Group A Streptococcus Serotypes Research Articles

The Streptococcus pyogenes stand-alone regulator RofA exhibits characteristics of a PRD-containing virulence regulator.

Streptococcus pyogenes [group A streptococcus (GAS)] is a human pathogen capable of infecting diverse tissues. To successfully infect these sites, GAS must detect available nutrients and adapt accordingly. The phosphoenolpyruvate transferase system (PTS) mediates carbohydrate uptake and metabolic gene regulation to adapt to the nutritional environment. Regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several PRD-containing stand-alone regulators with regulons encoding both metabolic genes and virulence factors [PRD-containing virulence regulators (PCVRs)]. One is RofA, which regulates the expression of virulence genes in multiple GAS serotypes. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate virulence gene expression. In this study, the RofA regulon of M1T1 strain 5448 was determined using RNA sequencing. Two operons were consistently differentially expressed across growth in the absence of RofA; the pilus operon was downregulated, and the capsule operon was upregulated. This correlated with increased capsule production and decreased adherence to keratinocytes. Purified RofA-His was phosphorylated in vitro by PTS proteins EI and HPr, and phosphorylated RofA-FLAG was detected in vivo when GAS was grown in low-glucose C medium. Phosphorylated RofA was not observed when C medium was supplemented 10-fold with glucose. Mutations of select histidine residues within the putative PRDs contributed to the in vivo phosphorylation of RofA, although phosphorylation of RofA was still observed, suggesting other phosphorylation sites exist in the protein. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar metabolism with virulence regulation.

Conserved molecular chaperone PrsA stimulates protective immunity against group A Streptococcus

Group A Streptococcus (GAS) is a significant human pathogen that poses a global health concern. However, the development of a GAS vaccine has been challenging due to the multitude of diverse M-types and the risk of triggering cross-reactive immune responses. Our previous research has identified a critical role of PrsA1 and PrsA2, surface post-translational molecular chaperone proteins, in maintaining GAS proteome homeostasis and virulence traits. In this study, we aimed to further explore the potential of PrsA1 and PrsA2 as vaccine candidates for preventing GAS infection. We found that PrsA1 and PrsA2 are highly conserved among GAS isolates, demonstrating minimal amino acid variation. Antibodies specifically targeting PrsA1/A2 showed no cross-reactivity with human heart proteins and effectively enhanced neutrophil opsonophagocytic killing of various GAS serotypes. Additionally, passive transfer of PrsA1/A2-specific antibodies conferred protective immunity in infected mice. Compared to alum, immunization with CFA-adjuvanted PrsA1/A2 induced higher levels of Th1-associated IgG isotypes and complement activation and provided approximately 70% protection against invasive GAS challenge. These findings highlight the potential of PrsA1 and PrsA2 as universal vaccine candidates for the development of an effective GAS vaccine.

Mucosal Immunization Has Benefits over Traditional Subcutaneous Immunization with Group A Streptococcus Antigens in a Pilot Study in a Mouse Model.

Group A Streptococcus (GAS) is a major human pathogen for which there is no licensed vaccine. To protect against infection, a strong systemic and mucosal immune response is likely to be necessary to prevent initial colonization and any events that might lead to invasive disease. A broad immune response will be necessary to target the varied GAS serotypes and disease presentations. To this end, we designed a representative panel of recombinant proteins to cover the stages of GAS infection and investigated whether mucosal and systemic immunity could be stimulated by these protein antigens. We immunized mice sublingually, intranasally and subcutaneously, then measured IgG and IgA antibody levels and functional activity through in vitro assays. Our results show that both sublingual and intranasal immunization in the presence of adjuvant induced both systemic IgG and mucosal IgA. Meanwhile, subcutaneous immunization generated only a serum IgG response. The antibodies mediated binding and killing of GAS cells and blocked binding of GAS to HaCaT cells, particularly following intranasal and subcutaneous immunizations. Further, antigen-specific assays revealed that immune sera inhibited cleavage of IL-8 by SpyCEP and IgG by Mac/IdeS. These results demonstrate that mucosal immunization can induce effective systemic and mucosal antibody responses. This finding warrants further investigation and optimization of humoral and cellular responses as a viable alternative to subcutaneous immunization for urgently needed GAS vaccines.

Detoxification of reactive oxygen species by the hyaluronic acid capsule of group A Streptococcus.

The pro-inflammatory cytokine IL-6 regulates antimicrobial responses that are broadly crucial in the defense against infection. Our prior work shows that IL-6 promotes the killing of the M4 serotype group A Streptococcus (GAS) but does not impact the globally disseminated M1T1 serotype associated with invasive infections. Using in vitro and in vivo infection models, we show that IL-6 induces phagocyte reactive oxygen species (ROS) that are responsible for the differential susceptibility of M4 and M1T1 GAS to IL-6-mediated defenses. Clinical isolates naturally deficient in capsule, or M1T1 strains deficient in capsule production, are sensitive to this ROS killing. The GAS capsule is made of hyaluronic acid, an antioxidant that detoxifies ROS and can protect acapsular M4 GAS when added exogenously. During in vitro interactions with macrophages and neutrophils, acapsular GAS can also be rescued with the antioxidant N-acetylcysteine, suggesting this is a major virulence contribution of the capsule. In an intradermal infection model with gp91phox -/- (chronic granulomatous disease [CGD]) mice, phagocyte ROS production had a modest effect on bacterial proliferation and the cytokine response but significantly limited the size of the bacterial lesion in the skin. These data suggest that the capsule broadly provides enhanced resistance to phagocyte ROS but is not essential for invasive infection. Since capsule-deficient strains are observed across several GAS serotypes and are competent for transmission and both mild and invasive infections, additional host or microbe factors may contribute to ROS detoxification during GAS infections.

Different Group A Streptococcus pili lead to varying proinflammatory cytokine responses and virulence.

The human pathogen Streptococcus pyogenes, or Group A Streptococcus (GAS), is associated with a variety of diseases ranging from mild skin and soft tissue infections to invasive diseases and immune sequelae such as rheumatic heart disease. We have recently reported that one of the virulence factors of this pathogen, the pilus, has inflammatory properties and strongly stimulates the innate immune system. Here we used a range of nonpathogenic Lactococcus lactis gain-of-function mutants, each expressing one of the major pilus types of GAS, to compare the immune responses generated by various types of fully assembled pili. In vitro assays indicated variability in the inflammatory response induced by different pili, with the fibronectin-binding, collagen-binding, T antigen (FCT)-1-type pilus from GAS serotype M6/T6 inducing significantly stronger cytokine secretion than other pili. Furthermore, we established that the same trend of pili-mediated immune response could be modeled in Galleria mellonella larvae, which possess a similar innate immune system to vertebrates. Counterintuitively, across the panel of pili types examined in this study, we observed a negative correlation between the intensity of the immune response demonstrated in our experiments and the disease severity observed clinically in the GAS strains associated with each pilus type. This observation suggests that pili-mediated inflammation is more likely to promote bacterial clearance instead of causing disruptive damages that intensify pathogenesis. This also indicates that pili may not be the main contributor to the inflammatory symptoms seen in GAS diseases. Rather, the immune-potentiating properties of the pilus components could potentially be exploited as a vaccine adjuvant.

Attenuation of virulence in multiple serotypes (M1, M3, and M28) of Group A Streptococcus after the loss of secreted esterase.

Group A Streptococcus (GAS) can produce streptococcal secreted esterase (Sse), which inhibits neutrophil recruitment to the site of infection and is crucial for GAS pathogenesis. As an effective esterase, Sse hydrolyzes the sn-2 ester bond of human platelet-activating factor, inactivating it and abolishing its ability to recruit neutrophils. The purpose of this study was to investigate the effects of sse deletion on the virulence of multiple serotypes of GAS. Isogenic strains that lack the sse gene (Δsse) were derived from the parent strains MGAS5005 (serotype M1, CovRS mutant), MGAS2221 (serotype M1, wild-type CovRS), MGAS315 (serotype M3, CovRS mutant) and MGAS6180 (serotype M28, wild-type CovRS) and were used to study the differences in virulence and pathogenicity of GAS serotypes. In a subcutaneous infection model, mice infected with MGAS5005Δsse exhibited higher survival rates but decreased dissemination to the organs compared with mice infected with MGAS5005. When mice were infected with the four Δsse mutants, the MPO activity and IFN-γ, TNF-α, IL-2 and IL-6 levels increased, but the skin lesion sizes decreased. In an intraperitoneal infection model, the absence of Sse significantly reduced the virulence of GAS, leading to increased mouse survival rates and decreased GAS burdens in the organs in most of the challenge experiments. In addition, the numbers of the four Δsse mutants were greatly reduced 60 min after incubation with isolated rat neutrophils. Our results suggest that Sse participates in the pathogenesis of multiple GAS serotypes (MGAS5005, MGAS2221, MGAS315 and MGAS6180), particularly the hypervirulent CovS mutant strains MGAS5005 and MGAS315. These strain differences were positively correlated with the virulence of the serotype.

GMMA as an Alternative Carrier for a Glycoconjugate Vaccine against Group A Streptococcus.

Group A Streptococcus (GAS) causes about 500,000 annual deaths globally, and no vaccines are currently available. The Group A Carbohydrate (GAC), conserved across all GAS serotypes, conjugated to an appropriate carrier protein, represents a promising vaccine candidate. Here, we explored the possibility to use Generalized Modules for Membrane Antigens (GMMA) as an alternative carrier system for GAC, exploiting their intrinsic adjuvant properties. Immunogenicity of GAC-GMMA conjugate was evaluated in different animal species in comparison to GAC-CRM197; and the two conjugates were also compared from a techno-economic point of view. GMMA proved to be a good alternative carrier for GAC, resulting in a higher immune response compared to CRM197 in different mice strains, as verified by ELISA and FACS analyses. Differently from CRM197, GMMA induced significant levels of anti-GAC IgG titers in mice also in the absence of Alhydrogel. In rabbits, a difference in the immune response could not be appreciated; however, antibodies from GAC-GMMA-immunized animals showed higher affinity toward purified GAC antigen compared to those elicited by GAC-CRM197. In addition, the GAC-GMMA production process proved to be more cost-effective, making this conjugate particularly attractive for low- and middle-income countries, where this pathogen has a huge burden.

Immunity to Sda1 Protects against Infection by Sda1+ and Sda1- Serotypes of Group A Streptococcus.

Group A Streptococcus (GAS) causes a variety of diseases globally. The DNases in GAS promote GAS evasion of neutrophil killing by degrading neutrophil extracellular traps (NETs). Sda1 is a prophage-encoded DNase associated with virulent GAS strains. However, protective immunity against Sda1 has not been determined. In this study, we explored the potential of Sda1 as a vaccine candidate. Sda1 was used as a vaccine to immunize mice intranasally. The effect of anti-Sda1 IgG in neutralizing degradation of NETs was determined and the protective role of Sda1 was investigated with intranasal and systemic challenge models. Antigen-specific antibodies were induced in the sera and pharyngeal mucosal site after Sda1 immunization. The anti-Sda1 IgG efficiently prevented degradation of NETs by supernatant samples from different GAS serotypes with or without Sda1. Sda1 immunization promoted clearance of GAS from the nasopharynx independent of GAS serotypes but did not reduce lethality after systemic GAS challenge. Anti-Sda1 antibody can neutralize degradation of NETs by Sda1 and other phage-encoded DNases and decrease GAS colonization at the nasopharynx across serotypes. These results indicate that Sda1 can be a potential vaccine candidate for reduction in GAS reservoir and GAS tonsillitis-associated diseases.

Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation.

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Colonization of the oropharynx is the initial step in Group A Streptococcus (GAS) pharyngeal infection. We have previously reported that the highly virulent M1T1 GAS clone attaches to oral epithelial cells via M1 protein interaction with blood group antigen carbohydrate structures. Here, we have identified that colonization of human oral epithelial cells by GAS serotypes M3 and M12 is mediated by human blood group antigens [ABO(H)] and Lewis (Le) antigen expression. Removal of linkage-specific fucose, galactose, N-acetylgalactosamine, and sialic acid modulated GAS colonization, dependent on host ABO(H) blood group and Le expression profile. Furthermore, N-linked glycans from human salivary glycoproteins, when released and purified, were potent inhibitors of M1, M3, and M12 GAS colonization ex vivo. These data highlight the important role played by human protein glycosylation patterns in GAS attachment to oral epithelial cell surfaces.-De Oliveira, D. M. P., Everest-Dass, A., Hartley-Tassell, L., Day, C. J., Indraratna, A., Brouwer, S., Cleary, A., Kautto, L., Gorman, J., Packer, N. H., Jennings, M. P., Walker, M. J., Sanderson-Smith, M. L. Human glycan expression patterns influence Group A streptococcal colonization of epithelial cells.

Group A streptococcus endocarditis in children: 2 cases and a review of the literature

BackgroundInfective endocarditis (IE) is defined as endocarditis caused by microorganisms (bacteria or fungi) involving either the heart or great vessels. The clinical course of IE can be complicated by cardiac dysfunction and bacterial embolization to virtually any organ. Staphylococcus aureus and viridans group streptococci are the most common causative organisms, whereas group A Streptococcus (GAS) is less common. Although some GAS serotypes have been associated with severe disease, there are few reports of IE associated with GAS serotypes. Here, we report two cases of GAS endocarditis and review the associated literature.Case presentationsPatient 1 was a previously healthy 14-year-old girl who developed bacteremia and disseminated intravascular coagulation secondary to left foot cellulitis. She was administered intravenous antibiotics. Two of three blood cultures grew Streptococcus pyogenes (T6 M6, emm6.104). Three days later, a new systolic ejection murmur was heard and echocardiography showed mitral regurgitation with mitral valve vegetation. Because of the resultant severity of the mitral regurgitation, she underwent mitral valve repair after 10 weeks of antibiotic treatment. Patient 2 was a 17-month old boy who presented with a fever. He had a history of spontaneous closure of a ventricular septal defect (VSD). He was started on intravenous antibiotics for possible bacteremia. Two consecutive blood cultures with an interval of more than 12 h grew S. pyogenes (T4 M4, emm4.0). Five days later, echocardiography showed vegetation on a membranous ventricular septal aneurysm. The patient responded well to antibiotics, and recovered fully with no complications.ConclusionsAlthough both patients developed GAS endocarditis, patient 1 did not have any predisposing conditions for IE, and patient 2 had a only a low-risk predisposing condition, a VSD that had closed spontaneously at five months of age. We found twelve reports in the literature of GAS endocarditis with information on serotypes. All patients in these reports had GAS endocarditis caused by serotypes generally associated with milder infections, but no specific risk trends were identified. A greater accumulation of cases is necessary to more clearly elucidate the association between GAS IE and specific serotypes.

Streptococcal Lancefield polysaccharides are critical cell wall determinants for human Group IIA secreted phospholipase A2 to exert its bactericidal effects.

Human Group IIA secreted phospholipase A2 (hGIIA) is an acute phase protein with bactericidal activity against Gram-positive bacteria. Infection models in hGIIA transgenic mice have suggested the importance of hGIIA as an innate defense mechanism against the human pathogens Group A Streptococcus (GAS) and Group B Streptococcus (GBS). Compared to other Gram-positive bacteria, GAS is remarkably resistant to hGIIA activity. To identify GAS resistance mechanisms, we exposed a highly saturated GAS M1 transposon library to recombinant hGIIA and compared relative mutant abundance with library input through transposon-sequencing (Tn-seq). Based on transposon prevalence in the output library, we identified nine genes, including dltA and lytR, conferring increased hGIIA susceptibility. In addition, seven genes conferred increased hGIIA resistance, which included two genes, gacH and gacI that are located within the Group A Carbohydrate (GAC) gene cluster. Using GAS 5448 wild-type and the isogenic gacI mutant and gacI-complemented strains, we demonstrate that loss of the GAC N-acetylglucosamine (GlcNAc) side chain in the ΔgacI mutant increases hGIIA resistance approximately 10-fold, a phenotype that is conserved across different GAS serotypes. Increased resistance is associated with delayed penetration of hGIIA through the cell wall. Correspondingly, loss of the Lancefield Group B Carbohydrate (GBC) rendered GBS significantly more resistant to hGIIA-mediated killing. This suggests that the streptococcal Lancefield antigens, which are critical determinants for streptococcal physiology and virulence, are required for the bactericidal enzyme hGIIA to exert its bactericidal function.

Immunogenicity and Safety of a Chemically Synthesized Divalent Group A Streptococcal Vaccine.

Background Group A streptococcus (GAS) infections and poststreptococcal sequelae remain a health problem worldwide, which necessitates searching for an effective vaccine, while no licensed GAS vaccine is available. We have developed a divalent peptide vaccine composed of 84 amino acids to cover the main GAS serotypes (M1 and M12 streptococci) in China, and herein, we aimed to evaluate immunogenicity and safety of this vaccine. Methods Mice were immunized with the vaccine. ELISA, indirect bactericidal test, and immunofluorescent assay were used to study immunogenicity. GAS challenge assay was used to test the protective effect. Safety was tested by histopathological analysis. Results Immunized group mice (n=16) developed higher titer antibody after immunization than nonimmunized group mice (n=16) did. This antibody can deposit on the surface of GAS and promote killing of GAS, resulting in 93.1% decrease of M1 GAS and 89.5% of M12 GAS. When challenged with M1 and M12 streptococci, immunized group mice had a higher survival rate (87.5% and 75%) than nonimmunized group mice (37.5% and 25%). No autoimmune reactions were detected on organs of mice. Conclusion The results suggest that this vaccine shows fair immunogenicity and safety, which will lead our research on GAS vaccine into clinical trial.

Modular virus-like particles for sublingual vaccination against group A streptococcus

Infection with Group A streptococcus (GAS)—an oropharyngeal pathogen—leads to mortality and morbidity, primarily among developing countries and indigenous populations in developed countries. The development of safe and affordable GAS vaccines is challenging, due to the presence of various unique GAS serotypes, antigenic variation within the same serotype, and potential auto-immune responses. In the present study, we evaluated the use of a sublingual freeze-dried (FD) formulation based on immunogenic modular virus-like particles (VLPs) carrying the J8 peptide (J8-VLPs) as a potential safe and cost-effective GAS vaccine for inducing protective systemic and mucosal immunity. By using in vivo tracing of the sublingual J8-VLPs, we visualized the draining of J8-VLPs into the submandibular lymph nodes, in parallel with its rapid absorption into the systemic circulation, which support the induction of effective immune responses in both systemic and mucosal compartments. The sublingual administration of J8-VLPs resulted in a high serum IgG antibody level, with a good balance of Th1 and Th2 immune responses. Of note, sublingual vaccination with J8-VLPs elicited high levels of IgA antibody in the saliva. The co-administration of mucosal adjuvant cholera toxin (CT) further enhanced the increase in salivary IgA antibody levels induced by the J8-VLPs formulation. Moreover, the levels of salivary IgA and serum IgG observed following the administration of the CT-adjuvanted FD formulation of J8-VLPs (FD-J8-VLPs) and non-FD formulation of J8-VLPs were comparable. In fact, the saliva isolated from mice immunized with J8-VLPs and FD-J8-VLPs with CT demonstrated opsonizing activity against GAS in vitro. Thus, we observed that the sublingually delivered FD formulation of microbially produced modular VLPs could prevent and control GAS diseases in endemic areas in a cost-effective manner.

Interleukin-17A Contributes to the Control of Streptococcus pyogenes Colonization and Inflammation of the Female Genital Tract.

Postpartum women are at increased risk of developing puerperal sepsis caused by group A Streptococcus (GAS). Specific GAS serotypes, including M1 and M28, are more commonly associated with puerperal sepsis. However, the mechanisms of GAS genital tract infection are not well understood. We utilized a murine genital tract carriage model to demonstrate that M1 and M28 GAS colonization triggers TNF-α, IL-1β, and IL-17A production in the female genital tract. GAS-induced IL-17A significantly influences streptococcal carriage and alters local inflammatory responses in two genetically distinct inbred strains of mice. An absence of IL-17A or the IL-1 receptor was associated with reduced neutrophil recruitment to the site of infection; and clearance of GAS was significantly attenuated in IL-17A−/− mice and Rag1−/− mice (that lack mature lymphocytes) but not in mice deficient for the IL-1 receptor. Together, these findings support a role for IL-17A in contributing to the control of streptococcal mucosal colonization and provide new insight into the inflammatory mediators regulating host-pathogen interactions in the female genital tract.

Group A Streptococcus Prevents Initiation of the Oxidative Burst Response in Immune Cells

Group A Streptococcus (GAS) is a gram‐positive bacterium that produces infections ranging from mild self‐limiting infections to more severe infections such as necrotizing fasciitis and streptococcal toxic shock syndrome. In response to pathogens such as GAS, the host innate immune system produces antimicrobial factors such as reactive oxygen species that target microbes directly via oxidative stress, or indirectly via the initiation of other innate immune responses. Some bacteria, such as Lactococcus lactis and Staphylococcus aureus, contain pigment or catalase to detoxify these oxygen radicals. While GAS lacks these enzymes, it is still able to survive in innate immune cells, indicating that GAS may inhibit the oxidative burst response altogether. To examine this possibility, we infected macrophages with M1 and M49 serotype GAS, Staphylococcus aureus, Lactococcus lactis, and Group B Streptococcus and measured hydrogen peroxide production. Cells infected with either GAS serotypes produced significantly less peroxide compared with cells infected with other bacterial species, indicating that GAS suppresses the oxidative burst response. The GAS pore‐forming toxin streptolysin O (SLO) has similarity to listerolysin O (LLO), which prevents the assembly of the multi‐subunit NADPH oxidase on phagosome membranes. NADPH oxidase assembly is necessary to initiate the oxidative burst response. Peroxide generation was restored when cells were infected with GAS mutant bacteria of both M1 and M49 serotypes lacking SLO. We are currently examining the mechanism by which GAS uses SLO to prevents the oxidative burst response. Activation of the NADPH oxidase p47 subunit was verified Western blot for phosphorylated p47. Assembly of the NADPH oxidase is currently being assessed by cell fractionation of phagosomes and mass spectrometry. Preliminary data indicate that several NADPH oxidase subunits are missing from the phagosome. Thus, similar to LLO, SLO may inhibit the proper assembly of the NADPH oxidase, and prevent the generation of harmful reactive oxygen species.

The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype-specific targets.

Bacterial pathogens commonly show intra-species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T-antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT-types), and (c) the streptokinase-encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype-specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska-2, FCT-2), M2 (ska-1, FCT-6), M6 (ska-2, FCT-1) and M28 (ska-1, FCT-4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT-region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen-expressing mice. Our data are the first to identify and characterize serotype-specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.

Group A streptococcus expresses a trio of surface proteins containing protective epitopes.

Group A streptococci (GAS) (Streptococcus pyogenes) are common causes of infections in humans for which there is no licensed vaccine. Decades of work has focused on the role of the surface M protein in eliciting type-specific protective immunity. Recent studies have identified additional surface proteins of GAS that contain opsonic epitopes. In the present study, we describe a serotype M65 GAS originally isolated during an epidemiologic study in Bamako, Mali, which simultaneously expressed M, M-related protein (Mrp), and streptococcal protective antigen (Spa) on the bacterial surface. The emm, mrp, and spa genes were sequenced from PCR amplicons derived from the M65 chromosome. Rabbit antisera raised against synthetic peptides copying the N-terminal regions of M, Mrp, and Spa were highly specific for each peptide, reacted with the surface of M65 GAS, and promoted bactericidal activity against the organism. A mixture of antisera against all three peptides was most effective in the bactericidal assays. Immunofluorescence microscopy revealed that the M, Mrp, and Spa antisera bound to the bacterial surface in the presence of human plasma proteins and resulted in the deposition of complement. Five additional spa genes were identified in the Mrp-positive GAS serotypes, and their sequences were determined. Our results indicate that there are multiple antigens on the surface of GAS that evoke antibodies that promote bacterial killing. A more complete understanding of the relative contributions of M, Mrp, and Spa in eliciting protective immunity may aid in the development of GAS vaccines with enhanced coverage and efficacy.

The classical lancefield antigen of group a Streptococcus is a virulence determinant with implications for vaccine design.

Group A Streptococcus (GAS) is a leading cause of infection-related mortality in humans. All GAS serotypes express the Lancefield group A carbohydrate (GAC), comprising a polyrhamnose backbone with an immunodominant N-acetylglucosamine (GlcNAc) side chain, which is the basis of rapid diagnostic tests. No biological function has been attributed to this conserved antigen. Here we identify and characterize the GAC biosynthesis genes, gacA through gacL. An isogenic mutant of the glycosyltransferase gacI, which is defective for GlcNAc side-chain addition, is attenuated for virulence in two infection models, in association with increased sensitivity to neutrophil killing, platelet-derived antimicrobials in serum, and the cathelicidin antimicrobial peptide LL-37. Antibodies to GAC lacking the GlcNAc side chain and containing only polyrhamnose promoted opsonophagocytic killing of multiple GAS serotypes and protected against systemic GAS challenge after passive immunization. Thus, the Lancefield antigen plays a functional role in GAS pathogenesis, and a deeper understanding of this unique polysaccharide has implications for vaccine development.