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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 promotes resistance to macrophage killing.(A and B) BMDMs were inoculated at an MOI = 1 with GAS M1 and M49 strains expressing or lacking Sda1. A significantly higher survival rate, for both total (A) and in intracellular killing (B), was seen for GAS strains expressing Sda1. Differences in killing could not be observed in BMDMs derived from TLR9-deficient mice. (C) Oxidative burst of WT and TLR9-deficient BMDMs was measured as fluorescent units at 535 nm after 30 min stimulation with GAS M1 or GAS M49 strain either expressing or lacking Sda1. The oxidative burst fluorescent units of the non-stimulated control macrophages were set at 1. The presence of Sda1 decreased oxidative burst in WT BMDMs while no effect was detected in TLR9-deficient BMDMs. Data used in Fig. 5A and B were pooled from 3 experiments done in triplicate. Data shown in Fig. 5C is one out of three representative experiments each done in triplicate and presented as mean ± SEM (A and B) ± SD (C). * P<0.05 ** P<0.01.
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ppat-1002736-g005: GAS DNase Sda1 promotes resistance to macrophage killing.(A and B) BMDMs were inoculated at an MOI = 1 with GAS M1 and M49 strains expressing or lacking Sda1. A significantly higher survival rate, for both total (A) and in intracellular killing (B), was seen for GAS strains expressing Sda1. Differences in killing could not be observed in BMDMs derived from TLR9-deficient mice. (C) Oxidative burst of WT and TLR9-deficient BMDMs was measured as fluorescent units at 535 nm after 30 min stimulation with GAS M1 or GAS M49 strain either expressing or lacking Sda1. The oxidative burst fluorescent units of the non-stimulated control macrophages were set at 1. The presence of Sda1 decreased oxidative burst in WT BMDMs while no effect was detected in TLR9-deficient BMDMs. Data used in Fig. 5A and B were pooled from 3 experiments done in triplicate. Data shown in Fig. 5C is one out of three representative experiments each done in triplicate and presented as mean ± SEM (A and B) ± SD (C). * P<0.05 ** P<0.01.

Mentions: To date, DNase Sda1 has been appreciated to promote M1T1 GAS resistance to neutrophil extracellular killing due to its capacity to digest NETs [6], [7], [8]. Since we here identified a capacity of Sda1 to diminish TLR9-mediated macrophage responses, we hypothesized that the DNase activity could blunt the innate immune killing capacity of macrophages to kill GAS. Mice depleted of macrophages or treated with inhibitors of macrophage phagocytosis cannot clear GAS infections even at relatively low challenge doses [29], demonstrating the essential first line defense function of these immune cells against the pathogen. WT and TLR9-deficient BMDMs were challenged with live M1 or M49 GAS either expressing or not DNase and total, intra and extracellular bacterial killing was quantified. GAS strains expressing Sda1 survived significantly better in both the total and intracellular killing assays compared to the strains in which Sda1 was not expressed (Fig. 5A, B and S3). The Sda1-mediated survival advantages for GAS were much more pronounced in WT compared to TLR9-deficient BMDMs.


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 promotes resistance to macrophage killing.(A and B) BMDMs were inoculated at an MOI = 1 with GAS M1 and M49 strains expressing or lacking Sda1. A significantly higher survival rate, for both total (A) and in intracellular killing (B), was seen for GAS strains expressing Sda1. Differences in killing could not be observed in BMDMs derived from TLR9-deficient mice. (C) Oxidative burst of WT and TLR9-deficient BMDMs was measured as fluorescent units at 535 nm after 30 min stimulation with GAS M1 or GAS M49 strain either expressing or lacking Sda1. The oxidative burst fluorescent units of the non-stimulated control macrophages were set at 1. The presence of Sda1 decreased oxidative burst in WT BMDMs while no effect was detected in TLR9-deficient BMDMs. Data used in Fig. 5A and B were pooled from 3 experiments done in triplicate. Data shown in Fig. 5C is one out of three representative experiments each done in triplicate and presented as mean ± SEM (A and B) ± SD (C). * P<0.05 ** P<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3375267&req=5

ppat-1002736-g005: GAS DNase Sda1 promotes resistance to macrophage killing.(A and B) BMDMs were inoculated at an MOI = 1 with GAS M1 and M49 strains expressing or lacking Sda1. A significantly higher survival rate, for both total (A) and in intracellular killing (B), was seen for GAS strains expressing Sda1. Differences in killing could not be observed in BMDMs derived from TLR9-deficient mice. (C) Oxidative burst of WT and TLR9-deficient BMDMs was measured as fluorescent units at 535 nm after 30 min stimulation with GAS M1 or GAS M49 strain either expressing or lacking Sda1. The oxidative burst fluorescent units of the non-stimulated control macrophages were set at 1. The presence of Sda1 decreased oxidative burst in WT BMDMs while no effect was detected in TLR9-deficient BMDMs. Data used in Fig. 5A and B were pooled from 3 experiments done in triplicate. Data shown in Fig. 5C is one out of three representative experiments each done in triplicate and presented as mean ± SEM (A and B) ± SD (C). * P<0.05 ** P<0.01.
Mentions: To date, DNase Sda1 has been appreciated to promote M1T1 GAS resistance to neutrophil extracellular killing due to its capacity to digest NETs [6], [7], [8]. Since we here identified a capacity of Sda1 to diminish TLR9-mediated macrophage responses, we hypothesized that the DNase activity could blunt the innate immune killing capacity of macrophages to kill GAS. Mice depleted of macrophages or treated with inhibitors of macrophage phagocytosis cannot clear GAS infections even at relatively low challenge doses [29], demonstrating the essential first line defense function of these immune cells against the pathogen. WT and TLR9-deficient BMDMs were challenged with live M1 or M49 GAS either expressing or not DNase and total, intra and extracellular bacterial killing was quantified. GAS strains expressing Sda1 survived significantly better in both the total and intracellular killing assays compared to the strains in which Sda1 was not expressed (Fig. 5A, B and S3). The Sda1-mediated survival advantages for GAS were much more pronounced in WT compared to TLR9-deficient BMDMs.

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