The median patient age was 3.8 years (range: 0–18.6). One hundred eighteen (78.6%) isolates were from skin and soft tissue infections, 16 (10.7%) from bloodstream infections, 7 (4.7%) from bone/joint infections, 7 (4.7%) from pharyngitis and 2 (1.3%) from infections at other sites. Fifty emm-types were identified from 13 emm-clusters and 2 isolates were considered nontypeable (see Table, Supplemental Digital Content, https://links.lww.com/INF/C234). No novel emm-types were identified. The Simpson Reciprocal Index was 28.5 (95% confidence interval: 23.1–37.3) indicating considerable diversity, similar to that seen in other low-income countries.2 Potential coverage of the J8 vaccine was predicted to be excellent with 43 (28.7%) and 104 (69.3%) isolates predicted to have the J8 and J8.1 allele, respectively. Therefore, J8 vaccine coverage could be expected to be 98.0% (95% confidence interval: 94.2%–99.6%). Fifty isolates (33.3%) were of emm-types covered by the 30-valent vaccine, and an additional 42 isolates (28.0%) have been shown to be potentially covered by the vaccine as a result of cross-opsonization. Therefore, the potential coverage could be expected to be 61.3% (95% confidence interval: 53.0%–69.2%) but may be higher because 26.0% of the isolates belong to 16 emm-types, which have not yet been examined for evidence of cross-opsonization. Comparison with the only other available regional data set revealed considerably greater emm-type diversity in the Cambodian isolates compared with those isolated in Thailand between 1985 and 2004.5 Fifty-nine emm-types from 13 emm-clusters were represented in the combined data set. Only 8 of the 13 emm-clusters were found in Thailand. There were 10 shared emm-types, comprising 64.2% of the Thailand isolates but only 26.4% of the Cambodia isolates. Our study had several limitations. Pharyngeal isolates were not well represented because of clinician sampling practices and also there was an absence of adult sampling in the data set. Also, the population studied may not be representative of Cambodia as a whole. Finally, comparisons between the current data and the Thailand data set are limited by the differences between the study populations and nonoverlapping study periods. Overall, these data indicate a high diversity of circulating GAS strains in Cambodia, the potential high coverage of the J8 vaccine candidate and the need for complementary studies to assess the potential coverage of the 30-valent vaccine candidate. These results highlight the need for robust regional and country-level data for vaccine planning purposes. Paul Turner, MB BS, PhD, FRCPCH, FRCPath Cambodia-Oxford Medical Research Unit Angkor Hospital for Children, Siem Reap, Cambodia, Centre for Tropical Medicine and Global Health Nuffield Department of Medicine University of Oxford Oxford, United Kingdom Pises Ngeth, MD Angkor Hospital for Children Siem Reap, Cambodia Claudia Turner, MB BS, PhD, FRCPCH Cambodia-Oxford Medical Research Unit Angkor Hospital for Children, Siem Reap, Cambodia Centre for Tropical Medicine and Global Health Nuffield Department of Medicine University of Oxford Oxford, United Kingdom Sena Sao Angkor Hospital for Children Siem Reap, Cambodia Nicholas P. J. Day, MD, FRCP Centre for Tropical Medicine and Global Health Nuffield Department of Medicine University of Oxford Oxford, United Kingdom Mahidol-Oxford Tropical Medicine Research Unit Faculty of Tropical Medicine Mahidol University Bangkok, Thailand Ciara Baker, MSc Group A Streptococcal Research Group Murdoch Childrens Research Institute Melbourne, Australia Andrew C. Steer, MB BS, PhD, FRACP Pierre R. Smeesters, MD, PhD Group A Streptococcal Research Group Murdoch Childrens Research Institute Centre for International Child Health University of Melbourne Melbourne, Australia

Highly virulent M1 Streptococcus pyogenes isolates resistant to clindamycin

ContextEmm1-type group A Streptococcus (GAS), or Streptococcus pyogenes, is mostly responsible for invasive infections such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The recommended treatment of severe invasive GAS infections is a combination of clindamycin and penicillin. Until 2012, almost all emm1 isolates were susceptible to clindamycin. ObjectivesWe aimed to identify the phenotypic and genotypic characteristics of emm1 GAS clone resistant to clindamycin. MethodsGAS strains were characterized by emm sequence typing, detection of genes encoding pyrogenic exotoxins or superantigens. Cluster analysis was performed by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Antibiotic susceptibility was assessed using disk diffusion and resistance genes were detected by PCR. ResultsA total of 1321 GAS invasive isolates were analyzed between January 2011 and December 2012. The overall number of invasive isolates resistant to clindamycin was 52 (3.9%); seven of them were emm1 isolates. All isolates had the same genomic markers: macrolide resistance due to the presence of the erm(B) gene, emm subtype 1.0, the same toxin or superantigen profile, PFGE pattern and sequence type. ConclusionThis is the first description of highly virulent GAS emm1 isolates resistant to clindamycin in France. This article strengthens the need for monitoring the epidemiology of invasive GAS strains as they could lead to changes in treatment guidelines.

A Single Amino Acid Replacement in the Sensor Kinase LiaS Contributes to a Carrier Phenotype in Group A Streptococcus.

Despite the high frequency of asymptomatic carriage of bacterial pathogens, we understand little about the bacterial molecular genetic underpinnings of this phenomenon. To obtain new information about the molecular genetic mechanisms underlying carriage of group A Streptococcus (GAS), we performed whole-genome sequencing of GAS strains recovered from a single individual during acute pharyngitis and subsequent asymptomatic carriage. We discovered that compared to the initial infection isolate, the strain recovered during asymptomatic carriage contained three single nucleotide polymorphisms, one of which was in a highly conserved region of a gene encoding a sensor kinase, liaS, resulting in an arginine-to-glycine amino acid replacement at position 135 of LiaS (LiaS(R135G)). Using gene replacement, we demonstrate that introduction of the carrier allele (liaS(R135G)) into a serotype-matched invasive strain increased mouse nasopharyngeal colonization and adherence to cultured human epithelial cells. The carrier mutation also resulted in a reduced ability to grow in human blood and reduced virulence in a mouse model of necrotizing fasciitis. Repair of the mutation in the GAS carrier strain restored virulence and decreased adherence to cultured human epithelial cells. We also provide evidence that the carrier mutation alters the GAS transcriptome, including altered transcription of GAS virulence genes, providing a potential mechanism for the pleiotropic phenotypic effects. Our data obtained using isogenic strains suggest that the liaS(R135G) mutation in the carrier strain contributes to the transition from disease to asymptomatic carriage and provides new information about this poorly described regulatory system in GAS.

Effects of penicillin and erythromycin on adherence of invasive and noninvasive isolates of Streptococcus pyogenes to laminin

This study investigated the possible relationship between the invasiveness of group A Streptococcus (GAS) strains and their abilities to adhere to laminin and assessed the effects of subinhibitory concentrations of penicillin and erythromycin on the ability of GAS to adhere to laminin. The adherence of noninvasive and highly invasive isolates of GAS to laminin was significantly higher than the adherence displayed by isolates of low invasiveness. Antibiotic treatment caused significant reductions in adherence to laminin in all three groups of strains. Penicillin was more successful in reducing the adherence abilities of the tested GAS strains than erythromycin.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein

Streptokinase (SK), secreted by Group A Streptococcus (GAS), is a single-chain ∼47-kDa protein containing three consecutive primary sequence regions that comprise its α, β, and γ modules. Phylogenetic analyses of the variable β-domain sequences from different GAS strains suggest that SKs can be arranged into two clusters, SK1 and SK2, with a subdivision of SK2 into SK2a and SK2b. SK2b is secreted by skin-tropic Pattern D M-protein strains that also express plasminogen (human Pg (hPg)) binding Group A streptococcal M-protein (PAM) as its major cell surface M-protein. SK2a-expressing strains are associated with nasopharynx tropicity, and many of these strains express human fibrinogen (hFg) binding Pattern A-C M-proteins, e.g. M1. PAM interacts with hPg directly, whereas M1 binds to hPg indirectly via M1-bound hFg. Subsequently, SK is secreted by GAS and activates hPg to plasmin (hPm), thus generating a proteolytic surface on GAS that enhances its dissemination. Due to these different modes of hPg/hPm recognition by GAS, full characterizations of the mechanisms of activation of hPg by SK2a and SK2b and their roles in GAS virulence are important topics. To more fully examine these subjects, isogenic chimeric SK- and M-protein-containing GAS strains were generated, and the virulence of these chimeric strains were analyzed in mice. We show that SK and M-protein alterations influenced the virulence of GAS and were associated with the different natures of hPg activation and hPm binding. These studies demonstrate that GAS virulence can be explained by disparate hPg activation by SK2a and SK2b coupled with the coinherited M-proteins of these strains.

Phosphorylation events in the multiple gene regulator of group A Streptococcus significantly influence global gene expression and virulence.

Whole-genome sequencing analysis of ∼800 strains of group A Streptococcus (GAS) found that the gene encoding the multiple virulence gene regulator of GAS (mga) is highly polymorphic in serotype M59 strains but not in strains of other serotypes. To help understand the molecular mechanism of gene regulation by Mga and its contribution to GAS pathogenesis in serotype M59 GAS, we constructed an isogenic mga mutant strain. Transcriptome studies indicated a significant regulatory influence of Mga and altered metabolic capabilities conferred by Mga-regulated genes. We assessed the phosphorylation status of Mga in GAS cell lysates with Phos-tag gels. The results revealed that Mga is phosphorylated at histidines in vivo. Using phosphomimetic and nonphosphomimetic substitutions at conserved phosphoenolpyruvate:carbohydrate phosphotransferase regulation domain (PRD) histidines of Mga, we demonstrated that phosphorylation-mimicking aspartate replacements at H207 and H273 of PRD-1 and at H327 of PRD-2 are inhibitory to Mga-dependent gene expression. Conversely, non-phosphorylation-mimicking alanine substitutions at H273 and H327 relieved inhibition, and the mutant strains exhibited a wild-type phenotype. The opposing regulatory profiles observed for phosphorylation- and non-phosphorylation-mimicking substitutions at H273 extended to global gene regulation by Mga. Consistent with these observations, the H273D mutant strain attenuated GAS virulence, whereas the H273A strain exhibited a wild-type virulence phenotype in a mouse model of necrotizing fasciitis. Together, our results demonstrate phosphoregulation of Mga and its direct link to virulence in M59 GAS strains. These data also lay a foundation toward understanding how naturally occurring gain-of-function variations in mga, such as H201R, may confer an advantage to the pathogen and contribute to M59 GAS pathogenesis.

Matrix-assisted laser desorption ionization-time of flight and comparative genomic analysis of M-18 group a Streptococcus strains associated with an acute rheumatic fever outbreak in northeast Italy in 2012 and 2013.

Acute rheumatic fever (ARF) is a postsuppurative sequela caused by Streptococcus pyogenes infections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A Streptococcus (GAS) strains belonged to the M-18 serotype, including subtypes emm18.29 and emm18.32. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different S. pyogenes lineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that emm18.29 and emm18.32 GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between emm18.29 and emm18.32 strains that differed from noninvasive emm18.0 strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of emm18.29 and emm18.32 strains. Notably, phage ΦSPBO.1 carried erythrogenic toxin A gene (speA1) in six ARF-related M-18 GAS strains but not in emm18.0 strains. In addition, a phage-encoded hyaluronidase gene (hylP.2) presented different variants among M-18 GAS strains by showing internal deletions located in the α-helical and TSβH regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.

Application of the C3-binding motif of streptococcal pyrogenic exotoxin B to protect mice from invasive group a streptococcal infection.

Group A streptococcus (GAS) is an important human pathogen that produces several extracellular exotoxins to facilitate invasion and infection. Streptococcal pyrogenic exotoxin B (SPE B) has been demonstrated to be an important virulence factor of GAS. Our previous studies indicate that SPE B cleaves complement 3 (C3) and inhibits the activation of complement pathways. In this study, we constructed and expressed recombinant fragments of SPE B to examine the C3-binding site of SPE B. Using enzyme-linked immunosorbent assays and pull-down assays, we found that the C-terminal domain, containing amino-acid residues 345–398, of SPE B was the major binding site of human serum C3. We further identified a major, Ala376-Pro398, and a minor C3-binding motif, Gly346-Gly360, that both mediated the binding of C3 complement. Immunization with the C3-binding motifs protected mice against challenge with a lethal dose of non-invasive M49 strain GAS but not invasive M1 strains. To achieve higher efficiency against invasive M1 GAS infection, a combination of synthetic peptides derived from C-terminal epitope of streptolysin S (SLSpp) and from the major C3-binding motif of SPE B (PP6, Ala376-Pro398) was used to elicit specific immune response to those two important streptococcal exotoxins. Death rates and the severity of skin lesions decreased significantly in PP6/SLSpp-immunized mice that were infected with invasive M1 strains of GAS. These results indicate a combination of the C3-binding motif of SPE B and the protective epitope of SLS could be used as a subunit vaccine against invasive M1 strains group A streptococcal infection.

The change of macrolide resistance rates in group A Streptococcus isolates from children between 2002 and 2013 in Asahikawa city.

This study targeted patients in the Department of Pediatrics, Asahikawa Kosei Hospital, between January 2002 and December 2013. In patients suspected of having hemolytic streptococcal infection, Group A Streptococcus (GAS) strains isolated from a throat swab were examined for antimicrobial susceptibility testing. The MICs were measured by the broth microdilution method. The annual number of GAS strains examined for antimicrobial susceptibility testing ranged from 28 to 65 strains, for a total of 574 strains. Some of the isolates obtained from 2006 to 2009 and from 2011 to 2013 were analyzed to determine their emm types. An erythromycin (EM) resistant strain was not detected until 2004, but one EM-resistant strain appeared in 2005. Subsequently, EM-resistant strains rapidly increased, and 48 of 65 strains (73.8%) examined in 2009 were resistant. In 2010, the number of EM-resistant strains decreased to 12 of 36 strains (33.3%). However, it gradually increased afterwards, and 37 of 60 strains (61.7%) were resistant in 2013. Out of 574 strains examined, 184 exhibited EM-resistance, and the overall resistance rate was 31.9%. Partitioning the 124 strains examined between 2006 and 2008 according to emm types, only emm28 strains, which exhibited a high resistance rate, and emm12 strains demonstrated resistance. For the 142 strains examined between 2011 and 2013, the resistance rate of emm28 strains was similarly high; the resistance of emm12 strains significantly increased, and emm1 strains exhibited a high resistance rate. The number of emm types associated with the resistant strains increased.

Characterization of the Effect of the Histidine Kinase CovS on Response Regulator Phosphorylation in Group A Streptococcus

Two-component gene regulatory systems (TCSs) are a major mechanism by which bacteria respond to environmental stimuli and thus are critical to infectivity. For example, the control of virulence regulator/sensor kinase (CovRS) TCS is central to the virulence of the major human pathogen group A Streptococcus (GAS). Here, we used a combination of quantitative in vivo phosphorylation assays, isoallelic strains that varied by only a single amino acid in CovS, and transcriptome analyses to characterize the impact of CovS on CovR phosphorylation and GAS global gene expression. We discovered that CovS primarily serves to phosphorylate CovR, thereby resulting in the repression of virulence factor-encoding genes. However, a GAS strain selectively deficient in CovS phosphatase activity had a distinct transcriptome relative to that of its parental strain, indicating that both CovS kinase and phosphatase activities influence the CovR phosphorylation status. Surprisingly, compared to a serotype M3 strain, serotype M1 GAS strains had high levels of phosphorylated CovR, low transcript levels of CovR-repressed genes, and strikingly different responses to environmental cues. Moreover, the inactivation of CovS in the serotype M1 background resulted in a greater decrease in phosphorylated CovR levels and a greater increase in the transcript levels of CovR-repressed genes than did CovS inactivation in a serotype M3 strain. These data clarify the influence of CovS on the CovR phosphorylation status and provide insight into why serotype M1 GAS strains have high rates of spontaneous mutations in covS during invasive GAS infection, thus providing a link between TCS molecular function and the epidemiology of deadly bacterial infections.