Streptococcal Pyrogenic Exotoxin Research Articles

Mechanisms that potentially contribute to the development of post-streptococcal glomerulonephritis.

Post-streptococcal glomerulonephritis (PSGN) is primarily associated with preceding Group A streptococcal skin or throat infections, now mainly observed in economically disadvantaged communities. This condition significantly predisposes individuals to later-life chronic kidney disease and concurrent renal complications, with the elderly experiencing increased severity and less favourable outcomes. Streptococcal pyrogenic exotoxin B and nephritis associated plasmin receptor are identified nephritogenic antigens (nephritogens). Pathogenesis of PSGN is multifactorial. It can involve the formation of antigen-antibody immune complexes, causing inflammatory damage to renal glomeruli. Deposition of circulating immune complexes or in situ formation of immune complexes in glomeruli, or both, results in glomerulonephritis. Additionally, molecular mimicry is hypothesised as a mechanism, wherein cross-reactivity between anti-streptococcal antibodies and glomerular intrinsic matrix proteins leads to glomerulonephritis. Besides, as observed in clinical studies, streptococcal inhibitor of complement, a streptococcal-secreted protein, can also be associated with PSGN. However, the interplay between these streptococcal antigens in the pathogenesis of PSGN necessitates further investigation. Despite the clinical significance of PSGN, the lack of credible animal models poses challenges in understanding the association between streptococcal antigens and the disease process. This review outlines the postulated mechanisms implicated in the development of PSGN with possible therapeutic approaches.

The Emergence and Impact of the M1UK Lineage on Invasive Group A Streptococcus Disease in Aotearoa New Zealand.

M1UK is associated with current surges in invasive infection globally, partly due to increased production of superantigen streptococcal pyrogenic exotoxin A. We show that M1UK is now the dominant invasive emm1 lineage in Aotearoa New Zealand and is genomically related to community infections, suggesting that measures that effectively prevent group A Streptococcus pharyngitis in children could reduce invasive disease.

Interplay between group A Streptococcus and host innate immune responses.

SUMMARYGroup A Streptococcus (GAS), also known as Streptococcus pyogenes, is a clinically well-adapted human pathogen that harbors rich virulence determinants contributing to a broad spectrum of diseases. GAS is capable of invading epithelial, endothelial, and professional phagocytic cells while evading host innate immune responses, including phagocytosis, selective autophagy, light chain 3-associated phagocytosis, and inflammation. However, without a more complete understanding of the different ways invasive GAS infections develop, it is difficult to appreciate how GAS survives and multiplies in host cells that have interactive immune networks. This review article attempts to provide an overview of the behaviors and mechanisms that allow pathogenic GAS to invade cells, along with the strategies that host cells practice to constrain GAS infection. We highlight the counteractions taken by GAS to apply virulence factors such as streptolysin O, nicotinamide-adenine dinucleotidase, and streptococcal pyrogenic exotoxin B as a hindrance to host innate immune responses.

Toxic Shock Syndrome: A Literature Review.

Toxic shock syndrome (TSS) is a rare, life-threatening, toxin-mediated infectious process linked, in the vast majority of cases, to toxin-producing strains of Staphylococcus aureus or Streptococcus pyogenes. The pathophysiology, epidemiology, clinical presentation, microbiological features, management and outcome of TSS are described in this review. Bacterial superantigenic exotoxins induces unconventional polyclonal lymphocyte activation, which leads to rapid shock, multiple organ failure syndrome, and death. The main described superantigenic exotoxins are toxic shock syndrome toxin-1 (TSST-1) and enterotoxins for Staphylococcus aureus and Streptococcal pyrogenic exotoxins (SpE) A, B, and C and streptococcal superantigen A (SsA) for Streptococcus pyogenes. Staphylococcal TSS can be menstrual or nonmenstrual. Streptococcal TSS is linked to a severe group A streptococcal infection and, most frequently, to a necrotizing soft tissue infection. Management of TSS is a medical emergency and relies on early detection, immediate resuscitation, source control and eradication of toxin production, bactericidal antibiotic treatment, and protein synthesis inhibiting antibiotic administration. The interest of polyclonal intravenous immunoglobulin G administration as an adjunctive treatment for TSS requires further evaluation. Scientific literature on TSS mainly consists of observational studies, clinical cases, and in vitro data; although more data on TSS are required, additional studies will be difficult to conduct due to the low incidence of the disease.

Streptococcal pyrogenic exotoxin B is a superantigen that induces murine splenocyte proliferation and secretion of IL-2 and IFN-γ ex vivo.

Streptococcus pyogenes is a significant human pathogen, producing a range of virulence factors, including streptococcal pyrogenic exotoxin B (SpeB) that is associated with foodborne outbreaks. It was only known that this cysteine protease mediates cleavage of transmembrane proteins to permit bacterial penetration and is found in 25% of clinical isolates from streptococcal toxic shock syndrome patients with extreme inflammation. Its interaction with host and streptococcal proteins has been well characterized, but doubt remains about whether it constitutes a superantigen. In this study, for the first time it is shown that SpeB acts as a superantigen, similarly to other known superantigens such as staphylococcal enterotoxin A or streptococcal pyrogenic exotoxin type C, by inducing proliferation of murine splenocytes and cytokine secretion, primarily of interleukin-2 (IL-2), as shown by cytometric bead array analysis. IL-2 secretion was confirmed by enzyme-linked immunosorbent assay (ELISA) as well as secretion of interferon-γ. ELISA showed a dose-dependent relationship between SpeB concentration in splenocyte cells and IL-2 secretion levels, and it was shown that SpeB retains activity in milk pasteurized for 30 min at 63°C.

Group A Streptococcus Vaccine Targeting the Erythrogenic Toxins SpeA and SpeB Is Safe and Immunogenic in Rabbits and Does Not Induce Antibodies Associated with Autoimmunity.

Group A streptococcus (GAS) is a global pathogen associated with significant morbidity and mortality for which there is currently no licensed vaccine. Vaccine development has been slow, mostly due to safety concerns regarding streptococcal antigens associated with autoimmunity and related complications. For a GAS vaccine to be safe, it must be ensured that the antigens used in the vaccine do not elicit an antibody response that can cross-react with host tissues. In this study, we evaluated the safety of our GAS vaccine candidate called VaxiStrep in New Zealand White rabbits. VaxiStrep is a recombinant fusion protein comprised of streptococcal pyrogenic exotoxin A (SpeA) and exotoxin B (SpeB), also known as erythrogenic toxins, adsorbed to an aluminum adjuvant. The vaccine elicited a robust immune response against the two toxins in the rabbits without any adverse events or toxicity. No signs of autoimmune pathology were detected in the rabbits' brains, hearts, and kidneys via immunohistochemistry, and serum antibodies did not cross-react with cardiac or neuronal tissue proteins associated with rheumatic heart disease or Sydenham chorea (SC). This study further confirms that VaxiStrep does not elicit autoantibodies and is safe to be tested in a first-in-human trial.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes

BackgroundStreptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase.MethodsBacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry.ResultsHere, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation.ConclusionsOur findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

T-cell receptor Vβ8 for detection of biologically active streptococcal pyrogenic exotoxin type C

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPEs). The current method for detection cannot distinguish between the biologically active form of SPEs that has been reported to cause foodborne outbreaks and the inactivated toxin which poses no health risk. To measure the biological activity of SPE-C, one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vβ8. With this finding, we used a T-cell line natively expressing Vβ8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element (NFAT-RE) in combination with a B-cell line to present the rSPE-C toxin via MHC class II to the Vβ8 TCR in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross reactivity with SPE-B and significant loss of SPE-C biologically activity in spiked PBS while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.

The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4+ memory T cells

Severe bacterial or viral infections can induce a state of immune hyperactivation that can culminate in a potentially lethal cytokine storm. The classic example is toxic shock syndrome, a life-threatening complication of Staphylococcus aureus or Streptococcus pyogenes infection, which is driven by potent toxins known as superantigens (SAgs). SAgs are thought to promote immune evasion via the promiscuous activation of T cells, which subsequently become hyporesponsive, and act by cross-linking major histocompatibility complex class II molecules on antigen-presenting cells to particular β-chain variable (TRBV) regions of αβ T cell receptors (TCRs). Although some of these interactions have been defined previously, our knowledge of SAg-responsive TRBV regions is incomplete. In this study, we found that CD4+ and CD8+ T cells expressing TRBV12-3/12-4+ TCRs were highly responsive to streptococcal pyrogenic exotoxin C (SpeC) and toxic shock syndrome toxin-1 (TSST-1). In particular, SpeC and TSST-1 specifically induced effector cytokine production and the upregulation of multiple coinhibitory receptors among TRBV12-3/12-4+ CD4+ and CD8+ memory T cells, and importantly, these biological responses were dependent on human leukocyte antigen (HLA)-DR. Collectively, these data provided evidence of functionally determinative and therapeutically relevant interactions between SpeC and TSST-1 and CD4+ and CD8+ memory T cells expressing TRBV12-3/12-4+ TCRs, mediated via HLA-DR.

Agr Regulation of Streptococcal Pyrogenic Exotoxin A in Staphylococcus aureus.

Group A streptococcal pyrogenic exotoxins (SPEs A, B, and C) are superantigens. SPE A shares high sequence similarity with Staphylococcus aureus enterotoxins (SEs) B and C. Since SPE A is bacteriophage-encoded, we hypothesized that its gene ( speA ) was acquired from S. aureus . speA , when cloned into S. aureus , was stably expressed, its protein resistant to proteases, and the gene under accessory gene regulator control. speA was acquired by streptococci from cross-species transduction. speB was not expressed in S. aureus. SPE C was degraded by staphylococcal proteases. The genes speB and speC were not recently acquired from S. aureus.

Острый постстрептококковый гломерулонефрит у детей — реальность и парадоксы. Обзор литературы

Acute post-streptococcal glomerulonephritis (APSGN) is an immune-mediated disease associated with nephritogenic strains of group A beta-hemolytic streptococcus. The epidemiology of APSGN varies in different countries, due to the influence of social, racial, demographic, and climatic conditions. In the modern spectrum of progressive diseases, APSGN occupies a modest place, yielding to other immune glomerulopathies. The prevalence rate has been declining in recent decades due to the decrease in pyoderma, however, it remains endemic to the French Polynesians and Australian Aborigines. The review defines the features of the immunological processes of this disease: nephritogens were identified at the site of the formation of immune complexes (streptococcal plasmin receptor and streptococcal pyrogenic exotoxin B). An assessment of the clinical profile of patients with APSGN (mostly asymptomatic course with a self-limiting (self-limiting) outcome) was carried out. The role of genomic surveillance in tracking potential markers of APSGN is emphasized. Risk factors for the development of kidney damage were identified — hypoalbuminemia, hypocomplementemia, and an increase in inflammatory markers. The paper considers the factors that determine the prognosis in patients, presents the main approaches to treatment (predominantly symptomatic therapy, with the exception of severe and atypical cases of the disease). It is concluded that APSGN remains one of the reasons for the development of AKI in different countries of the world. The article gives recommendations on long-term follow-up of children after APSGN. The purpose is to analyze the prevalence and identify the factors contributing to OPSGN; to determine the specifics of the disease immunological processes of the (immunocomplexity, autoimmunity); to evaluate the clinical profile, diagnostic methods and features of OPSGN treatment; to discuss the possible progression of the disease and whether PSGN is associated with chronic kidney disease (CKD).

Acute Post-Streptococcal Glomerulonephritis in Children: A Comprehensive Review.

Acute post-streptococcal glomerulonephritis (APSGN) is an immune- complex (ICs) mediated glomerular disease triggered by group A β-hemolytic streptococcus (GAS) or Streptococcus pyogenes infections. APSGN represents a major cause of acquired kidney injury in children. This non-systematic review summarizes recent evidence on APSGN. We discuss the epidemiology, pathogenesis, clinical and laboratory findings, histopathology, treatment and prognosis of the disease. The median APSGN incidence in children in developing countries is estimated at 24.3/100,000 per year, compared with 6.2/100,000 per year in developed countries. Nephritis-associated plasmin receptor, identified as glyceraldehyde-3-phosphate dehydrogenase, and the cationic cysteine proteinase streptococcal pyrogenic exotoxin B are thought to be two leading streptococcal antigens involved in the pathogenesis of APSGN, which activate the complement system, mainly via the alternative but also the lectin pathway. This process is critical for the generation of inflammation by the ICs deposited in the glomerulus. The classic phenotype is an acute diffuse proliferative glomerulonephritis leading to features of the nephritic syndrome, including hematuria, oliguria, hypertension and edema. The histopathology shows that the glomeruli are diffusely affected, mostly presenting enlarged glomerular tuffs due to hypercellularity. Proliferative endothelial and mesangial cells and inflammation have also been observed. APSGN frequently has spontaneous recovery. There is no specific therapy, but its morbidity and mortality are drastically reduced by the prevention and/or treatment of complications. Despite recent advances, the pathogenesis of APSGN is not fully understood. There is no specific treatment for APSGN. The prognosis is generally good. However, some cases may evolve into chronic kidney disease.

Streptococcal pyrogenic exotoxin B cleaves GSDMA and triggers pyroptosis.

Gasdermins, a family of five pore-forming proteins (GSDMA-GSDME) in humans expressed predominantly in the skin, mucosa and immune sentinel cells, are key executioners of inflammatory cell death (pyroptosis), which recruits immune cells to infection sites and promotes protective immunity1,2. Pore formation is triggered by gasdermin cleavage1,2. Although the proteases that activate GSDMB, C, D and E have been identified, how GSDMA-the dominant gasdermin in the skin-is activated, remains unknown. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a major skin pathogen that causes substantial morbidity and mortality worldwide3. Here we show that the GAS cysteine protease SpeB virulence factor triggers keratinocyte pyroptosis by cleaving GSDMA after Gln246, unleashing an active N-terminal fragment that triggers pyroptosis. Gsdma1 genetic deficiency blunts mouse immune responses to GAS, resulting in uncontrolled bacterial dissemination and death. GSDMA acts as both a sensor and substrate of GAS SpeB and as an effector to trigger pyroptosis, adding a simple one-molecule mechanism for host recognition and control of virulence of a dangerous microbial pathogen.

Streptococcal Pyrogenic Exotoxin Genes SpeA and SpeB in Isolates of <i>Streptococcus pyogenes</i> from Children with Pharyngitis, Gezira State, Sudan

Background: Streptococcus pyogenes (group A streptococcus, GAS) is an important human bacterial pathogen. This organism possesses many virulence factors, Streptococcal pyrogenic exotoxinone of these. Aim: Detection of Streptococcal pyrogenic exotoxin SpeA and SpeB in isolated Streptococcus pyogenes. Methods: Tow hundred throat swab samples were collected from children with pharyngitis referred to Pediatric Teaching hospital and ENT hospital Wad medani, Sudan, from January to November 2021. The questionnaire was filled out to collect clinical and demographic data. Throat swabs were collected and processed with the standard microbiological procedure to isolate Streptococcus pyogenes. Antimicrobial susceptibility testing was done on all GAS isolates using the Kirby Bauer disk diffusion method according to clinical laboratory standard institute (CLSI) guidelines. Detection of Spy 1258 gene and Streptococcal pyrogenic exotoxins SpeA and SpeB were done by using Multiplex PCR. Results: Amongst the Tow hundred collected samples fifty-one isolates (25.5%) were identified as S. pyogenes. Antibiotic susceptibility testing showed that all the GAS isolates were sensitive to Azithromycin and Penicillin. Sensitivity to Erythromycin, Gentamicin, Clarithromycin, Amoxicillin and Cephalexin were 88.2%, 86.3%, 45.1%, 41.2%, 13.7%, respectively. SpeA was detected in 17 (33.3%) and SpeB in 48 (94.1%). Conclusion: Streptococcal pyrogenic exotoxin genes SpeA and SpeB were detected in 17 (33.3%) and 48 (94.1%) respectively of Streptococcus pyogenes isolates.

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches.

Streptococcal pyrogenic exotoxin B (SpeB) is a cysteine protease expressed during group A streptococcal infection that represents a major virulence factor. Although subject to several studies, its role during infection is still under debate, and its proteolytic properties remain insufficiently characterized. Here, we revisited this protease through a set of complementary approaches relying on state of-the-art HPLC-MS methods. After conceiving an efficient protocol to recombinantly express SpeB, the zymogen of the protease and its activation were characterized. Employing proteome-derived peptide libraries, a strong preference for hydrophobic and aromatic residues at P2 alongside negatively charged amino acids at P3′ to P6′ was revealed. To identify relevant in vivo substrates, native proteins were obtained from monocytic secretome and plasma to assess their cleavage under physiological conditions. Besides corroborating our findings concerning specificity, more than 200 cleaved proteins were identified, including proteins of the extracellular matrix, proteins of the immune system, and proteins involved in inflammation. Finally, the cleavage of IgG subclasses was studied in detail. This study precisely depicts the proteolytic properties of SpeB and provides a library of potential host substrates, including their exact cleavage positions, as a valuable source for further research to unravel the role of SpeB during streptococcal infection.

Improved Visual Detection of speB Gene in Streptococcus pyogenes Isolates by Real-time Loop-Mediated Isothermal Amplification Turbidimetry Method

Background: Group A Streptococcus (GAS) causes a wide array of clinical manifestations ranging from mild pharyngitis to suppurative and non-suppurative severe debilitating diseases. Hence, a simple, rapid detection method with high sensitivity and specificity is needed. Objectives: This study embarked on the visual detection of the streptococcal pyrogenic exotoxin B (speB) gene by real-time turbidimetry and loop-mediated isothermal amplification (RT-LAMP) methods. The real-time monitoring of the sigmoidal graph generated from a turbidimetry method was incorporated in the assay. Methods: The amplification of the speB gene was virtually observed in real-time monitoring of the graph (sigmoidal curve) generated via a turbidimeter, thus providing a “guide” to accurately estimate the time to positivity for the gene detection. Results: The targeted gene was detected at 15 min but was optimally amplified within 45 min at an isothermal temperature of 63°C with 100% specificity using an established set of primers. The formation of sigmoidal curves was correlated with other visual observations by the naked eye (from orange to green), ultra-violet light (green fluorescence), and agarose gel electrophoresis. The improved detection limit of the real-time RT-LAMP assay was also observed compared to conventional PCR assay (0.001 pg/µL versus 1 ng/µL). Conclusions: The improved visual detection of RT-LAMP assay could provide additional insight for rapid, cost-effective, and reliable identification of GAS via speB gene detection in low or middle-income countries. It could also be a very important tool to improve the healthcare management of patients infected with GAS in the future.

An Irreversible Inhibitor to Probe the Role of Streptococcus pyogenes Cysteine Protease SpeB in Evasion of Host Complement Defenses.

Members of the CA class of cysteine proteases have multifaceted roles in physiology and virulence for many bacteria. Streptococcal pyrogenic exotoxin B (SpeB) is secreted by Streptococcus pyogenes and implicated in the pathogenesis of the bacterium through degradation of key human immune effector proteins. Here, we developed and characterized a clickable inhibitor, 2S-alkyne, based on X-ray crystallographic analysis and structure-activity relationships. Our SpeB probe showed irreversible enzyme inhibition in biochemical assays and labeled endogenous SpeB in cultured S. pyogenes supernatants. Importantly, application of 2S-alkyne decreased S. pyogenes survival in the presence of human neutrophils and supports the role of SpeB-mediated proteolysis as a mechanism to limit complement-mediated host defense. We posit that our SpeB inhibitor will be a useful chemical tool to regulate, label, and quantitate secreted cysteine proteases with SpeB-like activity in complex biological samples and a lead candidate for new therapeutics designed to sensitize S. pyogenes to host immune clearance.

Pathogenesis of Streptococcus pyogenes and immune response

Streptococcus pyogenes is a Gram-positive beta-hemolytic bacteria, also known as group A streptococci, that causes a range of infections. The most common presentation is acute pharyngitis. GAS can be subdivided into >100 serotypes by the M-protein antigen that is located on the cell surface and by fimbriae (hairlike fuzz) that project from the outer edge of the cell. Classically, typing of the surface M protein relied upon available polyclonal antisera. GAS produce and release into the surrounding medium a large number of biologically active extracellular products. Some of these are toxic for human and other mammalian cells. Streptolysin S (SLS) is a small oxygen-stabile toxin responsible for β-hemolysis of GAS on blood agar, while streptolysin O (SLO) is an oxygen-labile, cholesterol-dependent toxin .Both SLS and SLO injure cell membranes, not only lysing red blood cells, but also damaging other eukaryotic cells and membranous subcellular organelles.15 Streptolysin O is antigenic; streptolysin S is not. Streptococcal pyrogenic exotox­ins (SPEs) are secreted factors with the capacity to act as superantigens and trigger T-cell proliferation and cytokine release.

Emergence of dominant toxigenic M1T1 Streptococcus pyogenes clone during increased scarlet fever activity in England: a population-based molecular epidemiological study

SummaryBackgroundSince 2014, England has seen increased scarlet fever activity unprecedented in modern times. In 2016, England's scarlet fever seasonal rise coincided with an unexpected elevation in invasive Streptococcus pyogenes infections. We describe the molecular epidemiological investigation of these events.MethodsWe analysed changes in S pyogenes emm genotypes, and notifications of scarlet fever and invasive disease in 2014–16 using regional (northwest London) and national (England and Wales) data. Genomes of 135 non-invasive and 552 invasive emm1 isolates from 2009–16 were analysed and compared with 2800 global emm1 sequences. Transcript and protein expression of streptococcal pyrogenic exotoxin A (SpeA; also known as scarlet fever or erythrogenic toxin A) in sequenced, non-invasive emm1 isolates was quantified by real-time PCR and western blot analyses.FindingsCoincident with national increases in scarlet fever and invasive disease notifications, emm1 S pyogenes upper respiratory tract isolates increased significantly in northwest London in the March to May period, from five (5%) of 96 isolates in 2014, to 28 (19%) of 147 isolates in 2015 (p=0·0021 vs 2014 values), to 47 (33%) of 144 in 2016 (p=0·0080 vs 2015 values). Similarly, invasive emm1 isolates collected nationally in the same period increased from 183 (31%) of 587 in 2015 to 267 (42%) of 637 in 2016 (p<0·0001). Sequences of emm1 isolates from 2009–16 showed emergence of a new emm1 lineage (designated M1UK)—with overlap of pharyngitis, scarlet fever, and invasive M1UK strains—which could be genotypically distinguished from pandemic emm1 isolates (M1global) by 27 single-nucleotide polymorphisms. Median SpeA protein concentration in supernatant was nine-times higher among M1UK isolates (190·2 ng/mL [IQR 168·9–200·4]; n=10) than M1global isolates (20·9 ng/mL [0·0–27·3]; n=10; p<0·0001). M1UK expanded nationally to represent 252 (84%) of all 299 emm1 genomes in 2016. Phylogenetic analysis of published datasets identified single M1UK isolates in Denmark and the USA.InterpretationA dominant new emm1 S pyogenes lineage characterised by increased SpeA production has emerged during increased S pyogenes activity in England. The expanded reservoir of M1UK and recognised invasive potential of emm1 S pyogenes provide plausible explanation for the increased incidence of invasive disease, and rationale for global surveillance.FundingUK Medical Research Council, UK National Institute for Health Research, Wellcome Trust, Rosetrees Trust, Stoneygate Trust.

Streptococcal Pyrogenic Exotoxin A-Stimulated Monocytes Mediate Regulatory T-Cell Accumulation through PD-L1 and Kynurenine

Bacterial superantigens (SAgs) are exotoxins that promote a fulminant activation of the immune system. The subsequent intense release of inflammatory cytokines often results in hypotension, shock, and organ failure with high mortality rates. In the current paradigm, the direct and simultaneous binding of SAgs with T-cell receptor (TCR)-bearing Vβ regions and conserved structures on major histocompatibility complex class II (MHC class II) on antigen-presenting cells (APCs) induces the activation of both cell types. However, by crosslinking MHC class II molecules, APCs can be activated by SAgs independently of T lymphocytes. Recently, we showed that streptococcal pyrogenic exotoxin A (SPEA) of Streptococcus pyogenes stimulates an immunogenic APC phenotype with upregulated costimulatory molecules and inflammatory cytokines. Additionally, we revealed that SPEA triggers immunosuppressive programs in monocytes that facilitate the accumulation of regulatory T cells (Tregs) in in vitro monocyte/CD4+ T-cell cocultures. Immunosuppressive factors include anti-inflammatory interleukin 10 (IL-10), co-inhibitory surface molecule programmed cell death 1 ligand 1 (PD-L1), and the inhibitory indoleamine 2,3-dioxygenase (IDO)/kynurenine effector system. In the present study, we investigated the underlying mechanism of SPEA-stimulated monocyte-mediated accumulation of Tregs. Blood-derived monocytes from healthy donors were stimulated with SPEA for 48 h (SPEA-monocytes). For the evaluation of SPEA-monocyte-mediated modulation of CD4+ T lymphocytes, SPEA was removed from the culture through extensive washing of cells before adding allogeneic CD3/CD28-activated T cells. Results: In coculture with allogeneic CD4+ T cells, SPEA-monocytes mediate apoptosis of CD4+Foxp3− lymphocytes and accumulation of CD4+Foxp3+ Tregs. PD-L1 and kynurenine are critically involved in the mediated cell death because blocking both factors diminished apoptosis and decreased the proportion of the CD25+/Foxp3+ Treg subpopulation significantly. Upregulation of PD-L1 and kynurenine as well as SPEA-monocyte-mediated effects on T cells depend on inflammatory IL-1β. Our study shows that monocytes activated by SPEA mediate apoptosis of CD4+Foxp3− T effector cells through PD-L1 and kynurenine. CD4+Foxp3+ T cells are resistant to apoptosis and accumulate in SPEA-monocyte/CD4+ T-cell coculture.