Jennifer C. Chang

Streptococcus pyogenes is a major human-restricted pathogen capable of both localized and systemic diseases, as well as causing post-infectious acute and chronic rheumatological conditions. Despite its well-documented clinical importance and over a century of extensive research, gaps persist in understanding this pathogen. We report the discovery of a novel pigment produced by S. pyogenes when cultured in a replete, chemically defined medium. Color development accumulates during growth, requires exposure to oxygen, and remains associated with the bacterial cell. Though only 20% of a small strain collection produced the pigment (8 of 40), positive cultures were overrepresented by M1 and M89 serotypes. Given that pigments are critical virulence and fitness determinants in pathogens like Staphylococcus aureus and Streptococcus agalactiae , here, we describe initial attempts to characterize S. pyogenes pigment biosynthesis, regulation, and potential benefits to the organism. A total of 20,405 transposon mutants were screened for pigment loss in liquid culture, and we identified 94 independent hits enriched in pathways associated with isoprenoid biosynthesis, purine biosynthesis, guanosine transport, and mixed acid fermentation. Although color development requires oxygen, the extracted pigment did not provide antioxidative activity as compared with non-pigmented extracts. Pigment production was inhibited when the Rgg2/Rgg3 quorum-sensing system was active, though by an unknown mechanism. Treatment of RAW-Blue macrophages with the extracted pigment significantly reduced NFκB activation, suggesting a potential anti-inflammatory effect. Overall, this study describes a previously uncharacterized pigment produced by S. pyogenes and provides insights into its oxygen-dependent production and associated metabolic pathways. IMPORTANCE: Streptococcus pyogenes is a ubiquitous pathogen responsible for ~1.8 million severe infections and 500,000 deaths annually. This organism has not been recognized as a pigment-producing bacterium, but here we identify and characterize the production of a previously unreported colorful compound generated when cultured in a chemically defined medium. This finding reveals an underexplored aspect of S. pyogenes biology and raises the question of its contribution to pathogenesis. Given the established roles of microbial pigments in virulence and immune modulation in other pathogens, this work provides a foundation for future investigations into its functional significance and potential as a target for therapeutic or vaccine development.

Streptococcus pyogenes is a major human-restricted pathogen capable of both localized and systemic diseases, as well as causing post-infectious acute and chronic rheumatological conditions. Despite its well-documented clinical importance and over a century of extensive research, gaps persist in understanding this pathogen. We report the discovery of a novel pigment produced by S. pyogenes when cultured in a replete, chemically defined medium. Color development accumulates during growth, requires exposure to oxygen, and remains associated with the bacterial cell. Though only 20% of a small strain collection produced the pigment (8 of 40), positive cultures were overrepresented by M1 and M89 serotypes. Given that pigments are critical virulence and fitness determinants in pathogens like Staphylococcus aureus and Streptococcus agalactiae , here we describe initial attempts to characterize S. pyogenes pigment biosynthesis, regulation, and potential benefits to the organism. 20,405 transposon mutants were screened for pigment loss in liquid culture, and we identified 94 independent hits enriched in pathways associated with isoprenoid biosynthesis, purine biosynthesis, guanosine transport, and mixed acid fermentation. Although color development requires oxygen, the extracted pigment did not provide antioxidative activity as compared to non-pigmented extracts. Pigment production was inhibited when the Rgg2/Rgg3 quorum-sensing system was active, though by unknown means. Treatment of RAW-Blue macrophages with the extracted pigment significantly reduced NFκB activation, suggesting a potential anti-inflammatory effect. Overall, this study describes a previously uncharacterized pigment produced by Streptococcus pyogenes and provides insights into its oxygen-dependent production and associated metabolic pathways. SIGNIFICANCE: Streptococcus pyogenes is a ubiquitous pathogen responsible for ∼1.8 million severe infections and 500,000 deaths annually. This organism has not been recognized as a pigment-producing bacterium, but here we identify and characterize the production of a previously unreported colorful compound generated when cultured in a chemically defined medium. This finding reveals an underexplored aspect of S. pyogenes biology and raises the question of its contribution to pathogenesis. Given the established roles of microbial pigments in virulence and immune modulation in other pathogens, this work provides a foundation for future investigations into its functional significance and potential as a target for therapeutic or vaccine development.

Inosine modification on transfer RNA (tRNA) anticodon (I34) is universally conserved in three kingdoms of life and critical to tRNA decoding capabilities. We found that tRNALeu(IAG) in commensal human bacterial families in Lactobaccilalles is concurrent with genome-wide synonymous leucine codon reprogramming. Pathway analysis reveals significant synonymous Leu codon changes in proteins in multiple KEGG pathways on cellular metabolism, where many genome-wide dominant UUA in families without tRNALeu(IAG) is reprogrammed to CUU, CUC, and UUG in families with tRNALeu(IAG). We provide biochemical and phenotypic results to support mechanisms that enable synonymous Leu codon substitutions to confer greater translation equivalency and growth fitness, indicating that a tRNA inosine modification can propel the genome-wide evolution of synonymous leucine codons.

Streptococcus pyogenes (group A streptococcus [GAS]) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum-sensing (QS) system, a cell-to-cell communication network, modifies the cell surface, resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon which is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wild type and operon mutants showed that QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like N -acetylglucosamine-linked ribitol (GlcNAc-Rbo) moiety. A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon QS. IMPORTANCE: Streptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections, and can lead to post-infection sequelae like rheumatic heart disease, whose prevalence on a global scale rivals that of the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long-term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host’s innate immune response. We find that modifications to the cell wall arise when this virulence system is activated, which has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

SUMMARY: Streptococcus pyogenes (Group A Streptococcus, GAS) is a human-restricted pathogen with a range of clinical manifestations and worldwide prevalence. The GAS Rgg2/Rgg3 quorum sensing (QS) system, a cell-to-cell communication network, modifies the cell surface resulting in increased lysozyme resistance, biofilm formation, and expression of the qim operon that is responsible for modulation of innate immune responses in macrophages. The operon encodes 10 genes with predicted homology to enzymes involved in bacterial cell surface-associated carbohydrate and teichoic acid biosynthesis pathways. Comparing extracts of GAS cell wall polysaccharides between wildtype and operon mutants determined that the QS-induced genes modify the S. pyogenes cell surface by adding a wall teichoic acid-like moiety of N -acetylglucosamine-linked ribitol (GlcNAc-Rbo). A fluorescently labeled phage receptor-binding protein, RBP-13-GFP, that recognizes GlcNAc-decorated ribitol phosphate repeats, bound to the GAS surface only when qim expression was induced. Deletion of the qim operon eliminated RBP-13-GFP binding, diminished bacterial colonization, and significantly attenuated GAS pathogenesis in a murine skin infection model. These findings indicate that GAS has evolved a strategy to evade innate immune response by presenting a previously unknown carbohydrate moiety upon quorum sensing. IMPORTANCE: Streptococcus pyogenes is a major human pathogen, responsible for diverse clinical manifestations of both superficial and invasive infections and can lead to post-infection sequelae like rheumatic heart disease whose prevalence on a global scale rivals the most serious pathogens. Invasive S. pyogenes infections are currently on the rise worldwide, notably correlating with increasing pediatric cases of scarlet fever and enhancing the concern for long term complications. There is much that remains unknown about S. pyogenes virulence and pathogenicity, and studies focused on understanding basic systems regulating virulence factors could lead to better therapeutics and translational research. We show here one such example, where a bacterial communication system regulating a virulence mechanism relevant to in vivo infection confers the ability to alter the host innate immune response. We find that modifications to the cell wall arise when this virulence system is activated that has a direct role in host-pathogen interactions. Further research into this system could provide a mechanism for disruption and serve to treat S. pyogenes infection.

The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology.

Cell-cell signaling mediated by Rgg-family transcription factors and their cognate pheromones is conserved in Firmicutes , including all streptococci. In Streptococcus pyogenes , or Group A strep, one of these systems, the Rgg2/3 quorum sensing (QS) system, has been shown to regulate phenotypes including cellular aggregation and biofilm formation, lysozyme resistance, and macrophage immunosuppression. Here, we show that the abundance of several secreted virulence factors (streptolysin O, SpyCEP, and M protein) decreases upon induction of QS. The main mechanism underlying the changes in protein levels appears to be transcriptional, occurs downstream of the QS circuit and is dysregulated by the deletion of an Rgg2/3 QS-regulated major facilitator superfamily (MFS) transporter. Additionally, we identify this MFS transporter as the factor responsible for a previously observed increase in aminoglycoside sensitivity in QS-induced cells. Importance: The production of virulence factors is a tightly regulated process in bacterial pathogens. Efforts to elucidate the mechanisms by which genes are regulated may advance the understanding of factors influencing pathogen behavior or cellular physiology. This work finds that expression of a major facilitator superfamily (MFS) transporter, which is governed by a quorum sensing (QS) system, impacts the expression of multiple secreted virulence factors and accounts for a documented QS-dependent antibiotic susceptibility. Although the mechanism underlying this effect is not clear, MFS orthologs with high sequence similarity from S. pneumoniae and S. porcinus were unable to substitute indicating substrate specificity of the GAS MFS gene. These findings demonstrate novel associations between the expression of a transmembrane transporter and virulence factor expression and aminoglycoside transport.

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N-acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

The cell wall of the human bacterial pathogen Group A Streptococcus (GAS) consists of peptidoglycan decorated with the Lancefield group A carbohydrate (GAC). GAC is a promising target for the development of GAS vaccines. In this study, employing chemical, compositional, and NMR methods, we show that GAC is attached to peptidoglycan via glucosamine 1-phosphate. This structural feature makes the GAC-peptidoglycan linkage highly sensitive to cleavage by nitrous acid and resistant to mild acid conditions. Using this characteristic of the GAS cell wall, we identify PplD as a protein required for deacetylation of linkage N -acetylglucosamine (GlcNAc). X-ray structural analysis indicates that PplD performs catalysis via a modified acid/base mechanism. Genetic surveys in silico together with functional analysis indicate that PplD homologs deacetylate the polysaccharide linkage in many streptococcal species. We further demonstrate that introduction of positive charges to the cell wall by GlcNAc deacetylation protects GAS against host cationic antimicrobial proteins.

Some bacterial pathogens utilize cell-cell communication systems, such as quorum sensing (QS), to coordinate genetic programs during host colonization and infection. The human-restricted pathosymbiont Streptococcus pyogenes (group A streptococcus [GAS]) uses the Rgg2/Rgg3 QS system to modify the bacterial surface, enabling biofilm formation and lysozyme resistance. Here, we demonstrate that innate immune cell responses to GAS are substantially altered by the QS status of the bacteria. We found that macrophage activation, stimulated by multiple agonists and assessed by cytokine production and NF-κB activity, was substantially suppressed upon interaction with QS-active GAS but not QS-inactive bacteria. Neither macrophage viability nor bacterial adherence, internalization, or survival were altered by the QS activation status, yet tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and interferon beta (IFN-β) levels and NF-κB reporter activity were drastically lower following infection with QS-active GAS. Suppression required contact between viable bacteria and macrophages. A QS-regulated biosynthetic gene cluster (BGC) in the GAS genome, encoding several putative enzymes, was also required for macrophage modulation. Our findings suggest a model wherein upon contact with macrophages, QS-active GAS produce a BGC-derived factor capable of suppressing inflammatory responses. The suppressive capability of QS-active GAS is abolished after treatment with a specific QS inhibitor. These observations suggest that interfering with the ability of bacteria to collaborate via QS can serve as a strategy to counteract microbial efforts to manipulate host defenses. IMPORTANCEStreptococcus pyogenes is restricted to human hosts and commonly causes superficial diseases such as pharyngitis; it can also cause severe and deadly manifestations including necrotizing skin disease or severe postinfectious sequelae like rheumatic heart disease. Understanding the complex mechanisms used by this pathogen to manipulate host defenses could aid in developing new therapeutics to treat infections. Here, we examine the impact of a bacterial cell-cell communication system, which is highly conserved across S. pyogenes, on host innate immune responses. We find that S. pyogenes uses this system to suppress macrophage proinflammatory cytokine responses in vitro. Interference with this communication system could serve as a strategy to disarm bacteria and maintain an effective immune response.