Sam Feldstein

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.

Streptococcus pyogenes or Group A Streptococcus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NFκB activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remain unknown. In this study, transcriptomic and phosphoproteomic approaches were used to address these unanswered questions. It was found that QS-ON GAS broadly suppressed most inflammatory transcriptional pathways, including those of NFκB, type I and type II interferon responses, and intracellular stress responses. Yet, we found that no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NFκB and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. A role for epigenetic regulation in QS-mediated immunomodulation was validated using pharmacological inhibitors of histone and chromatin modifiers. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ atypical methods of restricting inflammation. Uncovering this mechanism will offer invaluable insight into GAS itself, as well as into understudied immunological pathways. IMPORTANCE: Streptococcus pyogenes is a ubiquitous pathogen that causes over 600 million infections every year and 500,000 to 1 million fatalities. While in developed countries, it is generally known to cause mild conditions such as pharyngitis, it can also manifest as severe infections, such as necrotizing fasciitis and septic arthritis, and lead to post-infectious sequelae including rheumatic heart disease and glomerulonephritis. Elucidating new mechanisms of virulence in this organism, including how it evades and suppresses immune responses, is critical in understanding its pathogenicity and epidemiology, as well as identifying novel treatment avenues in this era of multidrug-resistant bacteria. In this study, we characterize the broad spectrum by which GAS modulates the host innate immune response and begin to uncover host pathways that the bacteria can use or inhibit for its survival.

Streptococcus pyogenes , or Group A Streptococus (GAS), a significant human pathogen, employs quorum sensing (QS) systems to coordinate its behavior and genetic regulation in order to enhance survival. Our previous research established that one such QS system, the Rgg2/3 system, can suppress macrophage NFκB activity and production of pro-inflammatory cytokines. Yet, the scope of suppression and the mechanism by which it occurs remains unknown. In this study, we used transcriptomic and phosphoproteomic approaches to address these unanswered questions. We found QS-ON GAS broadly suppressed most inflammatory transcriptional pathways including those of NFκB, type I and type II interferon responses, and intracellular stress responses. Yet, we found no alternative transcriptional programs were activated after QS-ON GAS infection. Additionally, phosphoproteomics showed no disruption in typical inflammatory pathways such as those related to NFκB and MAPK activation, which was confirmed by western blotting and translocation assays. Instead, the proteomic data highlighted a potential role for epigenetic mechanisms of inflammatory regulation. To determine if epigenetic regulation was involved in QS-mediated immunomodulation, DNA methylation was measured and studies were performed inhibiting various histone and chromatin modifiers. These studies also showed no dijerence between QS-ON compared with QS-OFF infected macrophages. These findings expand our understanding of QS-mediated suppression and of GAS virulence strategies that appear to employ unusual methods of restricting inflammation. Uncovering this mechanism will ojer invaluable insight into GAS, itself, as well as understudied immunological pathways. Importance: Streptococcus pyogenes is a ubiquitous pathogen that causes over 600 million infections every year and 500 thousand to 1 million fatalities. While in developed countries it is generally known to cause mild conditions such as pharyngitis, it can also manifest as severe infections such as necrotizing fasciitis, septic arthritis, and lead to post-infectious sequelae including rheumatic heart disease and glomerulonephritis. Elucidating new mechanisms of virulence in this organism, including how it evades and suppresses immune responses can be critical in understanding its pathogenicity, epidemiology, and identification of novel treatment avenues in this era of multi-drug-resistant bacteria. In this study, we characterize the broad spectrum by which GAS modulates the host innate immune response and begin to uncover host pathways that bacteria can use or inhibit for its survival.

Immunocytokines hold great potential as anticancer agents, as they use a specific antitumor antibody to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME). We have developed a novel immunocytokine (KD033) composed of a fully human, high-affinity antiprogrammed death-ligand 1 (PD-L1) linked to the sushi-domain of the human IL-15/IL-15 receptor alpha (IL-15/IL-15Rα) complex. A murine PD-L1 cross-reactive KD033 surrogate (srKD033) and a nontargeting antibody (ntKD033) were also developed to investigate mechanism of action in murine tumor models. Efficacy analyses showed a robust antitumor effect of single-dose srKD033 in several diverse syngeneic murine tumor models. In a CT26 murine colon tumor model, single-dose srKD033 produced durable antitumor immunity as evidenced by resistance to subsequent tumor rechallenges. Mice responding to srKD033 treatment showed increased retention of PD-L1/IL-15 in the TME which likely facilitated prolonged IL-15–induced expansion of cytotoxic cells. Importantly, target-based PD-L1/IL-15 delivery via srKD033 was well-tolerated and induced significant antitumor activity in murine carcinoma models that are non- or minimally responsive to IL-15 or anti–PD-L1/PD-1 monotherapy.

Immunocytokines hold great potential as anticancer agents, as they use a specific antitumor antibody to deliver an immune-activating cytokine directly to the immunosuppressive tumor microenvironment (TME). We have developed a novel immunocytokine (KD033) composed of a fully human, high-affinity antiprogrammed death-ligand 1 (PD-L1) linked to the sushi-domain of the human IL-15/IL-15 receptor alpha (IL-15/IL-15Rα) complex. A murine PD-L1 cross-reactive KD033 surrogate (srKD033) and a nontargeting antibody (ntKD033) were also developed to investigate mechanism of action in murine tumor models. Efficacy analyses showed a robust antitumor effect of single-dose srKD033 in several diverse syngeneic murine tumor models. In a CT26 murine colon tumor model, single-dose srKD033 produced durable antitumor immunity as evidenced by resistance to subsequent tumor rechallenges. Mice responding to srKD033 treatment showed increased retention of PD-L1/IL-15 in the TME which likely facilitated prolonged IL-15–induced expansion of cytotoxic cells. Importantly, target-based PD-L1/IL-15 delivery via srKD033 was well-tolerated and induced significant antitumor activity in murine carcinoma models that are non- or minimally responsive to IL-15 or anti–PD-L1/PD-1 monotherapy.

Analysis of KD033-Mediated Activation of Peripheral Immune Cell Populations in Cynomolgus Monkeys