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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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The recombinant GAS DNase Sda1 degrades genomic DNA.(A) Zymogram and (B and C) agarose gel electrophoresis were used to visualize degradation of DNA by rSda1. (B) The size of the GAS DNA treated with rSda1 for 0 to 6 hours was analyzed by agarose gel electrophoresis. (C) Supernatants of the GAS strains used in the assays were co-incubated with DNA. DNA degradation was assessed by agarose gel electrophoresis: the degradation efficiency displayed by GAS supernatants was directly compared to the degradation efficiency of different concentrations of rSda1. (D) GAS DNA was pre-incubated with rSda1 for 0 to 6 hours before the reaction mixture was tested for its capacity to induce cytokine secretion in BMDMs. The data were pooled from 3 experiments done in triplicates and presented as mean ± SEM., *P<0.05, **P<0.01 as compared to the 0 hour time point.
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ppat-1002736-g003: The recombinant GAS DNase Sda1 degrades genomic DNA.(A) Zymogram and (B and C) agarose gel electrophoresis were used to visualize degradation of DNA by rSda1. (B) The size of the GAS DNA treated with rSda1 for 0 to 6 hours was analyzed by agarose gel electrophoresis. (C) Supernatants of the GAS strains used in the assays were co-incubated with DNA. DNA degradation was assessed by agarose gel electrophoresis: the degradation efficiency displayed by GAS supernatants was directly compared to the degradation efficiency of different concentrations of rSda1. (D) GAS DNA was pre-incubated with rSda1 for 0 to 6 hours before the reaction mixture was tested for its capacity to induce cytokine secretion in BMDMs. The data were pooled from 3 experiments done in triplicates and presented as mean ± SEM., *P<0.05, **P<0.01 as compared to the 0 hour time point.

Mentions: An important characteristic of the hypervirulent globally disseminated M1T1 clone of GAS is the presence of a prophage-encoded secreted DNase, sda1[5]. Sda1 has been shown to promote M1T1 GAS virulence via degradation of NETs, allowing the bacteria to escape neutrophil killing and the tissue focus of infection, thus facilitating systemic spread of the pathogen [2], [6], [7]. Functional TLR9 is important in defense against GAS infection [14] and the DNA size required for optimal stimulation varies among host cells. Whereas B-cells are stimulated by small DNA fragments [26], macrophages show enhanced uptake and subsequent responses with increasing DNA length [26]. Having observed efficient BMDM activation by crude GAS DNA (above) we hypothesized that degradation by Sda1 could reduce stimulation of macrophage and thus be an additional immune evasion function of Sda1. To test this, we engineered recombinant GAS Sda1 (rSda1) in E. coli. Purification yielded a 45 kD recombinant protein which showed DNase activity at the expected size when analyzed by zymography (Fig. 3A). Recombinant Sda1 degraded GAS DNA in a time and concentration dependent manner (Fig. 3B–C). Recombinant Sda1 at around 4 µg/mL was similarly efficient in degrading DNA as the natively or overexpressed Sda1 in GAS supernatants (Fig. 3C). Degradation of GAS DNA by Sda1 abolished induction of TNF-α and IFN-α in BMDM's (Fig. 3D). DNase Sda1 on its own did not influence cytokine secretion (Fig. S2). Similarly DNase Sda1 treatment of GAS DNA did not affect the residual level of IFN-α and TNF-α induction when TLR9-deficient BMDMs were studied (Fig. 4A). We speculate that the decreased TLR9-dependent cytokine responses to Sda1-treated GAS DNA was mainly due to decreased average DNA size (Fig. 3B), which has also been shown by others to be crucial for cellular uptake of DNA and subsequent TLR9 stimulation [26]. In addition direct elimination of CpG motifs by the efficient enzymatic action of the bacterial DNase [10] could potentially further contribute to the differences observed. Since it has been reported that Sda1 can degrade RNA [27] and recent work shows that IFN-β is secreted by macrophages after challenge of GAS DNA complexed with RNA [16] we investigated the action of Sda1 against RNA, in addition to DNA, wondering if this could be a two-pronged approach to promote GAS infection. RNA co-incubated with our rSda1 showed no degradation when visualized by agarose gel electrophoresis (Fig. S1), suggesting that Sda1 possesses negligible or minor RNA-degrading activity under our assay conditions. To study the influence of Sda1 on TLR9-mediated macrophage responses to live GAS infection, BMDMs were challenged with the wild type M1TI GAS parent strain M1 5448 (M1WT), the isogenic GAS DNase sda1 knockout (M1Δsda1) and the sda1 complemented strain (M1Δsda1pDcsda1) using the pDcsda1 plasmid [6]. A significant increase in IFN-α and TNF-α secretion was observed from WT BMDMs challenged with the M1Δsda1 mutant strain compared to the parent and complemented strains (Fig. 4B). The observed Sda1-dependent reduction of cytokine responses to GAS was diminished in TLR9-deficient macrophages (Fig. 4B). Similarly, heterologous expression of sda1 in a less virulent M49 GAS strain diminished BMDM IFN-α and TNF-α secretion in a TLR9-dependent manner (Fig. 4C). Our paired loss- and gain-of-function analyses indicate that Sda1 is both necessary and sufficient to promote GAS avoidance of TLR9-dependent macrophage recognition [10].


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)

The recombinant GAS DNase Sda1 degrades genomic DNA.(A) Zymogram and (B and C) agarose gel electrophoresis were used to visualize degradation of DNA by rSda1. (B) The size of the GAS DNA treated with rSda1 for 0 to 6 hours was analyzed by agarose gel electrophoresis. (C) Supernatants of the GAS strains used in the assays were co-incubated with DNA. DNA degradation was assessed by agarose gel electrophoresis: the degradation efficiency displayed by GAS supernatants was directly compared to the degradation efficiency of different concentrations of rSda1. (D) GAS DNA was pre-incubated with rSda1 for 0 to 6 hours before the reaction mixture was tested for its capacity to induce cytokine secretion in BMDMs. The data were pooled from 3 experiments done in triplicates and presented as mean ± SEM., *P<0.05, **P<0.01 as compared to the 0 hour time point.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375267&req=5

ppat-1002736-g003: The recombinant GAS DNase Sda1 degrades genomic DNA.(A) Zymogram and (B and C) agarose gel electrophoresis were used to visualize degradation of DNA by rSda1. (B) The size of the GAS DNA treated with rSda1 for 0 to 6 hours was analyzed by agarose gel electrophoresis. (C) Supernatants of the GAS strains used in the assays were co-incubated with DNA. DNA degradation was assessed by agarose gel electrophoresis: the degradation efficiency displayed by GAS supernatants was directly compared to the degradation efficiency of different concentrations of rSda1. (D) GAS DNA was pre-incubated with rSda1 for 0 to 6 hours before the reaction mixture was tested for its capacity to induce cytokine secretion in BMDMs. The data were pooled from 3 experiments done in triplicates and presented as mean ± SEM., *P<0.05, **P<0.01 as compared to the 0 hour time point.
Mentions: An important characteristic of the hypervirulent globally disseminated M1T1 clone of GAS is the presence of a prophage-encoded secreted DNase, sda1[5]. Sda1 has been shown to promote M1T1 GAS virulence via degradation of NETs, allowing the bacteria to escape neutrophil killing and the tissue focus of infection, thus facilitating systemic spread of the pathogen [2], [6], [7]. Functional TLR9 is important in defense against GAS infection [14] and the DNA size required for optimal stimulation varies among host cells. Whereas B-cells are stimulated by small DNA fragments [26], macrophages show enhanced uptake and subsequent responses with increasing DNA length [26]. Having observed efficient BMDM activation by crude GAS DNA (above) we hypothesized that degradation by Sda1 could reduce stimulation of macrophage and thus be an additional immune evasion function of Sda1. To test this, we engineered recombinant GAS Sda1 (rSda1) in E. coli. Purification yielded a 45 kD recombinant protein which showed DNase activity at the expected size when analyzed by zymography (Fig. 3A). Recombinant Sda1 degraded GAS DNA in a time and concentration dependent manner (Fig. 3B–C). Recombinant Sda1 at around 4 µg/mL was similarly efficient in degrading DNA as the natively or overexpressed Sda1 in GAS supernatants (Fig. 3C). Degradation of GAS DNA by Sda1 abolished induction of TNF-α and IFN-α in BMDM's (Fig. 3D). DNase Sda1 on its own did not influence cytokine secretion (Fig. S2). Similarly DNase Sda1 treatment of GAS DNA did not affect the residual level of IFN-α and TNF-α induction when TLR9-deficient BMDMs were studied (Fig. 4A). We speculate that the decreased TLR9-dependent cytokine responses to Sda1-treated GAS DNA was mainly due to decreased average DNA size (Fig. 3B), which has also been shown by others to be crucial for cellular uptake of DNA and subsequent TLR9 stimulation [26]. In addition direct elimination of CpG motifs by the efficient enzymatic action of the bacterial DNase [10] could potentially further contribute to the differences observed. Since it has been reported that Sda1 can degrade RNA [27] and recent work shows that IFN-β is secreted by macrophages after challenge of GAS DNA complexed with RNA [16] we investigated the action of Sda1 against RNA, in addition to DNA, wondering if this could be a two-pronged approach to promote GAS infection. RNA co-incubated with our rSda1 showed no degradation when visualized by agarose gel electrophoresis (Fig. S1), suggesting that Sda1 possesses negligible or minor RNA-degrading activity under our assay conditions. To study the influence of Sda1 on TLR9-mediated macrophage responses to live GAS infection, BMDMs were challenged with the wild type M1TI GAS parent strain M1 5448 (M1WT), the isogenic GAS DNase sda1 knockout (M1Δsda1) and the sda1 complemented strain (M1Δsda1pDcsda1) using the pDcsda1 plasmid [6]. A significant increase in IFN-α and TNF-α secretion was observed from WT BMDMs challenged with the M1Δsda1 mutant strain compared to the parent and complemented strains (Fig. 4B). The observed Sda1-dependent reduction of cytokine responses to GAS was diminished in TLR9-deficient macrophages (Fig. 4B). Similarly, heterologous expression of sda1 in a less virulent M49 GAS strain diminished BMDM IFN-α and TNF-α secretion in a TLR9-dependent manner (Fig. 4C). Our paired loss- and gain-of-function analyses indicate that Sda1 is both necessary and sufficient to promote GAS avoidance of TLR9-dependent macrophage recognition [10].

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