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DNase Sda1 allows invasive M1T1 Group A Streptococcus to prevent TLR9-dependent recognition.

Uchiyama S, Andreoni F, Schuepbach RA, Nizet V, Zinkernagel AS - PLoS Pathog. (2012)

Bottom Line: Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background.Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity.Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.

ABSTRACT
Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

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GAS DNase Sda1 diminishes local secretion of IFN-α and TNF-α in a mouse necrotizing fasciitis model.(A and B) IFN-α and TNF-α concentrations were measured by ELISA in skin homogenates of mice infected with WT GASM1 or Δsda1 mutant strains 4 days previously. Samples derived from WT mice infected with GASM1 WT contained significantly lower levels of IFN-α and TNF-α compared to the samples derived from WT mice infected with GASM1 Δsda1. Low levels of IFN-α and TNF-α were detected in TLR-9 mice irrespective of the presence of Sda1. Data are displayed as box blots with n = 5 for the group of TLR9 mice injected with GAS Δsda1 and n = 6 for the other groups. Box and whiskers plot, box containing 50% with median, whiskers 2.5–97.5%. The data shown are pooled from two independent experiments. In (A) ANOVA was significant at *P<0.05, with the group of WT mice infected with GASM1 Δsda1 having stronger IFN-α induction (Bonferroni comparison). In (B) a factorial analysis revealed that both the mouse (P<0.01) and bacteria strain (P<0.02) as significant factors with significant interaction (P<0.01).
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ppat-1002736-g006: GAS DNase Sda1 diminishes local secretion of IFN-α and TNF-α in a mouse necrotizing fasciitis model.(A and B) IFN-α and TNF-α concentrations were measured by ELISA in skin homogenates of mice infected with WT GASM1 or Δsda1 mutant strains 4 days previously. Samples derived from WT mice infected with GASM1 WT contained significantly lower levels of IFN-α and TNF-α compared to the samples derived from WT mice infected with GASM1 Δsda1. Low levels of IFN-α and TNF-α were detected in TLR-9 mice irrespective of the presence of Sda1. Data are displayed as box blots with n = 5 for the group of TLR9 mice injected with GAS Δsda1 and n = 6 for the other groups. Box and whiskers plot, box containing 50% with median, whiskers 2.5–97.5%. The data shown are pooled from two independent experiments. In (A) ANOVA was significant at *P<0.05, with the group of WT mice infected with GASM1 Δsda1 having stronger IFN-α induction (Bonferroni comparison). In (B) a factorial analysis revealed that both the mouse (P<0.01) and bacteria strain (P<0.02) as significant factors with significant interaction (P<0.01).

Mentions: To provide in vivo corroboration of the ex vivo experiments carried out using BMDMs, we examined IFN-α and TNF-α levels in skin homogenates of mice infected subcutaneously with GAS. Despite the important contribution of Sda1 to GAS proliferation and necrotic ulcer development in this model [6], [7], WT mice infected with the GAS M1Δsda1 mutant showed higher levels of IFN-α and TNF-α in the skin samples than mice infected with the WT parent GAS expressing Sda1 (Fig. 6A, B). Parallel experiments performed in TLR9-deficient mice showed much lower cytokine levels in the infected skin compared to WT mice, again underlining the importance of TLR9 in mediating cytokine responses. However, in contrast to the WT mice, the presence or absence of Sda1 did not affect the level of cytokines produced in response to GAS in the TLR9-deficient mice (Fig. 6A, B). We had shown previously [14] that more bacteria are found in the skin of TLR9-deficient mice compared to WT mice, and that more surviving GASM1 WT bacteria compared to GASM1Δsda1 are present following experimental challenge of WT mice [6]. We also found more WT than ΔSda1 mutant bacteria present following injection into TLR9- deficient mice (Fig. S8). The observation that the TLR9-deficient mice injected with the GASM1Δsda1 mutant demonstrated similar bacterial counts compared to WT mice could be due to a large initial influx of neutrophils efficiently clearing the DNase-deficient mutant strain within extracellular traps. The increased cytokine levels detected in WT mice injected with GASM1Δsda1 mutant compared to WT bacteria are not explained by differences in bacterial counts, nor do bacterial levels account for increased cytokine levels in WT mice compared to TLR9- deficient mice. In sum, we documented that increased tissue expression of IFN-α and TNF-α in the mouse necrotizing skin infection model occurred in both a DNase- and TLR9-dependent manner.


DNase Sda1 allows invasive M1T1 Group A Streptococcus to prevent TLR9-dependent recognition.

Uchiyama S, Andreoni F, Schuepbach RA, Nizet V, Zinkernagel AS - PLoS Pathog. (2012)

GAS DNase Sda1 diminishes local secretion of IFN-α and TNF-α in a mouse necrotizing fasciitis model.(A and B) IFN-α and TNF-α concentrations were measured by ELISA in skin homogenates of mice infected with WT GASM1 or Δsda1 mutant strains 4 days previously. Samples derived from WT mice infected with GASM1 WT contained significantly lower levels of IFN-α and TNF-α compared to the samples derived from WT mice infected with GASM1 Δsda1. Low levels of IFN-α and TNF-α were detected in TLR-9 mice irrespective of the presence of Sda1. Data are displayed as box blots with n = 5 for the group of TLR9 mice injected with GAS Δsda1 and n = 6 for the other groups. Box and whiskers plot, box containing 50% with median, whiskers 2.5–97.5%. The data shown are pooled from two independent experiments. In (A) ANOVA was significant at *P<0.05, with the group of WT mice infected with GASM1 Δsda1 having stronger IFN-α induction (Bonferroni comparison). In (B) a factorial analysis revealed that both the mouse (P<0.01) and bacteria strain (P<0.02) as significant factors with significant interaction (P<0.01).
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ppat-1002736-g006: GAS DNase Sda1 diminishes local secretion of IFN-α and TNF-α in a mouse necrotizing fasciitis model.(A and B) IFN-α and TNF-α concentrations were measured by ELISA in skin homogenates of mice infected with WT GASM1 or Δsda1 mutant strains 4 days previously. Samples derived from WT mice infected with GASM1 WT contained significantly lower levels of IFN-α and TNF-α compared to the samples derived from WT mice infected with GASM1 Δsda1. Low levels of IFN-α and TNF-α were detected in TLR-9 mice irrespective of the presence of Sda1. Data are displayed as box blots with n = 5 for the group of TLR9 mice injected with GAS Δsda1 and n = 6 for the other groups. Box and whiskers plot, box containing 50% with median, whiskers 2.5–97.5%. The data shown are pooled from two independent experiments. In (A) ANOVA was significant at *P<0.05, with the group of WT mice infected with GASM1 Δsda1 having stronger IFN-α induction (Bonferroni comparison). In (B) a factorial analysis revealed that both the mouse (P<0.01) and bacteria strain (P<0.02) as significant factors with significant interaction (P<0.01).
Mentions: To provide in vivo corroboration of the ex vivo experiments carried out using BMDMs, we examined IFN-α and TNF-α levels in skin homogenates of mice infected subcutaneously with GAS. Despite the important contribution of Sda1 to GAS proliferation and necrotic ulcer development in this model [6], [7], WT mice infected with the GAS M1Δsda1 mutant showed higher levels of IFN-α and TNF-α in the skin samples than mice infected with the WT parent GAS expressing Sda1 (Fig. 6A, B). Parallel experiments performed in TLR9-deficient mice showed much lower cytokine levels in the infected skin compared to WT mice, again underlining the importance of TLR9 in mediating cytokine responses. However, in contrast to the WT mice, the presence or absence of Sda1 did not affect the level of cytokines produced in response to GAS in the TLR9-deficient mice (Fig. 6A, B). We had shown previously [14] that more bacteria are found in the skin of TLR9-deficient mice compared to WT mice, and that more surviving GASM1 WT bacteria compared to GASM1Δsda1 are present following experimental challenge of WT mice [6]. We also found more WT than ΔSda1 mutant bacteria present following injection into TLR9- deficient mice (Fig. S8). The observation that the TLR9-deficient mice injected with the GASM1Δsda1 mutant demonstrated similar bacterial counts compared to WT mice could be due to a large initial influx of neutrophils efficiently clearing the DNase-deficient mutant strain within extracellular traps. The increased cytokine levels detected in WT mice injected with GASM1Δsda1 mutant compared to WT bacteria are not explained by differences in bacterial counts, nor do bacterial levels account for increased cytokine levels in WT mice compared to TLR9- deficient mice. In sum, we documented that increased tissue expression of IFN-α and TNF-α in the mouse necrotizing skin infection model occurred in both a DNase- and TLR9-dependent manner.

Bottom Line: Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background.Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity.Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

View Article: PubMed Central - PubMed

Affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital Zurich, University of Zurich, Zurich, Switzerland.

ABSTRACT
Group A Streptococcus (GAS) has developed a broad arsenal of virulence factors that serve to circumvent host defense mechanisms. The virulence factor DNase Sda1 of the hyperinvasive M1T1 GAS clone degrades DNA-based neutrophil extracellular traps allowing GAS to escape extracellular killing. TLR9 is activated by unmethylated CpG-rich bacterial DNA and enhances innate immune resistance. We hypothesized that Sda1 degradation of bacterial DNA could alter TLR9-mediated recognition of GAS by host innate immune cells. We tested this hypothesis using a dual approach: loss and gain of function of DNase in isogenic GAS strains and presence and absence of TLR9 in the host. Either DNA degradation by Sda1 or host deficiency of TLR9 prevented GAS induced IFN-α and TNF-α secretion from murine macrophages and contributed to bacterial survival. Similarly, in a murine necrotizing fasciitis model, IFN-α and TNF-α levels were significantly decreased in wild type mice infected with GAS expressing Sda1, whereas no such Sda1-dependent effect was seen in a TLR9-deficient background. Thus GAS Sda1 suppressed both the TLR9-mediated innate immune response and macrophage bactericidal activity. Our results demonstrate a novel mechanism of bacterial innate immune evasion based on autodegradation of CpG-rich DNA by a bacterial DNase.

Show MeSH
Related in: MedlinePlus