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Autoinducer-2 can mediate inter- and intra-species communication signal between bacteria and these signals from AI-2 is noted from limited species of bacteria. In humans, S. pyogenes is a pathogen that causes a wide range of illnesses and can survive in the host system and transmit infection. The process by which S. pyogenes acquires the competence to live and disseminate infection remains unknown. We hypothesized that AI-2 and their receptors would play a significant role during infection, and for that present investigation provides the experimental and molecular insights. In the absence of details about the receptor LuxP and LuxQ, the screening approach provides supporting insights. The evolutionary relationship and similarities of the PBP domain (Spy 1535) and the signal transmission PDZ domain (Spy 1536) were studied in relation to their counterparts in other bacteria. Molecular docking and modeling confirmed the domain-enhanced specificity for AI-2 binding. In vitro studies showed that AI-2, which is present in the cell-free supernatant of S. pyogenes , regulates luminescence in P. luminous and biofilm development in E. coli using the LuxS reporter genes. Examination of S. pyogenes gene expression revealed modulation of virulence genes when the pathogen was exposed to V. harveyi HSL and AI-2. Therefore, S. pyogenes pathogenicity is sequentially regulated by AI-2 it acquires from other commensal bacteria. Overall, this study lays the groundwork for understanding the signalling mechanism from AI-2, which are critical to the pathogenic mechanism of S. pyogenes .Communicated by Ramaswamy H. Sarma.

Tissue injury, including extracellular matrix (ECM) degradation, is a hallmark of group A Streptococcus (GAS) skin infection and is partially mediated by M proteins which possess lectin-like properties. Hyaluronic acid is a glycosaminoglycan enriched in the cutaneous ECM, yet an interaction with M proteins has yet to be explored. This study revealed that hyaluronic acid binding was conserved across phylogenetically diverse M proteins, mediated by RR/SR motifs predominantly localized in the C repeat region. Keratinocyte wound healing was decreased through the recruitment of hyaluronic acid by M proteins in an M type-specific manner. GAS strains 5448 (M1 serotype) and ALAB49 (M53 serotype) also bound hyaluronic acid via M proteins, but hyaluronic acid could increase bacterial adherence independently of M proteins. The identification of host-pathogen mechanisms that affect ECM composition and cell repair responses may facilitate the development of nonantibiotic therapeutics that arrest GAS disease progression in the skin., (© 2024 Federation of American Societies for Experimental Biology.)

In Streptococcus pyogenes, the type II fatty acid (FA) synthesis pathway FASII is feedback-controlled by the FabT repressor bound to an acyl-Acyl carrier protein. Although FabT defects confer reduced virulence in animal models, spontaneous fabT mutants arise in vivo. We resolved this paradox by characterizing the conditions and mechanisms requiring FabT activity, and those promoting fabT mutant emergence. The fabT defect leads to energy dissipation, limiting mutant growth on human tissue products, which explains the FabT requirement during infection. Conversely, emerging fabT mutants show superior growth in biotopes rich in saturated FAs, where continued FASII activity limits their incorporation. We propose that membrane alterations and continued FASII synthesis are the primary causes for increased fabT mutant mortality in nutrient-limited biotopes, by failing to stop metabolic consumption. Our findings elucidate the rationale for emerging fabT mutants that improve bacterial survival in lipid-rich biotopes, but lead to a genetic impasse for infection., (© 2024. The Author(s).)

Streptococcus pyogenes is responsible for a range of diseases in humans contributing significantly to morbidity and mortality. Among more than 200 serotypes of S. pyogenes , serotype M1 strains hold the greatest clinical relevance due to their high prevalence in severe human infections. To enhance our understanding of pathogenesis and discovery of potential therapeutic approaches, we have developed the first genome-scale metabolic model (GEM) for a serotype M1 S. pyogenes strain, which we name iYH543. The curation of iYH543 involved cross-referencing a draft GEM of S. pyogenes serotype M1 from the AGORA2 database with gene essentiality and autotrophy data obtained from transposon mutagenesis-based and growth screens. We achieved a 92.6% (503/543 genes) accuracy in predicting gene essentiality and a 95% (19/20 amino acids) accuracy in predicting amino acid auxotrophy. Additionally, Biolog Phenotype microarrays were employed to examine the growth phenotypes of S. pyogenes, which further contributed to the refinement of iYH543. Notably, iYH543 demonstrated 88% accuracy (168/190 carbon sources) in predicting growth on various sole carbon sources. Discrepancies observed between iYH543 and the actual behavior of living S. pyogenes highlighted areas of uncertainty in the current understanding of S. pyogenes metabolism. iYH543 offers novel insights and hypotheses that can guide future research efforts and ultimately inform novel therapeutic strategies.IMPORTANCEGenome-scale models (GEMs) play a crucial role in investigating bacterial metabolism, predicting the effects of inhibiting specific metabolic genes and pathways, and aiding in the identification of potential drug targets. Here, we have developed the first GEM for the S. pyogenes highly virulent serotype, M1, which we name iYH543. The iYH543 achieved high accuracy in predicting gene essentiality. We also show that the knowledge obtained by substituting actual measurement values for iYH543 helps us gain insights that connect metabolism and virulence. iYH543 will serve as a useful tool for rational drug design targeting S. pyogenes metabolism and computational screening to investigate the interplay between inhibiting virulence factor synthesis and growth., Competing Interests: The authors declare no conflict of interest.

Cancer is one of the main causes of death in the world. Resistance to anticancer treatments in patients with advanced solid tumors leads to new treatments. Therefore, more alternative anticancer methods have been found over time with greater specificity against tumor cells and with less or no adverse effects on normal cells. Bacterial spores of obligate anaerobes exclusively germinate in the hypoxic/necrotic areas and not in the well oxygenated areas of the body. This unique phenomenon has been exploited in using bacterial spores as a remedy for cancer. Bacterial toxins also play a significant role in either directly killing tumor cells or altering the cellular processes of the tumor cells which ultimately leads to the inhibition and regression of the solid tumor. In the microbial environment, pathogens such as Staphylococcus aureus, Bacillus cereus, or Streptococcus pyogenes produce hemolysin. This protein is used as an anti-cancer protein. To identify the production of hemolysin by bacteria, which can destroy cancer cells more effectively, different bacterial strains were first cultured in blood agar culture medium. The Strains that completely lysed red blood cells, creating transparent zones, were selected for further investigation. Then, to find out which strains have more ability to lyse red blood cells, the qualitative method of halo diameter measurement was used. Also, using quantitative methods, hemolysin strength in microtubes was determined compared to control samples. The results of the hemolysis in the microtube and the qualitative test results showed similar results. In the next step, the cell viability test was performed with the partially purified proteins. Then, bioinformatics studies such as secondary structure investigation, physicochemical properties, pseudo amino acid composition, and molecular docking were performed. The results of molecular docking showed that the hemolysin protein has the highest affinity for the cholesterol of the cytoplasmic membrane, respectively, of Bacillus subtilis, Bacillus cereus, and Staphylococcus aureus bacteria which play a significant role in either directly killing tumor cells or altering the cellular processes of the tumor cells which ultimately leads to the inhibition and regression of the solid tumor., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The hyaluronic acid capsule is crucial in protecting group A Streptococcus (GAS) against phagocytic killing. However, there have been reported outbreaks caused by capsule-deficient GAS strains, and the mechanisms underlying their evasion of immune clearance remain unclear. This study demonstrated that the capsule-deficient mutant [Cap(-)] of the emm1 strain increased survival within phagocytic cells compared to the wild-type strain [Cap(+)]. Although both Cap(+) and Cap(-) strains exhibited similar abilities to disrupt the phagosome, only the Cap(+) strain was colocalized with lysosomes and acidified compartments in phagocytic cells, indicating its susceptibility to autophagosome elimination. In contrast, the Cap(-) mutant evaded the recognition of galectin-8 and ubiquitin, impairing selective autophagy-mediated elimination. These findings suggest that a deficiency in the capsule could impair the intracellular elimination of GAS in macrophages, revealing previously unknown aspects of the host's recognition of the GAS capsule in macrophages., Importance: Group A Streptococcus (GAS) is a Gram-positive bacterium that causes diseases ranging from mild pharyngitis to severe necrotizing fasciitis. Phagocytic cells serve as the primary defense against bacterial infections, exhibiting remarkable efficiency in eliminating intracellular pathogens. The hyaluronic acid capsule is a critical virulence factor that contributes to the resistance of phagocytosis in GAS. Nevertheless, the outbreaks caused by GAS strains that lack the hyaluronic acid capsule have been reported, and the selective advantage of capsule-deficient strains during infection is not fully understood. This study showed that the autophagic adaptor proteins recognize the capsulated GAS strain but not the capsule-deficient mutant, indicating that the hyaluronic acid capsule could be the autophagic target in macrophages. These findings imply that the hyaluronic acid capsule of GAS actually enhances its elimination within phagocytic cells, subverting the understanding of the capsule in GAS pathogenesis., Competing Interests: The authors declare no conflict of interest.

The interplay between host nutritional immune mechanisms and bacterial nutrient uptake systems has a major impact on the disease outcome. The host immune factor calprotectin (CP) limits the availability of essential transition metals, such as manganese (Mn) and zinc (Zn), to control the growth of invading pathogens. We previously demonstrated that the competition between CP and the human pathogen group A streptococcus (GAS) for Zn impacts GAS pathogenesis. However, the contribution of Mn sequestration by CP in GAS infection control and the role of GAS Mn acquisition systems in overcoming host-imposed Mn limitation remain unknown. Using a combination of in vitro and in vivo studies, we show that GAS-encoded mtsABC is a Mn uptake system that aids bacterial evasion of CP-imposed Mn scarcity and promotes GAS virulence. Mn deficiency caused by either the inactivation of mtsC or CP also impaired the protective function of GAS-encoded Mn-dependent superoxide dismutase. Our ex vivo studies using human saliva show that saliva is a Mn-scant body fluid, and Mn acquisition by MtsABC is critical for GAS survival in human saliva. Finally, animal infection studies using wild-type (WT) and CP-/ - mice showed that MtsABC is critical for GAS virulence in WT mice but dispensable in mice lacking CP, indicating the direct interplay between MtsABC and CP in vivo . Together, our studies elucidate the role of the Mn import system in GAS evasion of host-imposed metal sequestration and underscore the translational potential of MtsABC as a therapeutic or prophylactic target., Competing Interests: The authors declare no conflict of interest.

DNA manipulation is an essential tool in molecular microbiology research that is dependent on the ability of bacteria to take up and preserve foreign DNA by horizontal gene transfer. This process can be significantly impaired by the activity of bacterial restriction modification systems; bacterial operons comprising paired enzymatic activities that protectively methylate host DNA, while cleaving incoming unmodified foreign DNA. Ocr is a phage-encoded protein that inhibits Type I restriction modification systems, the addition of which significantly improves bacterial transformation efficiency. We recently established an improved and highly efficient transformation protocol for the important human pathogen group A Streptococcus using commercially available recombinant Ocr protein, manufacture of which has since been discontinued. In order to ensure the continued availability of Ocr protein within the research community, we have generated tools and methods for in-house Ocr production and validated the activity of the purified recombinant protein.

Antigenically sequence variable M proteins of the major bacterial pathogen Streptococcus pyogenes (Strep A) are responsible for recruiting human C4b-binding protein (C4BP) to the bacterial surface, which enables Strep A to evade destruction by the immune system. The most sequence divergent portion of M proteins, the hypervariable region (HVR), is responsible for binding C4BP. Structural evidence points to the conservation of two C4BP-binding sequence patterns (M2 and M22) in the HVR of numerous M proteins, with this conservation applicable to vaccine immunogen design. These two patterns, however, only partially explain C4BP binding by Strep A. Here, we identified several M proteins that lack these patterns but still bind C4BP and determined the structures of two, M68 and M87 HVRs, in complex with a C4BP fragment. Mutagenesis of these M proteins led to the identification of amino acids that are crucial for C4BP binding, enabling formulation of new C4BP-binding patterns. Mutagenesis was also carried out on M2 and M22 proteins to refine or generate experimentally grounded C4BP-binding patterns. The M22 pattern was the most prevalent among M proteins, followed by the M87 and M2 patterns, while the M68 pattern was rare. These patterns, except for M68, were also evident in numerous M-like Enn proteins. Binding of C4BP via these patterns to Enn proteins was verified. We conclude that C4BP-binding patterns occur frequently in Strep A strains of differing M types, being present in their M or Enn proteins, or frequently both, providing further impetus for their use as vaccine immunogens., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Kinetoplastids are unicellular eukaryotic flagellated parasites found in a wide range of hosts within the animal and plant kingdoms. They are known to be responsible in humans for African sleeping sickness (Trypanosoma brucei), Chagas disease (Trypanosoma cruzi), and various forms of leishmaniasis (Leishmania spp.), as well as several animal diseases with important economic impact (African trypanosomes, including Trypanosoma congolense). Understanding the biology of these parasites necessarily implies the ability to manipulate their genomes. In this study, we demonstrate that transfection of a ribonucleoprotein complex, composed of recombinant Streptococcus pyogenes Cas9 (SpCas9) and an in vitro-synthesized guide RNA, results in rapid and efficient genetic modifications of trypanosomatids, in marker-free conditions. This approach was successfully developed to inactivate, delete, and mutate candidate genes in various stages of the life cycle of T. brucei and T. congolense, and Leishmania promastigotes. The functionality of SpCas9 in these parasites now provides, to the research community working on these parasites, a rapid and efficient method of genome editing, without requiring plasmid construction and selection by antibiotics but requires only cloning and PCR screening of the clones. Importantly, this approach is adaptable to any wild-type parasite., (© 2024 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Bacterial or viral antigens can contain subdominant protein regions that elicit weak antibody responses upon vaccination or infection although there is accumulating evidence that antibody responses against subdominant regions can enhance the protective immune response. One proposed mechanism for subdominant protein regions is the binding of host proteins that prevent antibody production against epitopes hidden within the protein binding interfaces. Here, we used affinity purification combined with quantitative mass spectrometry (AP-MS) to examine the level of competition between antigen-specific antibodies and host-pathogen protein interaction networks using the M1 protein from Streptococcus pyogenes as a model system. As most humans have circulating antibodies against the M1 protein, we first used AP-MS to show that the M1 protein interspecies protein network formed with human plasma proteins is largely conserved in naïve mice. Immunizing mice with the M1 protein generated a time-dependent increase of anti-M1 antibodies. AP-MS analysis comparing the composition of the M1-plasma protein network from naïve and immunized mice showed significant enrichment of 292 IgG peptides associated with 56 IgG chains in the immune mice. Despite the significant increase of bound IgGs, the levels of interacting plasma proteins were not significantly reduced in the immune mice. The results indicate that the antigen-specific polyclonal IgG against the M1 protein primarily targets epitopes outside the other plasma protein binding interfaces. In conclusion, this study demonstrates that AP-MS is a promising strategy to determine the relationship between antigen-specific antibodies and host-pathogen interaction networks that could be used to define subdominant protein regions of relevance for vaccine development., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an NGG protospacer-adjacent motif (PAM) sequence immediately next to the DNA target limits the number of editable genes. Recently developed Cas9 variants have been engineered with relaxed PAM requirements, including SpG-Cas9 (SpG) and the nearly PAM-less SpRY-Cas9 (SpRY). However, the molecular mechanisms of how SpRY recognizes all potential PAM sequences remains unclear. Here, we combine structural and biochemical approaches to determine how SpRY interrogates DNA and recognizes target sites. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region, which facilitates tight binding to off-target DNA sequences. Nuclease activation occurs ~1000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Experiments with SpG position it as an intermediate enzyme between Cas9 and SpRY. Our findings shed light on the molecular mechanisms of PAMless genome editing., (© 2024. The Author(s).)

Streptococcus pyogenes is a human-specific pathogen that commonly colonizes the upper respiratory tract and skin, causing a wide variety of diseases ranging from pharyngitis to necrotizing fasciitis and toxic shock syndrome. S. pyogenes has a repertoire of secreted virulence factors that promote infection and evasion of the host immune system including the cytolysins streptolysin O (SLO) and streptolysin S (SLS). S. pyogenes does not naturally infect the upper respiratory tract of mice although mice transgenic for MHC class II human leukocyte antigens (HLA) become highly susceptible. Here we used HLA-transgenic mice to assess the role of both SLO and SLS during both nasopharyngeal and skin infection. Using S. pyogenes MGAS8232 as a model strain, we found that an SLS-deficient strain exhibited a 100-fold reduction in bacterial recovery from the nasopharynx and a 10-fold reduction in bacterial burden in the skin, whereas an SLO-deficient strain did not exhibit any infection defects in these models. Furthermore, depletion of neutrophils significantly restored the bacterial burden of the SLS-deficient bacteria in skin, but not in the nasopharynx. In mice nasally infected with the wildtype S. pyogenes, there was a marked change in localization of the tight junction protein ZO-1 at the site of infection, demonstrating damage to the nasal epithelia that was absent in mice infected with the SLS-deficient strain. Overall, we conclude that SLS is required for the establishment of nasopharyngeal infection and skin infection in HLA-transgenic mice by S. pyogenes MGAS8232 and provide evidence that SLS contributes to nasopharyngeal infection through the localized destruction of nasal epithelia., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shannon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Group A Streptococcus (GAS) is a major human pathogen, causing diseases ranging from mild superficial infections of the skin and pharyngeal epithelium to severe systemic and invasive diseases. Moreover, post infection auto-immune sequelae arise by a yet not fully understood mechanism. The ability of GAS to cause a wide variety of infections is linked to the expression of a large set of virulence factors and their transcriptional regulation in response to various physiological environments. The use of transcriptomics, among others -omics technologies, in addition to traditional molecular methods, has led to a better understanding of GAS pathogenesis and host adaptation mechanisms. This review focusing on bacterial transcriptomic provides new insight into gene-expression patterns in vitro, ex vivo and in vivo with an emphasis on metabolic shifts, virulence genes expression and transcriptional regulators role.

Group A Streptococcal M-related proteins (Mrps) are dimeric α-helical-coiled-coil cell membrane-bound surface proteins. During infection, Mrp recruit the fragment crystallizable region of human immunoglobulin G via their A-repeat regions to the bacterial surface, conferring upon the bacteria enhanced phagocytosis resistance and augmented growth in human blood. However, Mrps show a high degree of sequence diversity, and it is currently not known whether this diversity affects the Mrp-IgG interaction. Herein, we report that diverse Mrps all bind human IgG subclasses with nanomolar affinity, with differences in affinity which ranged from 3.7 to 11.1 nM for mixed IgG. Using surface plasmon resonance, we confirmed Mrps display preferential IgG-subclass binding. All Mrps were found to have a significantly weaker affinity for IgG3 (p < 0.05) compared to all other IgG subclasses. Furthermore, plasma pulldown assays analyzed via Western blotting revealed that all Mrp were able to bind IgG in the presence of other serum proteins at both 25 °C and 37 °C. Finally, we report that dimeric Mrps bind to IgG with a 1:1 stoichiometry, enhancing our understanding of this important host-pathogen interaction., Competing Interests: Conflict of interest The authors declare they have no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Group A Streptococcus (GAS) has a fantastically wide tissue tropism in humans, manifesting as different diseases depending on the strain's virulence factor repertoire and the tissue involved. Activation of immune cells and pro-inflammatory signaling has historically been considered an exclusively host-protective response that a pathogen would seek to avoid. However, recent advances in human and animal models suggest that in some tissues, GAS will activate and manipulate specific pro-inflammatory pathways to promote growth, nutrient acquisition, persistence, recurrent infection, competition with other microbial species, dissemination, and transmission. This review discusses molecular interactions between the host and pathogen to summarize how infection varies across tissue and stages of inflammation. A need for inflammation for GAS survival during common, mild infections may drive selection for mechanisms that cause pathological and excess inflammation severe diseases such as toxic shock syndrome, necrotizing fasciitis, and rheumatic heart disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

The standalone regulator RofA is a positive regulator of the pilus locus in Streptococcus pyogenes . Found in only certain emm genotypes, RofA has been reported to regulate other virulence factors, although its role in the globally dominant emm 1 S. pyogenes is unclear. Given the recent emergence of a new emm 1 (M1 UK ) toxigenic lineage that is distinguished by three non-synonymous SNPs in rofA , we characterized the rofA regulon in six emm 1 strains that are representative of the two contemporary major emm 1 lineages (M1 global and M1 UK ) using RNAseq analysis, and then determined the specific role of the M1 UK -specific rofA SNPs. Deletion of rofA in three M1 global strains led to altered expression of 14 genes, including six non-pilus locus genes. In M1 UK strains, deletion of rofA led to altered expression of 16 genes, including nine genes that were unique to M1 UK . Only the pilus locus genes were common to the RofA regulons of both lineages, while transcriptomic changes varied between strains even within the same lineage. Although introduction of the three SNPs into rofA did not impact gene expression in an M1 global strain, reversal of three SNPs in an M1 UK strain led to an unexpected number of transcriptomic changes that in part recapitulated transcriptomic changes seen when deleting RofA in the same strain. Computational analysis predicted that interactions with a key histidine residue in the PRD domain of RofA would differ between M1 UK and M1 global . RofA is a positive regulator of the pilus locus in all emm 1 strains but effects on other genes are strain- and lineage-specific, with no clear, common DNA binding motif. The SNPs in rofA that characterize M1 UK may impact regulation of RofA; whether they alter phosphorylation of the RofA PRD domain requires further investigation.

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR + CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (K M ) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar K M of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2200-fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity., (© 2023. The Author(s).)

The important bacterial pathogen Streptococcus pyogenes secretes IdeS (immunoglobulin G-degrading enzyme of S. pyogenes), a proteinase that cleaves human immunoglobulin G (IgG) antibodies in the hinge region resulting in Fc (fragment crystallizable) and F(ab') 2 (fragment antigen-binding) fragments and protects the bacteria against phagocytic killing. Experiments with radiolabeled IdeS and flow cytometry demonstrated that IdeS binds to the surface of S. pyogenes, and the interaction was most prominent in conditions resembling those in the pharynx (acidic pH and low salt), the habitat for S. pyogenes. SpnA (S. pyogenes nuclease A) is a cell wall-anchored DNase. A dose-dependent interaction between purified SpnA and IdeS was demonstrated in slot binding and surface plasmon resonance spectroscopy experiments. Gel filtration showed that IdeS forms proteolytically active complexes with SpnA in solution, and super-resolution fluorescence microscopy revealed the presence of SpnA-IdeS complexes at the surface of S. pyogenes. Finally, specific IgG antibodies binding to S. pyogenes surface antigens were efficiently cleaved by surface-associated IdeS. IdeS is secreted by all S. pyogenes isolates and cleaves IgG antibodies with a unique degree of specificity and efficiency. These properties and the finding here that the proteinase is present and fully active at the bacterial surface in complex with SpnA implicate an important role for IdeS in S. pyogenes biology and pathogenesis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Using Sanger sequencing and high-throughput genome sequencing of DNA cleavage reactions, we find that the Streptococcus pyogenes SpCas9 complex responds to internal mechanical strain by robustly generating a distribution of overhanging, rather than blunt, DNA ends. Internal mechanical strain is generated by shifting (increasing or decreasing) the spacing between the RNA-DNA hybrid and the downstream canonical PAM. Up to 2-base 3' overhangs can be robustly generated via a 2-base increase in the distance between hybrid and PAM. We also use single-molecule experiments to reconstruct the full course of the CRISPR-SpCas9 reaction in real-time, structurally and kinetically monitoring and quantifying R-loop formation, the first and second DNA-incision events, and dissociation of the post-catalytic complex. Complex dissociation and release of broken DNA ends is a rate-limiting step of the reaction, and shifted SpCas9 is sufficiently destabilized so as to rapidly dissociate after formation of broken DNA ends., (© 2023. The Author(s).)

Membranes are a universal barrier to all cells. Phospholipids, essential bacterial membrane components, are composed of a polar head and apolar fatty acid (FA) chains. Most bacterial FAs are synthesized by the Type II FA synthesis pathway (FASII). In Streptococcaceae, Enterococci, and Lactococcus lactis , a unique feedback mechanism controls the FASII gene expression. FabT, encoded in the FASII main locus, is the repressor, and it is activated by long-chain acyl-acyl carrier protein (acyl-ACP). Many Streptococci, Enterococcus faecalis , but not L. lactis , possess two ACPs. The AcpA-encoding gene is within the FASII locus and is coregulated with the FASII genes. Acyl-AcpA is the end product of FASII. The AcpB-encoding gene is in operon with plsX encoding an acyl-ACP:phosphate acyltransferase. The role of acyl-AcpB as FabT corepressor is controversial. Streptococcus pyogenes , which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possesses AcpB. In this study, by comparing the expression of FabT-controlled genes in an acpB -deleted mutant with those in a wild-type and in a fabT mutant strain, grown in the presence or absence of exogenous FAs, we show that AcpB is the S. pyogenes FabT main corepressor. Its deletion impacts membrane FA composition and bacterial adhesion to eucaryotic cells, highlighting the importance of FASII control. Importance Membrane composition is crucial for bacterial growth or interaction with the environment. Bacteria synthesize fatty acids (FAs), membrane major constituents, via the Type II FAS (FASII) pathway. Streptococci control the expression of the FASII genes via a transcriptional repressor, FabT, with acyl-acyl carrier proteins (ACPs) as corepressor. Streptococcus pyogenes that causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections possesses two ACPs. acpA , but not acpB , is a FASII gene. In this study, we show that acyl-AcpBs are FabT main corepressors. Also, AcpB deletion has consequences on the membrane FA composition and bacterial adhesion to host cells. In addition to highlighting the importance of FASII control in the presence of exogeneous FAs for the adaptation of bacteria to their environment, our data indicate that FASII gene repression is mediated by a corepressor whose gene expression is not repressed in the presence of exogenous FAs., Competing Interests: The authors declare no conflict of interest.

Immunoglobulin (Ig) A functions as monomeric IgA in the serum and Secretory (S) IgA in mucosal secretions. Host IgA Fc receptors (FcαRs), including human FcαR1/CD89, mediate IgA effector functions; however, human pathogen Streptococcus pyogenes has evolved surface-protein virulence factors, including M4, that also engage the CD89-binding site on IgA. Despite human mucosa serving as a reservoir for pathogens, SIgA interactions with CD89 and M4 remain poorly understood. Here we report cryo-EM structures of M4-SIgA and CD89-SIgA complexes, which unexpectedly reveal different SIgA-binding stoichiometry for M4 and CD89. Structural data, supporting experiments, and modeling indicate that copies of SIgA bound to S. pyogenes M4 will adopt similar orientations on the bacterium surface and leave one host FcαR binding site open. Results suggest unappreciated functional consequences associated with SIgA binding to host and bacterial FcαRs relevant to understanding host-microbe co-evolution, IgA effector functions and improving the outcomes of group A Streptococcus infection., (© 2023. Springer Nature Limited.)

Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS]) expresses two homologous CDF efflux transporters, MntE and CzcD, which selectively transport Mn and Zn, respectively. We discovered that the MntE- and CzcD-deficient strains exhibited a marked decrease in the viability of macrophage-differentiated THP-1 cells and neutrophils. In addition, the viability of mice infected with both deficient strains markedly increased. Consistent with a previous study, our results suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular Mn levels and contributing to the growth of GAS. The maturation and proteolytic activity of streptococcal cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zinc and copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the CzcD-deficient strain. Furthermore, Mn inhibited SpeB maturation and proteolytic activity in a MntE-deficient strain. Since the host pathogenicity of the SpeB-deficient strain was significantly reduced, maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also play an essential role in its virulence. These findings provide new insights into the molecular mechanisms of pathogenicity, which allow pathogens to survive under stressful conditions associated with elevated metal ion concentrations during host infection., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Posttranslational modifications of proteins can impact their therapeutic efficacy, stability, and potential for pharmaceutical development. The Group A Streptococcus pyogenes C5a peptidase (ScpA) is a multi-domain protein composed of an N-terminal signal peptide, a catalytic domain (including propeptide), three fibronectin domains, and cell membrane-associated domains. It is one of several proteins produced by Group A S. pyogenes known to cleave components of the human complement system. After signal peptide removal, ScpA undergoes autoproteolysis and cleaves its propeptide for full maturation. The exact location and mechanism of the propeptide cleavage, and the impact of this cleavage on stability and activity, are not clearly understood, and the exact primary sequence of the final enzyme is not known. A form of ScpA with no autoproteolysis fragments of propeptide present may be more desirable for pharmaceutical development from a regulatory and a biocompatibility in the body perspective. The current study describes an in-depth structural and functional characterization of propeptide truncated variants of ScpA expressed in Escherichia coli cells. All three purified ScpA variants, ScpA, 79ΔPro, and 92ΔPro, starting with N32, D79, and A92 positions, respectively, showed similar activity against C5a, which suggests a propeptide-independent activity profile of ScpA. CE-SDS and MALDI top-down sequencing analyses highlight a time-dependent propeptide autoproteolysis of ScpA at 37 °C with a distinct end point at A92 and/or D93. In comparison, all three variants of ScpA exhibit similar stability, melting temperatures, and secondary structure orientation. In summary, this work not only highlights propeptide localization but also provides a strategy to recombinantly produce a final mature and active form of ScpA without any propeptide-related fragments.

The host transcriptional activator Early growth response 1 (EGR1) plays a vital role in cell cycle and differentiation, cell proliferation, and regulation of cytokines and several growth factors. It is an immediate-early gene that is expressed as an initial response to various environmental stimuli. Bacterial infection is one such factor that can trigger the expression of EGR1 in host. Therefore, it is imperative to understand expression of EGR1 during early stages of host-pathogen interaction. Streptococcus pyogenes is an opportunistic bacteria causing skin and respiratory tract infections in humans. The quorum-sensing molecule, N-(3-oxododecanoyl)-l-homoserine lactone (Oxo-C12), not synthesised by S. pyogenes, can be sensed by S. pyogenes leading to molecular changes in the pathogen. In this study, we investigated the role of Oxo-C12 on EGR1 regulation in lung epithelial and murine macrophage cell line upon S. pyogenes infection. We report that Oxo-C12 sensitised S. pyogenes upregulates the transcriptional expression of EGR1 through ERK1/2 pathway. It was observed that EGR1 was not involved in the intial attachment of S. pyogenes to A549 cells. However, inhibition of EGR1 in macrophage cell line, J774A.1, through the ERK1/2 pathway resulted in decreased adhesion of S. pyogenes. The EGR1 upregulation by Oxo-C12 sensitised S. pyogenes plays a vital role in enhancing the survival of S. pyogenes in murine macrophages, leading to persistent infection. Thus, understanding the molecular modulation in the host during bacterial infection will further help develop therapeutics to target specific sites., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate against Group A Streptococcus infections. Native GAC consists of a polyrhamnose (polyRha) backbone with N-acetylglucosamine (GlcNAc) at every second rhamnose residue. Both native GAC and the polyRha backbone have been proposed as vaccine components. Here, chemical synthesis and glycoengineering were used to generate a panel of different length GAC and polyrhamnose fragments. Biochemical analyses were performed confirming that the epitope motif of GAC is composed of GlcNAc in the context of the polyrhamnose backbone. Conjugates from GAC isolated and purified from a bacterial strain and polyRha genetically expressed in E. coli and with similar molecular size to GAC were compared in different animal models. The GAC conjugate elicited higher anti-GAC IgG levels with stronger binding capacity to Group A Streptococcus strains than the polyRha one, both in mice and in rabbits. This work contributes to the development of a vaccine against Group A Streptococcus suggesting GAC as preferable saccharide antigen to include in the vaccine., Competing Interests: Declaration of competing interest This work was undertaken at the request of and was sponsored by GlaxoSmithKlineBiologicals SA. GVGH is an affiliate of GlaxoSmithKline Biologicals SA. Roberta Di Benedetto, Gianmarco Gasperini, Francesca Mancini, Martina Carducci, Luisa Massai, Omar Rossi, Francesco Berlanda Scorza, Danilo Gomes Moriel, Francesca Necchi, Roberto Adamo and Francesca Micoli are employees of the GSK group of companies. Roberta Di Benedetto, Omar Rossi, Francesco Berlanda Scorza and Danilo Gomes Moriel report shares in the GSK group of companies. Outside the submitted work, Francesco Berlanda Scorza and Danilo Gomes Moriel reports funding from CARB-X. Olimpia Pitirollo and Pedro Henriques participated in PhD programs at GSK at the time of the experimental work., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Streptococcus pyogenes displays a wide variety of pili, which is largely dependent on serotype. A distinct subset of S. pyogenes strains that possess the Nra transcriptional regulator demonstrates thermoregulated pilus production. Findings obtained in the present study of an Nra-positive serotype M49 strain revealed involvement of conserved virulence factor A (CvfA), also referred to as ribonuclease Y (RNase Y), in virulence factor expression and pilus production, while a cvfA deletion strain showed reduced pilus production and adherence to human keratinocytes as compared with wild-type and revertant strains. Furthermore, transcript levels of pilus subunits and srtC2 genes were decreased by cvfA deletion, which was remarkable at 25°C. Likewise, both messenger RNA (mRNA) and protein levels of Nra were remarkably decreased by cvfA deletion. Whether the expression of other pilus-related regulators, including fasX and CovR, was subject to thermoregulation was also examined. While the mRNA level of fasX, which inhibits cpa and fctA translation, was decreased by cvfA deletion at both 37°C and 25°C, CovR mRNA and protein levels, as well as its phosphorylation level were not significantly changed, suggesting that neither fasX nor CovR is necessarily involved in thermosensitive pilus production. Phenotypic analysis of the mutant strains revealed that culture temperature and cvfA deletion had varied effects on streptolysin S and SpeB activities. Furthermore, bactericidal assay data showed that cvfA deletion decreased the rate of survival in human blood. Together, the present findings indicate that CvfA is involved in regulation of pilus production and virulence-related phenotypes of the serotype M49 strain of S. pyogenes., (© 2023 The Societies and John Wiley & Sons Australia, Ltd.)

Microbial pathogens balance growth against tissue damage to achieve maximum fitness. Central carbon metabolism is connected to growth, but how it influences growth/damage balance is largely unknown. Here we examined how carbon flux through the exclusively fermentative metabolism of the pathogenic lactic acid bacterium Streptococcus pyogenes impacts patterns of growth and tissue damage. Using a murine model of soft tissue infection, we systematically examined single and pair-wise mutants that constrained carbon flux through the three major pathways that S. pyogenes employs for reduction of the glycolytic intermediate pyruvate, revealing distinct disease outcomes. Its canonical lactic acid pathway (via lactate dehydrogenase) made a minimal contribution to virulence. In contrast, its two parallel pathways for mixed-acid fermentation played important, but non-overlapping roles. Anaerobic mixed acid fermentation (via pyruvate formate lyase) was required for growth in tissue, while aerobic mixed-acid pathway (via pyruvate dehydrogenase) was not required for growth, but instead regulated levels of tissue damage. Infection of macrophages in vitro revealed that pyruvate dehydrogenase was required to prevent phagolysosomal acidification, which altered expression of the immunosuppressive cytokine IL-10. Infection of IL-10 deficient mice confirmed that the ability of aerobic metabolism to regulate levels of IL-10 plays a key role in the ability of S. pyogenes to modulate levels of tissue damage. Taken together, these results show critical non-overlapping roles for anaerobic and aerobic metabolism in soft tissue infection and provide a mechanism for how oxygen and carbon flux act coordinately to regulate growth/damage balance. Therapies targeting carbon flux could be developed to mitigate tissue damage during severe S. pyogenes infection., Competing Interests: The authors declare no competing interests., (Copyright: © 2023 Merriman et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The c ontrol o f v irulence two-component gene regulatory system (CovRS) is critical to the pathogenesis of many medically important streptococci. In emm1 group A streptococci (GAS), CovR directly binds the promoters of numerous GAS virulence factor-encoding genes. Elimination of CovS phosphatase activity increases CovR phosphorylation (CovR~P) levels and abrogates GAS virulence. Given the emm type-specific diversity of CovRS function, in this study we used chromatin immunoprecipitation sequencing (ChIP-seq) to define global CovR DNA occupancy in the wild-type emm3 strain MGAS10870 (medium CovR~P) and its CovS phosphatase-negative derivative 10870-CovS-T284A (high CovR~P). In the wild-type emm3 strain, 89% of the previously identified emm1 CovR binding sites present in the emm3 genome were also enriched; additionally, we ascertained unique CovR binding, primarily to genes in mobile genetic elements and other sites of interstrain chromosomal differences. Elimination of CovS phosphatase activity specifically increased CovR occupancy at the promoters of a broad array of CovR repressed virulence factor-encoding genes, including those encoding the key GAS regulator Mga and M protein. However, a limited number of promoters had augmented enrichment at low CovR~P levels. Differential motif searches using sequences enriched at high versus low CovR~P levels revealed two distinct binding patterns. At high CovR~P, a pseudopalindromic AT-rich consensus sequence (WTWTTATAAWAAAAWNATDA) consistent with CovR binding as a dimer was determined. Conversely, sequences specifically enriched at low CovR~P contained isolated ATTARA motifs suggesting an interaction with a monomer. These data extend understanding of global CovR DNA occupancy beyond emm1 GAS and provide a mechanism for previous observations regarding hypovirulence induced by CovS phosphatase abrogation. IMPORTANCE Given its key role in pathogenesis of Gram-positive bacteria, CovR is one of the most important members of the OmpR/PhoB family of transcriptional regulators. Herein we extend recent GAS CovR global binding analyses done in emm1 to a non- emm1 strain, which is important considering the known inter- emm -type heterogeneity in GAS CovRS function. Our data provide mechanistic understanding for variation in CovRS function between emm types and the profound hypovirulence of CovS phosphatase-negative strains in addition to indicating differential targeting by phosphorylated and nonphosphorylated CovR isoforms at specific CovR binding sites. These findings advance knowledge regarding how a key bacterial virulence regulator impacts pathogenesis and add to the growing appreciation of the function of nonphosphorylated OmpR/PhoB family members., Competing Interests: The authors declare no conflict of interest.

Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interactions with the target-spacer duplex in a sequence-independent manner. We revealed here that intramolecular interactions within the single guide RNA (sgRNA), between the spacer and the scaffold, cause most of these preferences. By using in cellulo and in vitro SpCas9 activity assays with systematically designed spacer and scaffold sequences and by analysing activity data from a large SpCas9 sequence library, we show that some long (>8 nucleotides) spacer motifs, that are complementary to the RAR unit of the scaffold, interfere with sgRNA loading, and that some motifs of more than 4 nucleotides, that are complementary to the SL1 unit, inhibit DNA binding and cleavage. Furthermore, we show that intramolecular interactions are present in the majority of the inactive sgRNA sequences of the library, suggesting that they are the most important intrinsic determinants of the activity of the SpCas9 ribonucleoprotein complex. We also found that in pegRNAs, sequences at the 3' extension of the sgRNA that are complementary to the SL2 unit are also inhibitory to prime editing, but not to the nuclease activity of SpCas9., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Streptococcus pyogenes (GAS) is a human bacterial pathogen that generates various mild to severe diseases. Worldwide, there are approximately 700 million cases of GAS infections per year. In some strains of GAS, the surface-resident M-protein, plasminogen-binding group A streptococcal M-protein (PAM), binds directly to human host plasminogen (hPg), where it is activated to plasmin through a mechanism involving a Pg/bacterial streptokinase (SK) complex as well as endogenous activators. Binding to Pg and its activation are dictated by selected sequences within the human host Pg protein, making it difficult to generate animal models to study this pathogen., Objectives: To develop a murine model for studying GAS infection by minimally modifying mouse Pg to enhance the affinity to bacterial PAM and sensitivity to GAS-derived SK., Methods: We used a targeting vector that contained a mouse albumin-promoter and mouse/human hybrid plasminogen cDNA targeted to the Rosa26 locus. Characterization of the mouse strain consisted of both gross and histological techniques and determination of the effects of the modified Pg protein through surface plasmon resonance measurements, Pg activation analyses, and mouse survival post-GAS infection., Results: We generated a mouse line expressing a chimeric Pg protein consisting of 2 amino acid substitutions in the heavy chain of Pg and a complete replacement of the mouse Pg light chain with the human Pg light chain., Conclusion: This protein demonstrated an enhanced affinity for bacterial PAM and sensitivity to activation by the Pg-SK complex, making the murine host susceptible to the pathogenic effects of GAS., Competing Interests: Declaration of competing interests All authors declare no conflict of interest., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Membranes contain lipids that are composed of fatty acids (FA) and a polar head. Membrane homeostasis is crucial for optimal bacterial growth and interaction with the environment. Bacteria synthesize their FAs via the FASII pathway. Gram-positive bacteria can incorporate exogenous FAs which need to be phosphorylated to become substrate of the lipid biosynthetic pathway. In many species including staphylococci, streptococci and enterococci, this phosphorylation is carried out by the Fak complex, which is composed of two subunits, FakA and FakB. FakA is the kinase. FakB proteins are members of the DegV family, proteins known to bind FAs. Two or three FakB types have been identified depending on the bacterial species and characterized by their affinity for saturated and/or unsaturated FAs. Some species such as Streptococcus pyogenes, which causes a wide variety of diseases ranging from mild non-invasive to severe invasive infections, possess an uncharacterized additional DegV protein. We identify here this DegV member as a fourth FakB protein, named FakB4. The fakB4 gene is co-regulated with FASII genes suggesting an interaction with endogenous fatty acids. fakB4 deletion has no impact on membrane phospholipid composition nor on the percentage of other major lipids. However, the fakB4 mutant strain produced more lipids and more extracellular membrane vesicles than the wild-type strain. This suggests that FakB4 is involved in endogenous FA binding and controls FA storage or catabolism resulting in a limitation of extracellular FA release via membrane vesicles., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lambert et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases ranging from mild noninvasive to severe invasive infections. To identify possible causes of colonization-to-invasive switches, we determined the genomic sequences of 10 isolates from five pairs each composed of an invasive strain and a carriage strain originating from five infectious clusters. Among them, one pair displayed a single-nucleotide difference in covS , encoding the sensor histidine kinase of the two-component CovRS system that controls the expression of 15% of the genome. In contrast to previously described cases where the invasive strains harbor nonfunctional CovS proteins, the carriage strain possessed the mutation covST115C , leading to the replacement of the tyrosine at position 39 by a histidine. The CovSY39H mutation affected the expression of the genes from the CovR regulon in a unique fashion. Genes usually overexpressed in covS mutant strains were underexpressed and vice versa. Furthermore, the covS mutant strain barely responded to the addition of the CovS-signaling compounds Mg 2+ and LL-37. The variations in the accumulation of two virulence factors paralleled the transcription modifications. In addition, the covST115C mutant strain showed less survival than its wild-type counterpart in murine macrophages. Finally, in two murine models of infection, the covS mutant strain was less virulent than the wild-type strain. Our study suggests that the CovSY39H protein compromises CovS phosphatase activity and that this yields a noninvasive strain. IMPORTANCE Streptococcus pyogenes, also known as group A Streptococcus, causes a wide variety of diseases, leading to 517,000 deaths yearly. The two-component CovRS system, which responds to MgCl 2 and the antimicrobial peptide LL-37, controls the expression of 15% of the genome. Invasive strains may harbor nonfunctional CovS sensor proteins that lead to the derepression of most virulence genes. We isolated a colonization strain that harbors a novel covS mutation. This mutant strain harbored a transcriptome profile opposite that of other covS mutant strains, barely responded to environmental signals, and was less virulent than the wild-type strain. This supports the importance of the derepression of the expression of most virulence genes, via mutations that impact the phosphorylation of the regulator CovR, for favoring S. pyogenes invasive infections.

RopB is a quorum-sensing regulator that binds to the SpeB-inducing peptide (SIP) under acidic conditions. SIP is known to be degraded by the endopeptidase PepO, whose transcription is repressed by the CovR/CovS two-component regulatory system. Both SIP-bound RopB (RopB-SIP) and SIP-free RopB (apo-RopB) can bind to the speB promoter; however, only RopB-SIP activates speB transcription. In this study, we found that the SpeB expression was higher in the ropB mutant than in the SIP-inactivated ( SIP *) mutant. Furthermore, the deletion of ropB in the SIP * mutant derepressed speB expression, suggesting that apo-RopB is a transcriptional repressor of speB Up-regulation of PepO in the covS mutant degraded SIP, resulting in the down-regulation of speB We demonstrate that deleting ropB in the covS mutant derepressed the speB expression, suggesting that the speB repression in this mutant was mediated not only by PepO-dependent SIP degradation but also by apo-RopB. These findings reveal a crosstalk between the CovR/CovS and RopB-SIP systems and redefine the role of RopB in regulating speB expression in group A Streptococcus ., (© 2023 Chiang-Ni et al.)

Maintenance of intracellular metal homeostasis during interaction with host niches is critical to the success of bacterial pathogens. To prevent infection, the mammalian innate immune response employs metal-withholding and metal-intoxication mechanisms to limit bacterial propagation. The first-row transition metal ion copper serves critical roles at the host-pathogen interface and has been associated with antimicrobial activity since antiquity. Despite lacking any known copper-utilizing proteins, streptococci have been reported to accumulate significant levels of copper. Here, we report that loss of CopA, a copper-specific exporter, confers increased sensitivity to copper in Streptococcus pyogenes strain HSC5, with prolonged exposure to physiological levels of copper resulting in reduced viability during stationary phase cultivation. This defect in stationary phase survival was rescued by supplementation with exogeneous amino acids, indicating the pathogen had altered nutritional requirements during exposure to copper stress. Furthermore, S. pyogenes HSC5 ΔcopA was substantially attenuated during murine soft-tissue infection, demonstrating the importance of copper efflux at the host-pathogen interface. Collectively, these data indicate that copper can severely reduce the viability of stationary phase S. pyogenes and that active efflux mechanisms are required to survive copper stress in vitro and during infection., Competing Interests: Declaration of Competing Interest None., (Copyright © 2023. Published by Elsevier Inc.)

Anginosus group streptococci (AGS) are opportunistic pathogens of the human oral cavity; however, their pathogenicity has not been discussed in detail. Oral streptococci live in the gingival sulcus, from where they can easily translocate into the bloodstream due to periodontal diseases and dental treatment and cause hazardous effects on the host through their virulence factors. Streptolysin S (SLS), a pathogenic factor produced by β-hemolytic species/strains belonging to AGS, plays an important role in damaging host cells. Therefore, we investigated the SLS-dependent cytotoxicity of β-hemolytic Streptococcus anginosus subsp. anginosus (SAA), focusing on different growth conditions such as in the bloodstream. Consequently, SLS-dependent hemolytic activity/cytotoxicity in the culture supernatant of β-hemolytic SAA was stabilized by blood components, particularly human serum albumin (HSA). The present study suggests that the secreted SLS, not only from β-hemolytic SAA, but also from other SLS-producing streptococci, is stabilized by HSA. As HSA is the most abundant protein in human plasma, the results of this study provide new insights into the risk of SLS-producing streptococci which can translocate into the bloodstream., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

Rheumatic heart disease (RHD) is a post-streptococcal sequela caused by Streptococcus pyogenes. The global burden of disease is high among people with low socio-economic status, with significant cases emerging every year despite global eradication efforts. The current treatment includes antibiotic therapies to target strep throat and rheumatic fever and valve replacement strategies as a corrective measure for chronic RHD patients. Valvular damage and valve calcification are considered to be the end-stage processes of the disease resulting from impairment of the endothelial arrangement due to immune infiltration. This immune infiltration is mediated by a cascade of events involving NLRP3 inflammasome activation. NLRP3 inflammasome is activated by wide range of stimuli including bacterial cell wall components like M proteins and leukocidal toxins like nicotinamide dehydrogenase (NADase) and streptolysin O (SLO) and these play a major role in sustaining the virulence of Streptococcus pyogenes and progression of RHD. In this review, we are discussing NLRP3 inflammasome and its plausible role in the pathogenesis of RHD by exploiting the host-pathogen interaction mainly focusing on the NLRP3 inflammasome-mediated cytokines IL-1β and IL-18. Different therapeutic approaches involving NLRP3 inflammasome inactivation, caspase-1 inhibition, and blockade of IL-1β and IL-18 are discussed in this review and may be promising for treating RHD patients.

Mass photometry (MP) is a single molecule technique that enables the characterization of individual proteins. Here we show a detailed workflow using the Refeyn One MP to investigate molecular complexes, using the M53 protein, a plasminogen-binding group A streptococcal M-like protein (PAM), and human plasminogen as exemplar proteins. The methodology described herein confirmed a 1:1 binding stoichiometry for the M53-plasminogen complex. Additionally, MP was used to identify the oligomerization state, homogeneity, purity, and approximate molecular weights of each protein., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Group A Streptococcus (GAS, Streptococcus pyogenes) is an exclusively human pathogen that causes a range of diseases, including pharyngitis, tonsillitis, impetigo, erysipelas, necrotizing fasciitis, and toxic shock syndrome. Post-streptococcal sequelae include acute rheumatic fever and rheumatic heart disease. The bacterium produces a large arsenal of virulence factors that contribute to host tissue adhesion/colonization, bacterial spread, and host immune evasion. Immune evasion factors include proteins that interfere with complement, a system of plasma proteins that are activated by pathogens resulting in a variety of reactions on the surface of the pathogen. This leads to the activation of active components with a variety of effector functions, such as cell lysis, opsonization, and chemotaxis of phagocytes to the site of infection. We have recently identified a novel "complement evasion factor" (CEF) in S. pyogenes. CEF directly interacts with complement proteins C1r, C1s, C3, and C5, interrupts all three complement pathways, and prevents opsonization of the bacterial surface with C3b. We here present methods used to analyze the complement interference of CEF., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Inflammasomes are large multiprotein complexes that assemble mainly in innate immune cells after detection of microbial or sterile insults. Activation of inflammasomes is a key proinflammatory event during infection, and many pathogens have evolved specific evasion mechanisms to evade or inhibit inflammasome activation. One such pathogen is the common bacterium group A Streptococcus (GAS), which causes a wide range of diseases of varying severity. GAS secretes a multitude of virulence factors whereof the pore-forming protein streptolysin O (SLO) is the main inflammasome activation determinant. Here we provide a protocol for reliable evaluation of inflammasome activation in murine bone marrow-derived macrophages (BMDM) infected with GAS, including instructions for generating BMDMs and growing the bacterium. This protocol can easily be modified to other bacterial pathogens, or human macrophages., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Group A Streptococcus (GAS) M and M-like proteins are essential virulence factors and represent the primary epidemiological marker of this pathogen. Protein sequences encoding 1054 M, Mrp and Enn proteins, from 1668 GAS genomes, were analysed by SplitsTree4, partitioning around medoids and co-occurrence. The splits network and groups-based analysis of all M and M-like proteins revealed four large protein groupings, with multiple evolutionary histories as represented by multiple edges for most splits, leading to 'M-family-groups' (FG) of protein sequences: FG I, Mrp; FG II, M protein and Protein H; FG III, Enn; and FG IV, M protein. M and Enn proteins formed two groups with nine sub-groups and Mrp proteins formed four groups with ten sub-groups. Discrete co-occurrence of M and M-like proteins were identified suggesting that while dynamic, evolution may be constrained by a combination of functional and virulence attributes. At a granular level, four distinct family-groups of M, Enn and Mrp proteins are observable, with Mrp representing the most genetically distinct of the family-group of proteins. While M and Enn protein families generally group into three distinct family-groups, horizontal and vertical gene flow between distinct GAS strains is ongoing.

The RNA-guided Cas9s serve as powerful tools for programmable gene editing and regulation; their targeting scopes and efficacies, however, are always constrained by the PAM sequence stringency. Most Streptococci Cas9s, including the prototype SpCas9 from S. pyogenes, specifically recognize a canonical NGG PAM via a conserved RxR PAM-binding motif within the PAM-interaction (PI) domain. Here, SpCas9-based mining unveils three distinct and rarely presented PAM-binding motifs (QxxxR, QxQ and RxQ) among Streptococci Cas9 orthologs. With the catalytically-dead QxxxR-containing SedCas9 from S. equinus, we dissect its NAG PAM specificity and elucidate its underlying recognition mechanism via computational prediction and mutagenesis analysis. Replacing the SedCas9 PI domain with alternate PAM-binding motifs rewires its PAM specificity to NGG or NAA. Moreover, a semi-rational design with minimal mutation creates a SedCas9-NQ variant showing robust activity towards expanded NNG and NAA PAMs, based upon which we engineered a compact ω-SedCas9-NQ transcriptional regulator for PAM-directed bifunctional and titratable gene control. The ω-SedCas9-NQ mediated metabolic reprogramming of endogenous genes in Escherichia coli affords a 2.6-fold increase of 4-hydroxycoumarin production. This work reveals new Cas9 scaffolds with distinct PAM-binding motifs for PAM relaxation and creates a new PAM-diverse Cas9 variant for versatile gene control in bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Enzymatic cleavage of IgG antibodies is a common strategy used by pathogenic bacteria to ablate immune effector function. The Streptococcus pyogenes bacterium secretes the protease IdeS and the glycosidase EndoS, which specifically catalyse cleavage and deglycosylation of human IgG, respectively. IdeS has received clinical approval for kidney transplantation in hypersensitised individuals, while EndoS has found application in engineering antibody glycosylation. We present crystal structures of both enzymes in complex with their IgG1 Fc substrate, which was achieved using Fc engineering to disfavour preferential Fc crystallisation. The IdeS protease displays extensive Fc recognition and encases the antibody hinge. Conversely, the glycan hydrolase domain in EndoS traps the Fc glycan in a "flipped-out" conformation, while additional recognition of the Fc peptide is driven by the so-called carbohydrate binding module. In this work, we reveal the molecular basis of antibody recognition by bacterial enzymes, providing a template for the development of next-generation enzymes., (© 2022. The Author(s).)

Streptococcus pyogenes is a globally prominent human-specific pathogen responsible for an enormous burden of human illnesses, including >600 million pharyngeal and >100 million skin infections each year. Despite intensive efforts that focus on invasive indications, much remains unknown about this bacterium in its natural state during colonization of the nasopharynx and skin. Using acute experimental infection models in HLA-transgenic mice, we evaluated how the hyaluronic acid (HA) capsule contributes to S. pyogenes MGAS8232 infection within these limited biological niches. Herein, we demonstrate that HA capsule expression promotes bacterial burden in murine nasal turbinates and skin lesions by resisting neutrophil-mediated killing. HA capsule production is encoded by the hasABC operon and compared to wildtype S. pyogenes infections, mice infected with a ΔhasA mutant exhibited over a 1000-fold CFU reduction at 48-hours post-nasal challenge, and a 10,000-fold CFU reduction from skin lesions 72-hours post-skin challenge. HA capsule expression contributed substantially to skin lesion size development following subdermal inoculations. In the absence of capsule expression, S. pyogenes revealed drastically impeded growth in whole human blood and increased susceptibility to killing by isolated neutrophils ex vivo, highlighting its important role in resisting phagocytosis. Furthermore, we establish that neutrophil depletion in mice recovered the reduced burden by the ΔhasA mutant in both the nasopharynx and skin. Together, this work confirms that the HA capsule is a key virulence determinant during acute infections by S. pyogenes and demonstrates that its predominant function is to protect S. pyogenes against neutrophil-mediated killing., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Hurst et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Streptococcus pyogenes, otherwise known as Group A Streptococcus (GAS), is an important and highly adaptable human pathogen with the ability to cause both superficial and severe diseases. Understanding how S. pyogenes senses and responds to its environment will likely aid in determining how it causes a breadth of diseases. One regulatory network involved in GAS's ability to sense and respond to the changing environment is the Rgg2/3 quorum sensing (QS) system, which responds to metal and carbohydrate availability and regulates changes to the bacterial surface. To better understand the impact of Rgg2/3 QS on S. pyogenes physiology, we performed RNA-seq and tandem mass tag (TMT)-LC-MS/MS analysis on cells in which this system was induced with short hydrophobic peptide (SHP) pheromone or disrupted. Primary findings confirmed that pheromone stimulation in wild-type cultures is limited to the induction of operons whose promoters contain previously determined Rgg2/3 binding sequences. However, a deletion mutant of rgg3 , a strain that endogenously produces elevated amounts of pheromone, led to extended alterations of the transcriptome and proteome, ostensibly by stress-induced pathways. Under such exaggerated pheromone conditions, a connection was identified between Rgg2/3 and the stringent response. Mutation of relA , the bifunctional guanosine tetra- and penta-phosphate nucleoside synthetase/hydrolase, and alarmone synthase genes sasA and sasB , impacted culture doubling times and disabled induction of Rgg2/3 in response to mannose, while manipulation of Rgg2/3 signaling modestly altered nucleotide levels. Our findings indicate that excessive pheromone production or exposure places stress on GAS resulting in an indirect altered proteome and transcriptome beyond primary pheromone signaling. IMPORTANCE Streptococcus pyogenes causes several important human diseases. This study evaluates how the induction or disruption of a cell-cell communication system alters S. pyogenes's gene expression and, in extreme conditions, its physiology. Using transcriptomic and proteomic approaches, the results define the pheromone-dependent regulon of the Rgg2/3 quorum sensing system. In addition, we find that excessive pheromone stimulation, generated by genetic disruption of the Rgg2/3 system, leads to stress responses that are associated with the stringent response. Disruption of stringent response affects the ability of the cell-cell communication system to respond under normally inducing conditions. These findings assist in the determination of how S. pyogenes is impacted by and responds to nontraditional sources of stress.

The group A Streptococcus (GAS; Streptococcus pyogenes) causes an elaborate array of human diseases. In part, such variability in disease potential is a consequence of GAS manipulating the expression of a catalogue of virulence factors, with regulation occurring at both the transcriptional and posttranscriptional levels. The GAS small regulatory RNA (sRNA) FasX contributes to this regulatory activity, enhancing expression of the thrombolytic agent streptokinase, and reducing expression of collagen (pili) and fibronectin (PrtF1 and PrtF2) -binding adhesins. Here, we expand insight into the regulatory targets of FasX by identifying the M-related protein (Mrp), a fibrinogen-binding adhesin with anti-phagocytic activity, as a negatively-regulated target of FasX. Importantly, investigation of the consequences of FasX-mediated regulation led to the discovery that FasX is a major positive regulator of GAS survival and proliferation in non-immune whole human blood, with a 30-fold difference in GAS cell numbers between a fasX mutant strain and isogenic parental and complemented mutant strains. No difference in cell numbers were observed when these strains were grown in human serum, consistent with the protective phenotype associated with FasX occurring due to the inhibition of cell (e.g., neutrophil) - mediated GAS killing. The FasX-regulated factor/s responsible for the blood survival phenotype remain to be defined. In summary, we expand the known FasX regulon and identify a new phenotype associated with the regulatory activity of this key GAS sRNA. IMPORTANCE Small regulatory RNAs (sRNAs) represent a major class of regulatory molecule that promotes the ability of the group A Streptococcus (GAS) and other pathogens to regulate virulence factor expression. Despite FasX being the best-described sRNA in GAS, there remains much to be learned. Here, we highlight the importance of FasX, identifying for the first time that the loss of this sRNA results in a major reduction in the ability of GAS to survive in human blood, a phenotype critical to the ability of this human-specific pathogen to cause severe invasive infections. We also identified a novel regulatory target of FasX, thereby expanding the known regulon of this key sRNA.

Genome editing tools based on CRISPR-Cas systems can repair genetic mutations in situ; however, off-target effects and DNA damage lesions that result from genome editing remain major roadblocks to its full clinical implementation. Protein and chemical inhibitors of CRISPR-Cas systems may reduce off-target effects and DNA damage. Here we describe the identification of several lead chemical inhibitors that could specifically inhibit the activity of Streptococcus pyogenes Cas9 (SpCas9). In addition, we obtained derivatives of lead inhibitors that could penetrate the cell membrane and inhibit SpCas9 in cellulo . Two of these compounds, SP2 and SP24, were able to improve the specificity of SpCas9 in cellulo at low-micromolar concentration. Furthermore, microscale thermophoresis (MST) assays showed that SP24 might inhibit SpCas9 activity by interacting with both the SpCas9 protein and the SpCas9-gRNA ribonucleoprotein complex. Taken together, SP24 is a novel chemical inhibitor of SpCas9 which has the potential to enhance therapies that utilize SpCas9.

Nicotinamide adenine dinucleotide (NAD+) is an essential co-factor for cellular metabolism and serves as a substrate in enzymatic processes. NAD+ is produced by de novo synthesis or salvage pathways in nearly all bacterial species. Haemophilus influenzae lacks the capacity for de novo synthesis, so it is dependent on import of NAD+ from the external environment or salvage biosynthetic pathways for recycling of NAD+ precursors and breakdown products. However, the actual sources of NAD+ utilized by H. influenzae in the respiratory tract are not well defined. In this study, we found that a variety of bacteria, including species found in the upper airway of humans, released NAD+ that was readily detectable in extracellular culture fluid, and which supported growth of H. influenzae in vitro. By contrast, certain strains of Streptococcus pyogenes (group A streptococcus or GAS) inhibited growth of H. influenzae in vitro by secreting NAD+-glycohydrolase (NADase), which degraded extracellular NAD+. Conversely, GAS strains that lacked enzymatically active NADase released extracellular NAD+, which could support H. influenzae growth. Our results suggest that many bacterial species, including normal flora of the upper airway, release NAD+ into the environment. GAS is distinctive in its ability to both release and degrade NAD+. Thus, colonization of the airway with H. influenzae may be promoted or restricted by co-colonization with GAS in a strain-specific manner that depends, respectively, on release of NAD+ or secretion of active NADase. We suggest that, in addition to its role as a cytotoxin for host cells, NADase may serve a separate function by restricting growth of H. influenzae in the human respiratory tract., Competing Interests: The authors have declared that no competing interests exist.