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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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P. aeruginosa PAO1 is trapped by human and mouse neutrophil extracellular traps.(A) PMA-induced NETs trapped P. aeruginosa Tn7::gfp and contained myeloperoxidase (MPO), DNA and histones when visualized by immunofluorescence with antibodies from autoimmune patient sera (see Methods). Representative images of the NET components (left), Gfp-tagged PAO1 (middle), and the merged (right) are presented. (B) NETosis in the skin of mice infected with ChFP-labeled P. aeruginosa as visualized by Sytox green stained extracellular DNA structures. Scale bar: 40 μm. (C) Arrows indicate ChFP-labeled P. aeruginosa trapped by Sytox green-stained NETs in vivo. (D) ChFP-labeled P. aeruginosa is phagocytosed by neutrophils during a skin infection model. Neutrophils (blue) are visualized with anti-mouse GR-1 antibody. Scale bar: 25 μm.
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ppat.1004593.g001: P. aeruginosa PAO1 is trapped by human and mouse neutrophil extracellular traps.(A) PMA-induced NETs trapped P. aeruginosa Tn7::gfp and contained myeloperoxidase (MPO), DNA and histones when visualized by immunofluorescence with antibodies from autoimmune patient sera (see Methods). Representative images of the NET components (left), Gfp-tagged PAO1 (middle), and the merged (right) are presented. (B) NETosis in the skin of mice infected with ChFP-labeled P. aeruginosa as visualized by Sytox green stained extracellular DNA structures. Scale bar: 40 μm. (C) Arrows indicate ChFP-labeled P. aeruginosa trapped by Sytox green-stained NETs in vivo. (D) ChFP-labeled P. aeruginosa is phagocytosed by neutrophils during a skin infection model. Neutrophils (blue) are visualized with anti-mouse GR-1 antibody. Scale bar: 25 μm.

Mentions: P. aeruginosa has been shown to induce the formation of neutrophil extracellular traps in purified human neutrophils [18] and NETs have been observed in CF sputum [16, 17], where P. aeruginosa is a predominant pathogen. PMA-induced NETs contained known neutrophil proteins embedded in the DNA lattice, including myeloperoxidase (MPO) and histones [2] (Fig. 1A). We also show that Gfp-tagged P. aeruginosa is efficiently trapped and aggregated in NETs (Fig. 1A). However, in vivo NETosis during P. aeruginosa infection has not been reported. Therefore intravital confocal microscopy was used to determine whether P. aeruginosa elicited NETosis in the mouse skin infection model [26]. Infection with P. aeruginosa led to the production of large NET-like structures that stained with the DNA-binding dye Sytox green and entrapped ChFP-labeled P. aeruginosa (Fig. 1B and C). In addition to the presence of NETs, neutrophils remained chemotactic and phagocytosed bacteria, suggesting that multiple neutrophil clearance mechanisms are employed in vivo to combat P. aeruginosa (Fig. 1D and S1 Movie).


DNA is an antimicrobial component of neutrophil extracellular traps.

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

P. aeruginosa PAO1 is trapped by human and mouse neutrophil extracellular traps.(A) PMA-induced NETs trapped P. aeruginosa Tn7::gfp and contained myeloperoxidase (MPO), DNA and histones when visualized by immunofluorescence with antibodies from autoimmune patient sera (see Methods). Representative images of the NET components (left), Gfp-tagged PAO1 (middle), and the merged (right) are presented. (B) NETosis in the skin of mice infected with ChFP-labeled P. aeruginosa as visualized by Sytox green stained extracellular DNA structures. Scale bar: 40 μm. (C) Arrows indicate ChFP-labeled P. aeruginosa trapped by Sytox green-stained NETs in vivo. (D) ChFP-labeled P. aeruginosa is phagocytosed by neutrophils during a skin infection model. Neutrophils (blue) are visualized with anti-mouse GR-1 antibody. Scale bar: 25 μm.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004593.g001: P. aeruginosa PAO1 is trapped by human and mouse neutrophil extracellular traps.(A) PMA-induced NETs trapped P. aeruginosa Tn7::gfp and contained myeloperoxidase (MPO), DNA and histones when visualized by immunofluorescence with antibodies from autoimmune patient sera (see Methods). Representative images of the NET components (left), Gfp-tagged PAO1 (middle), and the merged (right) are presented. (B) NETosis in the skin of mice infected with ChFP-labeled P. aeruginosa as visualized by Sytox green stained extracellular DNA structures. Scale bar: 40 μm. (C) Arrows indicate ChFP-labeled P. aeruginosa trapped by Sytox green-stained NETs in vivo. (D) ChFP-labeled P. aeruginosa is phagocytosed by neutrophils during a skin infection model. Neutrophils (blue) are visualized with anti-mouse GR-1 antibody. Scale bar: 25 μm.
Mentions: P. aeruginosa has been shown to induce the formation of neutrophil extracellular traps in purified human neutrophils [18] and NETs have been observed in CF sputum [16, 17], where P. aeruginosa is a predominant pathogen. PMA-induced NETs contained known neutrophil proteins embedded in the DNA lattice, including myeloperoxidase (MPO) and histones [2] (Fig. 1A). We also show that Gfp-tagged P. aeruginosa is efficiently trapped and aggregated in NETs (Fig. 1A). However, in vivo NETosis during P. aeruginosa infection has not been reported. Therefore intravital confocal microscopy was used to determine whether P. aeruginosa elicited NETosis in the mouse skin infection model [26]. Infection with P. aeruginosa led to the production of large NET-like structures that stained with the DNA-binding dye Sytox green and entrapped ChFP-labeled P. aeruginosa (Fig. 1B and C). In addition to the presence of NETs, neutrophils remained chemotactic and phagocytosed bacteria, suggesting that multiple neutrophil clearance mechanisms are employed in vivo to combat P. aeruginosa (Fig. 1D and S1 Movie).

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