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Group A Streptococcus (GAS) causes a wide range of human infections, ranging from simple pharyngitis to life-threatening necrotizing fasciitis and toxic shock syndrome. A globally disseminated clone of M1T1 GAS has been associated with an increase in severe, invasive GAS infections in recent decades. The secreted GAS pore-forming toxin streptolysin O (SLO), which induces eukaryotic cell lysis in a cholesterol-dependent manner, is highly upregulated in the GAS M1T1 clone during bloodstream dissemination. SLO is known to promote GAS resistance to phagocytic clearance by neutrophils, a critical first element of host defense against invasive bacterial infection. Here, we examine the role of SLO in modulating specific neutrophil functions during their early interaction with GAS. We find that SLO at subcytotoxic concentrations and early time points is necessary and sufficient to suppress neutrophil oxidative burst, in a manner reversed by free cholesterol and anti-SLO blocking antibodies. In addition, SLO at subcytotoxic concentrations blocked neutrophil degranulation, interleukin-8 secretion and responsiveness, and elaboration of DNA-based neutrophil extracellular traps, cumulatively supporting a key role for SLO in GAS resistance to immediate neutrophil killing. A non-toxic SLO derivate elicits protective immunity against lethal GAS challenge in a murine infection model. We conclude that SLO exerts a novel cytotoxic-independent function at early stages of invasive infections (<30 min), contributing to GAS escape from neutrophil clearance.

Background. The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation has been linked to mutations in the covR/S 2- component regulator. We investigated whether a fitness cost is associated with the covS mutation that counterbalances hypervirulence. Methods. Wild-type M1T1 group A Streptococcus and an isogenic covS-mutant strain derived from animal passage were compared for adherence to human laryngeal epithelial cells, human keratinocytes, or fibronectin; biofilm formation; and binding to intact mouse skin. Targeted mutagenesis of capsule expression of both strains was performed for analysis of its unique contribution to the observed phenotypes. Results. The covS-mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS-mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS-mutant strain was demonstrated in a murine model. Conclusions. Reduced colonization capacity provides a potential explanation for why the covS mutation, which confers hypervirulence, has not become fixed in the globally disseminated M1T1 group A Streptococcus clone, but rather may arise anew under innate immune selection in individual patients.

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures.

The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington's disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections.

Pathways dependent on the transcription factor NF-κB provide innate immunity against microbes. Karin and colleagues show that mice lacking the kinase IKKβ have IL-1-dependent neutrophilia. Loss of IL-1 signaling restores blood cellularity but severely compromises antimicrobial defense. Transcription factor NF-κB and its activating kinase IKKβ are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKKβ–NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKKβ ( Ikkβ Δ mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikkβ Δ mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikkβ Δ Il1r1 −/− mice, unlike Ikkβ Δ mice, were highly susceptible to bacterial infection, which indicated that signaling via IKKβ–NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

Transcription factor NF-κB and its activating kinase IKK[beta] are associated with inflammation and are believed to be critical for innate immunity. Despite the likelihood of immune suppression, pharmacological blockade of IKK[beta]-NF-κB has been considered as a therapeutic strategy. However, we found neutrophilia in mice with inducible deletion of IKK[beta] (Ikk[beta](Δ) mice). These mice had hyperproliferative granulocyte-macrophage progenitors and pregranulocytes and a prolonged lifespan of mature neutrophils that correlated with the induction of genes encoding prosurvival molecules. Deletion of interleukin 1 receptor 1 (IL-1R1) in Ikk[beta](Δ) mice normalized blood cellularity and prevented neutrophil-driven inflammation. However, Ikk[beta](Δ)Il1r1(-/-) mice, unlike Ikk[beta](Δ) mice, were highly susceptible to bacterial infection, which indicated that signaling via IKK[beta]-NF-κB or IL-1R1 can maintain antimicrobial defenses in each other's absence, whereas inactivation of both pathways severely compromises innate immunity.

The role of sirtuin-1 (SIRT1) in innate immunity, and in particular the influence of SIRT1 on antimicrobial defense against infection, has yet to be reported but is important to define since SIRT1 inhibitors are being investigated as therapeutic agents in the treatment of cancer, Huntington's disease, and autoimmune diseases. Given the therapeutic potential of SIRT1 suppression, we sought to characterize the role of SIRT1 in host defense. Utilizing both pharmacologic methods and a genetic knockout, we demonstrate that SIRT1 expression has little influence on macrophage and neutrophil antimicrobial functions. Myeloid SIRT1 expression does not change mortality in gram-negative toxin-induced shock or gram-positive bacteremia, suggesting that therapeutic suppression of SIRT1 may be done safely without suppression of myeloid cell-specific immune responses to severe bacterial infections.

Group A Streptococcus (GAS) is a leading human pathogen infecting millions annually. Colonization is mediated through bacterial attachment to host epithelium. Internalization of epithelial cells may offer the bacteria protection from immune cells and antibiotics, and allow for penetration of epithelial barriers. We investigated the role of surface-anchored protein serum opacity factor (SOF) in the invasion of epithelial cells and virulence. Our data demonstrate that SOF plays a critical role in invasion of epithelial cells, mediated through its activity domain, independent of its fibronectin binding capacity. To this end, elimination of SOF in GAS produced smaller lesions and yielded significantly less bacteria per lesion than wild-type (WT) in a mouse model of necrotizing fasciitis. To cause invasive disease GAS must circumvent host immune defenses. Neutrophils circulate in the bloodstream until chemokine signals recruit them to sites of infections. GAS produce a protease, SpyCEP, which cleaves and inactivates one of the most potent neutrophil chemoattractants interleukin-8 (IL-8). We observe that this leads to decreased neutrophil migration compared to a GAS mutant lacking the SpyCEP gene. In addition, we demonstrate that SpyCEP inhibits the formation of neutrophil NETs and contributes to GAS-induced neutrophil apoptosis. The SpyCEP mutant was significantly attenuated for virulence in a mouse model of necrotizing fasciitis. Macrophages are necessary defenders against GAS infections. We have found that live GAS trigger significant accelerated caspase-dependent macrophage apoptosis upon phagocytosis. This apoptosis disrupts mitochondrial integrity and promotes membrane remodeling, leading to the release of cytochrome c into the cytosol. Apoptosis-induction is dependent upon the GAS pore-forming cytolysin streptolysin O (SLO), which is necessary and sufficient for this phenotype. Accelerated apoptosis hampers the ability of macrophages to kill the bacteria and release cytokines, blunting immune activation. An SLO mutant was attenuated in a mouse systemic infection model, with less bacteria surviving in the blood and reduced killing of mice. This work demonstrates the complexity of GAS interactions with the host during disease and the multitude of virulence mechanisms employed to escape immune defenses. A deeper understanding of GAS disease pathogenesis can lead to novel therapeutic strategies in the treatment of this disease.