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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 DNA stimulation of murine macrophages is TLR9-dependent.(A, B and C) To test for TLR9-dependency of GAS DNA-mediated BMDMs stimulation, the known inhibitors of the TLR9 pathway cloroquine (100 µM) and G-ODN (10 µM) as well as the stimulator ODN2395 (1 µM) were used together with BMDMs derived from wild type and TLR9-deficient mice. LPS (10 ng/ml) was used as a positive control in experiments with perturbed TLR9 signalling. The addition of chloroquine or G-ODN and the use of TLR9-deficient BMDMs significantly reduced the GAS DNA and ODN2395-mediated production of IFN-α and TNF-α while stimulation mediated by LPS was unaffected. Data were pooled from 3 experiments done in triplicate and presented as mean ± SEM. *P<0.05 ** P<0.01 *** P<0.001.
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ppat-1002736-g002: GAS DNA stimulation of murine macrophages is TLR9-dependent.(A, B and C) To test for TLR9-dependency of GAS DNA-mediated BMDMs stimulation, the known inhibitors of the TLR9 pathway cloroquine (100 µM) and G-ODN (10 µM) as well as the stimulator ODN2395 (1 µM) were used together with BMDMs derived from wild type and TLR9-deficient mice. LPS (10 ng/ml) was used as a positive control in experiments with perturbed TLR9 signalling. The addition of chloroquine or G-ODN and the use of TLR9-deficient BMDMs significantly reduced the GAS DNA and ODN2395-mediated production of IFN-α and TNF-α while stimulation mediated by LPS was unaffected. Data were pooled from 3 experiments done in triplicate and presented as mean ± SEM. *P<0.05 ** P<0.01 *** P<0.001.

Mentions: The experiments above showed that GAS DNA, containing unmethylated CpG motifs, but not human DNA, induced IFN-α and TNF-α release by BMDM. Since IFN type 1 secretion is partially mediated by TLR9 [19], we tested whether cytokine release in murine BMDMs expressing TLR9 [20], [21], [22], occurred in a TLR9-dependent manner. Chloroquine blocks endosomal acidification and is a known inhibitor of TLR9 [23], [24]. We observed a significant decrease of IFN-α and TNF-α secretion in response to GAS DNA and to the TLR9 agonist ODN2395 in BMDMs pretreated with chloroquine, whereas TLR4-mediated responses to LPS were unaffected (Fig. 2A). Similar results were obtained with the synthetic TLR9 antagonist G-ODN (Fig. 2B). To further corroborate the TLR9 dependency, experiments were repeated with BMDMs extracted from TLR9-deficient mice. Stimulation using the TLR9 agonist ODN2395 induced BMDM secretion of IFN-α and TNF-α only in the presence of a functional TLR9 pathway, whereas responses to LPS were not influenced (Fig. 2C). Similarly, after stimulation with GAS DNA, a significantly lower release of IFN-α and TNF-α was observed from TLR9-deficient compared to WT BMDMs (Fig. 2C). The stimulation of IFN-α secretion from TLR9-deficient BMDMs, albeit at a reduced level, is most likely explained by a ubiquitous interferon response to immunostimmulatory nucleic acids, mediated by cytosolic DNA sensors amongst others [25]. Similarly, recent work shows that IFN-β is secreted after challenge of TLR9-deficient macrophages with live GAS or GAS DNA complexed with RNA [16].


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 DNA stimulation of murine macrophages is TLR9-dependent.(A, B and C) To test for TLR9-dependency of GAS DNA-mediated BMDMs stimulation, the known inhibitors of the TLR9 pathway cloroquine (100 µM) and G-ODN (10 µM) as well as the stimulator ODN2395 (1 µM) were used together with BMDMs derived from wild type and TLR9-deficient mice. LPS (10 ng/ml) was used as a positive control in experiments with perturbed TLR9 signalling. The addition of chloroquine or G-ODN and the use of TLR9-deficient BMDMs significantly reduced the GAS DNA and ODN2395-mediated production of IFN-α and TNF-α while stimulation mediated by LPS was unaffected. Data were pooled from 3 experiments done in triplicate and presented as mean ± SEM. *P<0.05 ** P<0.01 *** P<0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375267&req=5

ppat-1002736-g002: GAS DNA stimulation of murine macrophages is TLR9-dependent.(A, B and C) To test for TLR9-dependency of GAS DNA-mediated BMDMs stimulation, the known inhibitors of the TLR9 pathway cloroquine (100 µM) and G-ODN (10 µM) as well as the stimulator ODN2395 (1 µM) were used together with BMDMs derived from wild type and TLR9-deficient mice. LPS (10 ng/ml) was used as a positive control in experiments with perturbed TLR9 signalling. The addition of chloroquine or G-ODN and the use of TLR9-deficient BMDMs significantly reduced the GAS DNA and ODN2395-mediated production of IFN-α and TNF-α while stimulation mediated by LPS was unaffected. Data were pooled from 3 experiments done in triplicate and presented as mean ± SEM. *P<0.05 ** P<0.01 *** P<0.001.
Mentions: The experiments above showed that GAS DNA, containing unmethylated CpG motifs, but not human DNA, induced IFN-α and TNF-α release by BMDM. Since IFN type 1 secretion is partially mediated by TLR9 [19], we tested whether cytokine release in murine BMDMs expressing TLR9 [20], [21], [22], occurred in a TLR9-dependent manner. Chloroquine blocks endosomal acidification and is a known inhibitor of TLR9 [23], [24]. We observed a significant decrease of IFN-α and TNF-α secretion in response to GAS DNA and to the TLR9 agonist ODN2395 in BMDMs pretreated with chloroquine, whereas TLR4-mediated responses to LPS were unaffected (Fig. 2A). Similar results were obtained with the synthetic TLR9 antagonist G-ODN (Fig. 2B). To further corroborate the TLR9 dependency, experiments were repeated with BMDMs extracted from TLR9-deficient mice. Stimulation using the TLR9 agonist ODN2395 induced BMDM secretion of IFN-α and TNF-α only in the presence of a functional TLR9 pathway, whereas responses to LPS were not influenced (Fig. 2C). Similarly, after stimulation with GAS DNA, a significantly lower release of IFN-α and TNF-α was observed from TLR9-deficient compared to WT BMDMs (Fig. 2C). The stimulation of IFN-α secretion from TLR9-deficient BMDMs, albeit at a reduced level, is most likely explained by a ubiquitous interferon response to immunostimmulatory nucleic acids, mediated by cytosolic DNA sensors amongst others [25]. Similarly, recent work shows that IFN-β is secreted after challenge of TLR9-deficient macrophages with live GAS or GAS DNA complexed with RNA [16].

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