The unfolded protein response (UPR) is a central cellular stress pathway increasingly recognized as a target of microbial manipulation. While viral engagement of the UPR is well documented, far less is known about how bacterial pathogens, particularly extracellular ones, exploit this host stress machinery. Group A Streptococcus (GAS) is an exquisitely human-adapted pathogen capable of causing asymptomatic colonization as well as severe invasive diseases and provides a compelling example. GAS selectively activates the PKR-like endoplasmic reticulum kinase (PERK)-eukaryotic initiation factor 2 (eIF2α)-activating transcription factor 4 (ATF4) arm of the UPR, driving host asparagine (Asn) biosynthesis. The bacterium then imports this Asn to boost its metabolic activity, growth, and virulence, establishing a direct metabolic link between host ER stress and GAS pathogenicity. This Asn-driven regulatory circuit parallels the ATF4-Asn axis in cancer biology, where stress-induced Asn production supports metabolic adaptation, proliferation, and resistance to therapy. Together, these insights position Asn as a central metabolic signal at the intersection of host stress responses and GAS virulence.

Group A Streptococcus (GAS) causes various human diseases linked to virulome expression predominantly regulated by the two-component system (TCS), CovR/S. Here, we demonstrate that asparagine (Asn) presence in a minimal chemically defined medium increases virulence gene expression in a CovR-dependent fashion. It also decreases the transcription of asparagine synthetase (AsnA), the ABC transporter responsible for Asn uptake (GlnPQ), and that of the hemolysin toxins responsible for scavenging Asn from the host. Metabolomics data show that Asn availability increases intracellular ADP/ATP ratio, which enhances phosphatase activity in structurally related CovS sensors and is probably responsible for the Asn-mediated decrease in CovR phosphorylation. Mutants deficient in AsnA, GlnPQ, asparaginase, (AsnB) activities are attenuated in a mouse model of human GAS invasive soft tissue infection. The similarity between the mechanisms of Asn-mediated regulation of GAS virulence and tumor growth suggests that, as in cancer, components maintaining Asn homeostasis could be targeted for anti-GAS treatments.

Group A Streptococcus (GAS) causes a wide variety of diseases ranging from mild, noninvasive, such as pharyngitis and impetigo, to life-threatening infections, such as necrotizing fasciitis (NF) and streptococcal toxic shock syndrome (STSS). The two-component CovR/S system, comprising the sensor kinase CovS and transcription factor CovR, is a central regulator of GAS virulence. An attenuated pharyngeal colonizing variant (S126) possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H) was recovered in France from a member of a family in which another individual developed NF and STSS caused by the M1T1 WT strain (S119). We employed transcriptome analyses (RNA-seq), quantitative determinations of CovR phosphorylation, measurements of virulence factor activity, and a murine model of human NF to demonstrate that CovS of strain S126 almost lost its entire phosphatase activity but retained its kinase and phosphotransfer activities. Moreover, we reversed its attenuated phenotype by ectopically expressing the cytosolic domain of wild-type CovS. Culturing the corresponding strain S126 cov S-3’ in a chemically defined medium (CDM) supplemented with asparagine (Asn), conditions that produce an excess of cytosolic ADP over ATP, stimulated the ectopically expressed phosphatase activity. Consequently, this led to dephosphorylation of CovR∼P and increased the expression of virulence factors. Most importantly, S126 cov S-3’ reverted to the wild-type phenotype of S119 in the mouse model of human GAS NF. Our study provides a new mechanistic tool that enables the manipulation of CovS phosphatase activity both in vitro and in vivo . IMPORTANCE The shift from high to low virulence and back in GAS is critical for understanding its pathogenesis and developing new treatments to control GAS infections. The two-component CovR/S system, comprising the sensor kinase/phosphatase CovS and the transcription regulator CovR, regulates the degree of GAS virulence. In the M1T1 serotype, cov R/S mutations are typically associated with a loss of CovR/S function, leading to hypervirulent phenotypes. However, an attenuated pharyngeal-colonizing variant possessing a single-nucleotide polymorphism (SNP) in CovS (Y39H strain S126) was isolated in France. Here, we demonstrate that S126 is attenuated because the mutation in CovS inhibits its phosphatase activity but preserves its kinase and phosphotransfer activities. By expressing the cytosolic domain of WT CovS in the S126 background, we endowed the resulting strain, S126 cov S-3’, with phosphatase activity, which was further stimulated by ADP when formed in chemically defined medium (CDM) supplemented with asparagine (Asn). Most importantly, S126 cov S-3’ switched to full virulence, similar to that of S119 in a mouse model of human GAS NF. These findings underscore the importance of comprehensive analyses of disease-related cov R/S mutants in understanding the virulence and persistence of GAS.

The M protein located on the surface of group A Streptococcus has been extensively researched as a promising vaccine candidate. However, issues such as potential cross-reactivity with human tissues and the impact of selection of M peptide sequences have raised concerns regarding the safety and efficacy of the M protein vaccine. In this study, we utilized a KSI (ketosteroid isomerase, 15.78 kDa) tag and conducted a comparative analysis of the N-terminal (M12-N, 28.14 kDa), C-terminal (M12-C, 30.24 kDa), and fusion form (M12-N+C, 29.19 kDa) derived from the M12 protein found in MGAS9429. Three vaccine candidates formulated with aluminum hydroxide adjuvant significantly increased specific antibody titers in serum following booster immunization. Furthermore, immunization with these vaccines improved the survival rates in mice challenged subcutaneously with MGAS9429 compared to control mice. The immune responses induced by our vaccine formulation were characterized by Th1 type responses marked by IFN-γ secretion rather than the Th2 type responses and a notable increase in effector memory T cells. Significantly, the vaccine candidate M12-C exhibited several advantages including shortened vaccination times, enhanced antibody levels, improved survival rates against non-vaccine serotype MGAS5005 challenge. Moreover, the M12-C antiserum demonstrated significant opsonization and killing effects on the non-vaccine strains of M1, M3, M6 and M18. This work identifies a promising fusion sequence of vaccine candidate when developing GAS vaccines based on M peptides to enhance immune responses and protective efficacy.

Streptococcus (GAS) is a highly adapted and human-restricted pathogen causing a wide variety of infections, some life-threatening[1][1]. This ability is linked to the expression of many virulence factors, whose transcription is regulated by the two-component system, CovR/S[2][2]–[5][3]. Here, we show that genome transcription of GAS cultured in a chemically defined medium (CDM) is globally affected when supplemented with asparagine (Asn), including increased expression of many virulence genes. For the first time, we report that GAS solely depends on asparagine synthetase (AsnA) for Asn synthesis, on the ABC transporter (GlnPQ) to import Asn, and on the asparaginase (AsnB) to maintain a precisely balanced intracellular Asn concentration. Furthermore, we show that mutants defective in either asn A, gln P, or asn B express significantly lower levels of virulence factors in CDM and are severely attenuated in the sublethal murine model of human GAS soft-tissue infection. We further show that the synthesis and import of Asn in GAS are ATP-dependent and negatively regulated by intracellular Asn. Thus, Asn availability controls the intracellular ATP level. When ATP becomes limiting, CovR phosphorylation decreases. This augments GAS growth rate, virulence production, metabolism, and vice versa when the ATP level increases. Furthermore, excess Asn accumulates inside GAS in AsnB mutant, destroying the balance between Asn and ATP. We discuss the high similarity between these mechanistic principles of the Asn-mediated control of GAS virulence and metabolism to the Asn-mediated control of tumor growth[6][4], indicating evolutionary significance. ### Competing Interest Statement The authors have declared no competing interest. [1]: #ref-1 [2]: #ref-2 [3]: #ref-5 [4]: #ref-6

ABSTRACT GBS may cause a devasting disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to understand better understand T7SS contribution to virulence in these different clinical scenarios. GBS isolates were obtained from colonized mother prior to delivery and from newborns with EOD. DNA was analyzed for T7SS genes. A mutant EOD strain (ST17) was created by knocking out the essC gene encoding a T7SS protein. Galleria mellonella larvae were used to compare virulence of colonizing, EOD, and mutant EOD isolates. 33 GBS genomes were tested, 17 EOD isolates and 16 colonizing isolates. The T7SS locus encoded 8 genes: essC , membrane-embedded proteins ( essA; essB ), modulators of T7SS activity (esaA; esaB; esaC ) and effectors: [ esxA (SAG1039); esxB (SAG1030). ST17 isolates encode two copies of the essC gene and esxA gene encoding putative effectors but were present only in 23.5% of isolates. In ST1 isolates three copies of esxA gene were identified, but in ST6 and ST19 isolates all T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella model compared to colonizing isolates. The 118659Δ essC strain was attenuated in its killing ability, and the larvae were more effective in eradicating 118659Δ essC infection. essC gene deletion was associated with reduced bacterial growth. We demonstrated that T7SS plays an essential role during infection and contributes to GBS pathogenicity. Author Summary Type VII secretion system (T7SS) is related to virulence in various bacteria but is not well characterized in Group B Streptococci (GBS). GBS may cause sepsis, meningitis, and death in newborns. The bacteria rarely cause disease in pregnant mothers. Newborns acquire GBS from the colonized mother during delivery. We studied the role of T7SS in GBS isolates obtained from newborns with GBS sepsis in the first week of life and in colonized pregnant mothers. By studying T7SS genes we discovered that the genetic structure of the T7SS differs between isolates causing severe disease and colonizing isolates. To study the virulence of different GBS isolates we injected them into larvae and monitored larvae survival. Isolates causing severe disease in the newborn caused a more severe disease in larvae compared to colonizing isolates. We then deleted T7SS genes in GBS isolates causing severe disease. The killing activity of GBS isolates without T7SS genes was attenuated. The larva responded to these bacteria similarly to the response found when injecting the larva with GBS isolates from colonized mothers. These results support our hypothesis that T7SS is important for causing severe infection in the newborn and that this system contributes to GBS pathogenicity.

IntroductionGBS may cause a devastating disease in newborns. In early onset disease of the newborn the bacteria are acquired from the colonized mother during delivery. We characterized type VII secretion system (T7SS), exporting small proteins of the WXG100 superfamily, in group B Streptococci (GBS) isolates from pregnant colonized women and newborns with early onset disease (EOD) to better understand T7SS contribution to virulence in these different clinical scenarios. MethodsGBS genomes [N=33, 17 EOD isolates (serotype III/ST17) and 16 colonizing isolates (12 serotype VI/ST1, one serotype VI/ST19, one serotype VI/ST6, and two serotype 3/ST19)] were analyzed for presence of T7SS genes and genes encoding WXG100 proteins. We also perform bioinformatic analysis. Galleria mellonella larvae were used to compare virulence between colonizing, EOD, and mutant EOD isolates. The EOD isolate number 118659 (III/ST17) was used for knocking out the essC gene encoding a membrane-bound ATPase, considered the driver of T7SS. ResultsMost GBS T7SS loci encoded core component genes: essC, membrane-embedded proteins (essA; essB), modulators of T7SS activity (esaA; esaB; esaC) and effectors: [esxA (SAG1039); esxB (SAG1030)].Bioinformatic analysis indicated that based on sequence type (ST) the clinicalGBS isolates encode at least three distinct subtypes of T7SS machinery. In all ST1isolates we identified two copies of esxA gene (encoding putative WXG100proteins), when only 23.5% of the ST17 isolates harbored the esxA gene. Five ST17isolates encoded two copies of the essC gene. Orphaned WXG100 molecule(SAG0230), distinct from T7SS locus, were found in all tested strains, except inST17 strains where the locus was found in only 23.5% of the isolates. In ST6 andST19 isolates most of the structure T7SS genes were missing. EOD isolates demonstrated enhanced virulence in G. mellonella modelcompared to colonizing isolates. The 118659DessC strain was attenuated in itskilling ability, and the larvae were more effective in eradicating 118659DessC. ConclusionsWe demonstrated that T7SS plays a role during infection. Knocking out the essC gene, considered the driver of T7SS, decreased the virulence of ST17 responsible for EOD, causing them to be less virulent comparable to the virulence observed in colonizing isolates.

Group A streptococcus (GAS) necrotizing fasciitis (NF) causes high morbidity and mortality despite prompt intravenous administration of antibiotics, surgical soft-tissue debridement, and supportive treatment in the intensive care unit. Since there is no effective vaccine against GAS infections, a comprehensive understanding of NF pathogenesis is required to design more efficient treatments. To increase our understanding of NF pathogenesis, we need a reliable animal model that mirrors, at least in part, the infectious process in humans. This chapter describes a reliable murine model of human NF that mimics the histopathology observed in humans, namely the destruction of soft tissue, a paucity of infiltrating neutrophils, and the presence of many gram-positive cocci at the center of the infection.

Group A streptococcus (GAS) is a Gram-positive human pathogen that causes invasive infections with mild to life-threatening severity, like toxic shock syndrome, rheumatic heart disease, and necrotizing fasciitis (NF). NF is characterized by a clinical presentation of widespread tissue destruction due to the rapid spread of GAS infection into fascial planes. Despite quick medical interventions, mortality from NF is high. The early onset of the disease is difficult to diagnose because of non-specific clinical symptoms. Moreover, the unavailability of an effective vaccine against GAS warrants a genuine need for alternative treatments against GAS NF. One endoplasmic reticulum stress signaling pathway (PERK pathway) gets triggered in the host upon GAS infection. Bacteria utilize asparagine release as an output of this pathway for its pathogenesis. We reported that the combination of sub-cutaneous (SC) and intraperitoneal (IP) administration of PERK pathway inhibitors (GSK2656157 and ISRIB) cures local as well as systemic GAS infection in a NF murine model, by reducing asparagine release at the infection site. This protocol's methodology is detailed below. This protocol was validated in: Sci Transl Med (2021), DOI: 10.1126/scitranslmed.abd7465.

Infectious diseases are one of the main grounds of death and disabilities in human beings globally. Lack of effective treatment and immunization for many deadly infectious diseases and emerging drug resistance in pathogens underlines the need to either develop new vaccines or sufficiently improve the effectiveness of currently available drugs and vaccines. In this review, we discuss the application of advanced tools like bioinformatics, genomics, proteomics and associated techniques for a rational vaccine design.

Group A streptococcus (GAS) is among the top 10 causes of mortality from an infectious disease, producing mild to invasive life-threatening manifestations. Necrotizing fasciitis (NF) is characterized by a rapid GAS spread into fascial planes followed by extensive tissue destruction. Despite prompt treatments of antibiotic administration and tissue debridement, mortality from NF is still high. Moreover, there is no effective vaccine against GAS, and early diagnosis of NF is problematic because its clinical presentations are not specific. Thus, there is a genuine need for effective treatments against GAS NF. Previously, we reported that GAS induces endoplasmic reticulum (ER) stress to gain asparagine from the host. Here, we demonstrate that GAS-mediated asparagine induction and release occur through the PERK-eIF2α-ATF4 branch of the unfolded protein response. Inhibitors of PERK or integrated stress response (ISR) blocked the formation and release of asparagine by infected mammalian cells, and exogenously added asparagine overcame this inhibition. Moreover, in a murine model of NF, we show that the inhibitors minimized mortality when mice were challenged with a lethal dose of GAS and reduced bacterial counts and lesion size when mice were challenged with a sublethal dose. Immunohistopathology studies demonstrated that PERK/ISR inhibitors protected mice by enabling neutrophil infiltration into GAS-infected fascia and reducing the pro-inflammatory response that causes tissue damage. Inhibitor treatment was also effective in mice when started at 12 hours after infection. We conclude that host metabolic alteration induced by PERK or ISR inhibitors is a promising therapeutic strategy to treat highly invasive GAS infections.

Group A Streptococcus (GAS) causes diverse human diseases, including life-threatening soft-tissue infections. It is accepted that the human antimicrobial peptide LL-37 protects the host by killing GAS. Here, we show that GAS extracellular protease ScpC N-terminally cleaves LL-37 into two fragments of 8 and 29 amino acids, preserving its bactericidal activity. At sub-bactericidal concentrations, the cleavage inhibits LL-37-mediated neutrophil chemotaxis, shortens neutrophil lifespan, and eliminates P2X7 and EGF receptors' activation. Mutations at the LL-37 cleavage site protect the peptide from ScpC-mediated splitting, maintaining all its functions. The mouse LL-37 ortholog CRAMP is neither cleaved by ScpC nor does it activate P2X7 or EGF receptors. Treating wild-type or CRAMP-null mice with sub-bactericidal concentrations of the non-cleavable LL-37 analogs promotes GAS clearance that is abolished by the administration of either P2X7 or EGF receptor antagonists. We demonstrate that LL-37-mediated activation of host receptors is critical for defense against GAS soft-tissue infections.

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.

Bacteria use quorum sensing (QS) to regulate gene expression. We identified a group A Streptococcus (GAS) strain possessing the QS system sil, which produces functional bacteriocins, through a sequential signaling pathway integrating host and bacterial signals. Host cells infected by GAS release asparagine (ASN), which is sensed by the bacteria to alter its gene expression and rate of proliferation. We show that upon ASN sensing, GAS upregulates expression of the QS autoinducer peptide SilCR. Initial SilCR expression activates the autoinduction cycle for further SilCR production. The autoinduction process propagates throughout the GAS population, resulting in bacteriocin production. Subcutaneous co-injection of mice with a bacteriocin-producing strain and the globally disseminated M1T1 GAS clone results in M1T1 killing within soft tissue. Thus, by sensing host signals, a fraction of a bacterial population can trigger an autoinduction mechanism mediated by QS, which acts on the entire bacterial community to outcompete other bacteria within the infection.

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotising fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions along with the host-pathogen interactions that might influence biofilm formation. Here, we establish and characterise an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a mouse model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a 3D architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) that induce endoplasmic reticulum (ER) stress in the host. In an in vivo mouse model, the streptolysin null mutant is attenuated in both microcolony formation and bacterial spread, but pretreatment of soft-tissue with an ER stressor restores the ability of the mutant to form wild-type-like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.

Group A Streptococcus (GAS; Streptococcus pyogenes) causes a wide range of infections, including pharyngitis, impetigo, and necrotizing fasciitis, and results in over half a million deaths annually. GAS ScpC (SpyCEP), a 180-kDa surface-exposed, subtilisin-like serine protease, acts as an essential virulence factor that helps S. pyogenes evade the innate immune response by cleaving and inactivating C-X-C chemokines. ScpC is thus a key candidate for the development of a vaccine against GAS and other pathogenic streptococcal species. Here, we report the crystal structures of full-length ScpC wild-type, the inactive mutant, and the ScpC-AEBSF inhibitor complex. We show ScpC to be a multi-domain, modular protein consisting of nine structural domains, of which the first five constitute the PR + A region required for catalytic activity. The four unique C-terminal domains of this protein are similar to collagen-binding and pilin proteins, suggesting an additional role for ScpC as an adhesin that might mediate the attachment of S. pyogenes to various host tissues. The Cat domain of ScpC is similar to subtilisin-like proteases with significant difference to dictate its specificity toward C-X-C chemokines. We further show that ScpC does not undergo structural rearrangement upon maturation. In the ScpC-inhibitor complex, the bound inhibitor breaks the hydrogen bond between active-site residues, which is essential for catalysis. Guided by our structure, we designed various epitopes and raised antibodies capable of neutralizing ScpC activity. Collectively, our results demonstrate the structure, maturation process, inhibition, and substrate recognition of GAS ScpC, and reveal the presence of functional domains at the C-terminal region.

Here, we report the complete genome sequence of the Streptococcus pyogenes emm14 strain JS95, isolated from a patient with necrotizing fasciitis. The streptococcal invasion locus (sil), the first quorum-sensing system characterized in S. pyogenes, was identified in this strain.

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotizing fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions and the host-pathogen interactions that might influence biofilm formation. Here we establish and characterize an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a murine model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a three-dimensional architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) resulting in the release of a host-associated biofilm promoting-factor(s). Supernatants from GAS-infected mammalian cells or from cells treated with endoplasmic reticulum (ER) stressors restore biofilm formation to an SLO and SLS null mutant that is otherwise attenuated in biofilm formation on cells, together suggesting a role for streptolysin-induced ER stress in this process. In an i n vivo mouse model, the streptolysin-null mutant is attenuated in both microcolony formation and bacterial spread, but pre-treatment of softtissue with an ER-stressor restores the ability of the mutant to form wild type like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression. Significance Statement Although it is well-accepted that bacterial biofilms are associated with many chronic infections, little is known about the mechanisms by which group A Streptococcus (GAS) biofilms contribute to acute soft tissue-invasive diseases like necrotizing fasciitis (NF). In this study, we establish a physiologically relevant in vitro model to study GAS biofilm formation on mammalian cells and validate our findings in a mouse model that mimics human NF. This study demonstrates a novel role of GAS streptolysin-mediated ER stress in the development and spread of GAS biofilms in acute softtissue infections. We also show that biofilm formation depends on the release of a host-associated factor that promotes microcolony formation and GAS dissemination in vivo .

The connection between bacterial pathogens and unfolded protein response (UPR) is poorly explored. In this review we highlight the evidence showing that group A streptococcus (GAS) induces endoplasmic reticulum (ER) stress and UPR through which it captures the amino acid asparagine (ASN) from the host. GAS acts extracellularly and during adherence to host cells it delivers the hemolysin toxins; streptolysin O (SLO) and streptolysin S (SLS). By poorly understood pathways, these toxins trigger UPR leading to the induction of the transcriptional regulator ATF4 and consequently to the upregulation of asparagine synthetase (ASNS) transcription leading to production and release of ASN. GAS senses ASN and alters gene expression profile accordingly, and increases the rate of multiplication. We suggest that induction of UPR by GAS and by other bacterial pathogens represent means through which bacterial pathogens gain nutrients from the host, obviating the need to become internalized or inflict irreversible cell damage.

Successful infection depends on the ability of the pathogen to gain nutrients from the host. The extracellular pathogenic bacterium group A Streptococcus (GAS) causes a vast array of human diseases. By using the quorum-sensing sil system as a reporter, we found that, during adherence to host cells, GAS delivers streptolysin toxins, creating endoplasmic reticulum stress. This, in turn, increases asparagine (ASN) synthetase expression and the production of ASN. The released ASN is sensed by the bacteria, altering the expression of similar to 17% of GAS genes of which about one-third are dependent on the two-component system TrxSR. The expression of the streptolysin toxins is strongly upregulated, whereas genes linked to proliferation are downregulated in ASN absence. Asparaginase, a widely used chemotherapeutic agent, arrests GAS growth in human blood and blocks GAS proliferation in a mouse model of human bacteremia. These results delineate a pathogenic pathway and propose a therapeutic strategy against GAS infections.