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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.)

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.)

The nasopharynx and the skin are the major oxygen-rich anatomical sites for colonization by the human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]). To establish infection, GAS must survive oxidative stress generated during aerobic metabolism and the release of reactive oxygen species (ROS) by host innate immune cells. Glutathione is the major host antioxidant molecule, while GAS is glutathione auxotrophic. Here, we report the molecular characterization of the ABC transporter substrate binding protein GshT in the GAS glutathione salvage pathway. We demonstrate that glutathione uptake is critical for aerobic growth of GAS and that impaired import of glutathione induces oxidative stress that triggers enhanced production of the reducing equivalent NADPH. Our results highlight the interrelationship between glutathione assimilation, carbohydrate metabolism, virulence factor production, and innate immune evasion. Together, these findings suggest an adaptive strategy employed by extracellular bacterial pathogens to exploit host glutathione stores for their own benefit. IMPORTANCE During infection, microbes must escape host immune responses and survive exposure to reactive oxygen species produced by immune cells. Here, we identify the ABC transporter substrate binding protein GshT as a key component of the glutathione salvage pathway in glutathione-auxotrophic GAS. Host-acquired glutathione is crucial to the GAS antioxidant defense system, facilitating escape from the host innate immune response. This study demonstrates a direct link between glutathione assimilation, aerobic metabolism, and virulence factor production in an important human pathogen. Our findings provide mechanistic insight into host adaptation that enables extracellular bacterial pathogens such as GAS to exploit the abundance of glutathione in the host cytosol for their own benefit.

The re-emergence of scarlet fever poses a new global public health threat. The capacity of North-East Asian serotype M12 (emm12) Streptococcus pyogenes (group A Streptococcus, GAS) to cause scarlet fever has been linked epidemiologically to the presence of novel prophages, including prophage ΦHKU.vir encoding the secreted superantigens SSA and SpeC and the DNase Spd1. Here, we report the molecular characterization of ΦHKU.vir-encoded exotoxins. We demonstrate that streptolysin O (SLO)-induced glutathione efflux from host cellular stores is a previously unappreciated GAS virulence mechanism that promotes SSA release and activity, representing the first description of a thiol-activated bacterial superantigen. Spd1 is required for resistance to neutrophil killing. Investigating single, double and triple isogenic knockout mutants of the ΦHKU.vir-encoded exotoxins, we find that SpeC and Spd1 act synergistically to facilitate nasopharyngeal colonization in a mouse model. These results offer insight into the pathogenesis of scarlet fever-causing GAS mediated by prophage ΦHKU.vir exotoxins.

Glycans, also known as carbohydrates, are abundant upon cell surfaces, where they often mediate host-pathogen interactions. The specific recognition of host glycans by pathogenic lectins is an important process that allows the adherence of bacteria to the host epithelial surface in many species, including Group A Streptococcus (GAS). Glycan microarrays present a sensitive, high-throughput approach for identifying novel lectin-glycan interactions and can be applied in the context of whole bacteria or purified bacterial proteins.

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

Streptococcus pyogenes (group A streptococcus [GAS]) is responsible for over 500,000 deaths worldwide each year. The highly virulent M1T1 GAS clone is one of the most frequently isolated serotypes from streptococcal pharyngitis and invasive disease. The oral epithelial tract is a niche highly abundant in glycosylated structures, particularly those of the ABO(H) blood group antigen family. Using a high-throughput approach, we determined that a strain representative of the globally disseminated M1T1 GAS clone 5448 interacts with numerous, structurally diverse glycans. Preeminent among GAS virulence factors is the surface-expressed M protein. M1 protein showed high affinity for several terminal galactose blood group antigen structures. Deletion mutagenesis shows that M1 protein mediates glycan binding via its B repeat domains. Association of M1T1 GAS with oral epithelial cells varied significantly as a result of phenotypic differences in blood group antigen expression, with significantly higher adherence to those cells expressing H antigen structures compared to cells expressing A, B, or AB antigen structures. These data suggest a novel mechanism for GAS attachment to host cells and propose a link between host blood group antigen expression and M1T1 GAS colonization., Importance: There has been a resurgence in group A streptococcal (GAS) invasive disease, which has been paralleled by the emergence of the highly virulent M1T1 GAS clone. Intensive research has focused on mechanisms that contribute to the invasive nature of this serotype, while the mechanisms that contribute to host susceptibility to disease and bacterial colonization and persistence are still poorly understood. The M1T1 GAS clone is frequently isolated from the throat, an environment highly abundant in blood group antigen structures. This work examined the interaction of the M1 protein, the preeminent GAS surface protein, against a wide range of host-expressed glycan structures. Our data suggest that susceptibility to infection by GAS in the oral tract may correlate with phenotypic differences in host blood group antigen expression. Thus, variations in host blood group antigen expression may serve as a selective pressure contributing to the dissemination and overrepresentation of M1T1 GAS., (Copyright © 2017 De Oliveira et al.)

Plasminogen (Plg) circulates in the host as two predominant glycoforms. Glycoform I Plg (GI-Plg) contains glycosylation sites at Asn289 and Thr346, whereas glycoform II Plg (GII-Plg) is exclusively glycosylated at Thr346. Surface plasmon resonance experiments demonstrated that Plg binding group A streptococcal M protein (PAM) exhibits comparative equal affinity for GI- and GII-Plg in the "closed" conformation (for GII-Plg, KD = 27.4 nM; for GI-Plg, KD = 37.0 nM). When Plg was in the "open" conformation, PAM exhibited an 11-fold increase in affinity for GII-Plg (KD = 2.8 nM) compared with that for GI-Plg (KD = 33.2 nM). The interaction of PAM with Plg is believed to be mediated by lysine binding sites within kringle (KR) 2 of Plg. PAM-GI-Plg interactions were fully inhibited with 100 mM lysine analogue ε-aminocaproic acid (εACA), whereas PAM-GII-Plg interactions were shown to be weakened but not inhibited in the presence of 400 mM εACA. In contrast, binding to the KR1-3 domains of GII-Plg (angiostatin) by PAM was completely inhibited in the presence 5 mM εACA. Along with PAM, emm pattern D GAS isolates express a phenotypically distinct SK variant (type 2b SK) that requires Plg ligands such as PAM to activate Plg. Type 2b SK was able to generate an active site and activate GII-Plg at a rate significantly higher than that of GI-Plg when bound to PAM. Taken together, these data suggest that GAS selectively recruits and activates GII-Plg. Furthermore, we propose that the interaction between PAM and Plg may be partially mediated by a secondary binding site outside of KR2, affected by glycosylation at Asn289.

Streptococcus pyogenes ranks among the main causes of mortality from bacterial infections worldwide. Currently there is no vaccine to prevent diseases such as rheumatic heart disease and invasive streptococcal infection. The streptococcal M protein that is used as the substrate for epidemiological typing is both a virulence factor and a vaccine antigen. Over 220 variants of this protein have been described, making comparisons between proteins difficult, and hindering M protein-based vaccine development. A functional classification based on 48 emm-clusters containing closely related M proteins that share binding and structural properties is proposed. The need for a paradigm shift from type-specific immunity against S. pyogenes to emm-cluster based immunity for this bacterium should be further investigated. Implementation of this emm-cluster-based system as a standard typing scheme for S. pyogenes will facilitate the design of future studies of M protein function, streptococcal virulence, epidemiological surveillance, and vaccine development., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

A new variant of Streptococcus pyogenes serotype M1 (designated 'M1UK') has been reported in the United Kingdom, linked with seasonal scarlet fever surges, marked increase in invasive infections, and exhibiting enhanced expression of the superantigen SpeA. The progenitor S. pyogenes 'M1global' and M1UK clones can be differentiated by 27 SNPs and 4 indels, yet the mechanism for speA upregulation is unknown. Here we investigate the previously unappreciated expansion of M1UK in Australia, now isolated from the majority of serious infections caused by serotype M1 S. pyogenes. M1UK sub-lineages circulating in Australia also contain a novel toxin repertoire associated with epidemic scarlet fever causing S. pyogenes in Asia. A single SNP in the 5' transcriptional leader sequence of the transfer-messenger RNA gene ssrA drives enhanced SpeA superantigen expression as a result of ssrA terminator read-through in the M1UK lineage. This represents a previously unappreciated mechanism of toxin expression and urges enhanced international surveillance. A variant of group A Streptococcus serotype M1 (UK) has been increasingly reported and can be differentiated from the global variant by its overexpression of the superantigen SpeA. Here, Davies et al probe the mechanism behind enhanced SpeA expression and demonstrate that a SNP in the 5' leader sequence of ssrA is responsible for this virulence phenotype. [ABSTRACT FROM AUTHOR]

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