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DNA is an antimicrobial component of neutrophil extracellular traps.

Halverson TW, Wilton M, Poon KK, Petri B, Lewenza S - PLoS Pathog. (2015)

Bottom Line: During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing.Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA.Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

View Article: PubMed Central - PubMed

Affiliation: University of Calgary, Snyder Institute for Chronic Diseases, Department of Microbiology, Immunology and Infectious Diseases, Calgary, Alberta, Canada.

ABSTRACT
Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

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Structurally intact NETs still possess histones and MPO after treatment with excess Mg2+ and PTase.Neutrophil extracellular traps were visualized with (A) Sytox Green staining of DNA and anti-histone primary antibodies. (B) Neutrophil extracellular traps were visualized with DAPI staining of DNA and anti-MPO primary antibodies. NETs were not observed in unactivated neutrophils, but were present in PMA-induced NETs that were treated with either excess 5 mM Mg2+ or exogenous PTase. Alexa Fluor 647-conjugated secondary antibodies were used to visualize histone H1 and MPO. Representative immunofluorescence images were merged to show overlap of histone H1 and MPO with structurally intact NETs. Scale bars, 10 µm.
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ppat.1004593.g006: Structurally intact NETs still possess histones and MPO after treatment with excess Mg2+ and PTase.Neutrophil extracellular traps were visualized with (A) Sytox Green staining of DNA and anti-histone primary antibodies. (B) Neutrophil extracellular traps were visualized with DAPI staining of DNA and anti-MPO primary antibodies. NETs were not observed in unactivated neutrophils, but were present in PMA-induced NETs that were treated with either excess 5 mM Mg2+ or exogenous PTase. Alexa Fluor 647-conjugated secondary antibodies were used to visualize histone H1 and MPO. Representative immunofluorescence images were merged to show overlap of histone H1 and MPO with structurally intact NETs. Scale bars, 10 µm.

Mentions: To determine whether the restored bacterial viability in the presence of NETs was due to preventing damage to the bacterial envelope, we performed flow cytometry to assess membrane integrity. Increased PI staining of NET-exposed bacteria is an indicator of membrane damage, which was completely blocked by addition of DNase (Fig. 5C). The addition of excess Mg2+ or treatment with exogenous PTase also limited membrane damage (Fig. 5C). Importantly, both Mg2+ and PTase treatments neutralized the antimicrobial activity of NETs (Fig. 5A and B) did not disrupt overall NET architecture, as MPO and histones were still present within the treated NET structures (Fig. 6). To assess the function of a NET-bound protein, we measured elastase activity in PMA-treated neutrophils and showed no difference in elastase activity when NETs were treated with exogenous PTase or Mg2+ (S5 Fig.). NET structures remain intact and contain functional proteins (elastase) after treatment with PTase or excess Mg2+, but are no longer antibacterial for E. coli and P. aeruginosa (Fig. 5). Together these results suggest an antibacterial mechanism wherein the DNA backbone of NETs target and destabilize the bacterial membrane and promotes cell death.


DNA is an antimicrobial component of neutrophil extracellular traps.

Halverson TW, Wilton M, Poon KK, Petri B, Lewenza S - PLoS Pathog. (2015)

Structurally intact NETs still possess histones and MPO after treatment with excess Mg2+ and PTase.Neutrophil extracellular traps were visualized with (A) Sytox Green staining of DNA and anti-histone primary antibodies. (B) Neutrophil extracellular traps were visualized with DAPI staining of DNA and anti-MPO primary antibodies. NETs were not observed in unactivated neutrophils, but were present in PMA-induced NETs that were treated with either excess 5 mM Mg2+ or exogenous PTase. Alexa Fluor 647-conjugated secondary antibodies were used to visualize histone H1 and MPO. Representative immunofluorescence images were merged to show overlap of histone H1 and MPO with structurally intact NETs. Scale bars, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4295883&req=5

ppat.1004593.g006: Structurally intact NETs still possess histones and MPO after treatment with excess Mg2+ and PTase.Neutrophil extracellular traps were visualized with (A) Sytox Green staining of DNA and anti-histone primary antibodies. (B) Neutrophil extracellular traps were visualized with DAPI staining of DNA and anti-MPO primary antibodies. NETs were not observed in unactivated neutrophils, but were present in PMA-induced NETs that were treated with either excess 5 mM Mg2+ or exogenous PTase. Alexa Fluor 647-conjugated secondary antibodies were used to visualize histone H1 and MPO. Representative immunofluorescence images were merged to show overlap of histone H1 and MPO with structurally intact NETs. Scale bars, 10 µm.
Mentions: To determine whether the restored bacterial viability in the presence of NETs was due to preventing damage to the bacterial envelope, we performed flow cytometry to assess membrane integrity. Increased PI staining of NET-exposed bacteria is an indicator of membrane damage, which was completely blocked by addition of DNase (Fig. 5C). The addition of excess Mg2+ or treatment with exogenous PTase also limited membrane damage (Fig. 5C). Importantly, both Mg2+ and PTase treatments neutralized the antimicrobial activity of NETs (Fig. 5A and B) did not disrupt overall NET architecture, as MPO and histones were still present within the treated NET structures (Fig. 6). To assess the function of a NET-bound protein, we measured elastase activity in PMA-treated neutrophils and showed no difference in elastase activity when NETs were treated with exogenous PTase or Mg2+ (S5 Fig.). NET structures remain intact and contain functional proteins (elastase) after treatment with PTase or excess Mg2+, but are no longer antibacterial for E. coli and P. aeruginosa (Fig. 5). Together these results suggest an antibacterial mechanism wherein the DNA backbone of NETs target and destabilize the bacterial membrane and promotes cell death.

Bottom Line: During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing.Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA.Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

View Article: PubMed Central - PubMed

Affiliation: University of Calgary, Snyder Institute for Chronic Diseases, Department of Microbiology, Immunology and Infectious Diseases, Calgary, Alberta, Canada.

ABSTRACT
Neutrophil extracellular traps (NETs) comprise an ejected lattice of chromatin enmeshed with granular and nuclear proteins that are capable of capturing and killing microbial invaders. Although widely employed to combat infection, the antimicrobial mechanism of NETs remains enigmatic. Efforts to elucidate the bactericidal component of NETs have focused on the role of NET-bound proteins including histones, calprotectin and cathepsin G protease; however, exogenous and microbial derived deoxyribonuclease (DNase) remains the most potent inhibitor of NET function. DNA possesses a rapid bactericidal activity due to its ability to sequester surface bound cations, disrupt membrane integrity and lyse bacterial cells. Here we demonstrate that direct contact and the phosphodiester backbone are required for the cation chelating, antimicrobial property of DNA. By treating NETs with excess cations or phosphatase enzyme, the antimicrobial activity of NETs is neutralized, but NET structure, including the localization and function of NET-bound proteins, is maintained. Using intravital microscopy, we visualized NET-like structures in the skin of a mouse during infection with Pseudomonas aeruginosa. Relative to other bacteria, P. aeruginosa is a weak inducer of NETosis and is more resistant to NETs. During NET exposure, we demonstrate that P. aeruginosa responds by inducing the expression of surface modifications to defend against DNA-induced membrane destabilization and NET-mediated killing. Further, we show induction of this bacterial response to NETs is largely due to the bacterial detection of DNA. Therefore, we conclude that the DNA backbone contributes both to the antibacterial nature of NETs and as a signal perceived by microbes to elicit host-resistance strategies.

Show MeSH
Related in: MedlinePlus