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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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Neutralizing the cation chelating activity of the DNA backbone of NETs protects bacteria.(A) Percent survival of P. aeruginosa PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg2+. Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P<0.05 or P<0.01, respectively) between NET-alone versus NET and enzymatic or excess cation treatments, as determined by one-way ANOVA with Bonferroni post tests. (B) Luminescence-based viability as a real-time measure of P. aeruginosa PAO1::p16Slux survival in the presence of NETs alone, or combined treatment of NETS with DNase I, PTase or Mg2+. ### denotes a statistically significant difference of P<0.001 between NET-challenged PAO1 versus PAO1 alone (white). ***P<0.001 versus NET killed samples (black). (C) Flow cytometry of P. aeruginosa PAO1 (2 × 107 CFU) coincubated for four hours with PMA-stimulated neutrophils alone (1 × 106; MOI: 10) or with the addition of DNase I, PTase and 5 mM Mg2+. N = 50 000 for each plot. Numbers in each corner represent the % of 50 000 events that fall into each quadrant gate.
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ppat.1004593.g005: Neutralizing the cation chelating activity of the DNA backbone of NETs protects bacteria.(A) Percent survival of P. aeruginosa PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg2+. Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P<0.05 or P<0.01, respectively) between NET-alone versus NET and enzymatic or excess cation treatments, as determined by one-way ANOVA with Bonferroni post tests. (B) Luminescence-based viability as a real-time measure of P. aeruginosa PAO1::p16Slux survival in the presence of NETs alone, or combined treatment of NETS with DNase I, PTase or Mg2+. ### denotes a statistically significant difference of P<0.001 between NET-challenged PAO1 versus PAO1 alone (white). ***P<0.001 versus NET killed samples (black). (C) Flow cytometry of P. aeruginosa PAO1 (2 × 107 CFU) coincubated for four hours with PMA-stimulated neutrophils alone (1 × 106; MOI: 10) or with the addition of DNase I, PTase and 5 mM Mg2+. N = 50 000 for each plot. Numbers in each corner represent the % of 50 000 events that fall into each quadrant gate.

Mentions: The bactericidal activity of neutrophil extracellular traps is attributed to direct contact and exposure of bacteria to the antimicrobial proteins embedded in the DNA scaffold of NETs [1, 2]. Given the antimicrobial activity of DNA, we propose that the DNA backbone of the NET itself is antibacterial. Therefore, if DNA contributes to bacterial killing, treatments that quench the cation chelation potential of the DNA backbone will block bactericidal activity of NETs. To address this possibility, PMA-activated neutrophils were treated with the addition of DNase, PTase or excess Mg2+ and bacterial viability was monitored. The DNA-targeted treatments completely protected P. aeruginosa and E. coli from killing by neutrophil extracellular traps (Fig. 5A). To confirm these results, we monitored the luminescence of P. aeruginosa PAO1::p16Slux co-incubated with PMA-activated neutrophils and observed that the antibacterial effects of NETs were neutralized by treatment with exogenous DNase, Mg2+ cations and PTase (Fig. 5B).


DNA is an antimicrobial component of neutrophil extracellular traps.

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

Neutralizing the cation chelating activity of the DNA backbone of NETs protects bacteria.(A) Percent survival of P. aeruginosa PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg2+. Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P<0.05 or P<0.01, respectively) between NET-alone versus NET and enzymatic or excess cation treatments, as determined by one-way ANOVA with Bonferroni post tests. (B) Luminescence-based viability as a real-time measure of P. aeruginosa PAO1::p16Slux survival in the presence of NETs alone, or combined treatment of NETS with DNase I, PTase or Mg2+. ### denotes a statistically significant difference of P<0.001 between NET-challenged PAO1 versus PAO1 alone (white). ***P<0.001 versus NET killed samples (black). (C) Flow cytometry of P. aeruginosa PAO1 (2 × 107 CFU) coincubated for four hours with PMA-stimulated neutrophils alone (1 × 106; MOI: 10) or with the addition of DNase I, PTase and 5 mM Mg2+. N = 50 000 for each plot. Numbers in each corner represent the % of 50 000 events that fall into each quadrant gate.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004593.g005: Neutralizing the cation chelating activity of the DNA backbone of NETs protects bacteria.(A) Percent survival of P. aeruginosa PAO1 and E. coli DH5α as determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils or combined treatment of NETS with DNase, PTase or Mg2+. Error bars are SEM from 6 replicates. ** or *** denotes a statistically significant difference (P<0.05 or P<0.01, respectively) between NET-alone versus NET and enzymatic or excess cation treatments, as determined by one-way ANOVA with Bonferroni post tests. (B) Luminescence-based viability as a real-time measure of P. aeruginosa PAO1::p16Slux survival in the presence of NETs alone, or combined treatment of NETS with DNase I, PTase or Mg2+. ### denotes a statistically significant difference of P<0.001 between NET-challenged PAO1 versus PAO1 alone (white). ***P<0.001 versus NET killed samples (black). (C) Flow cytometry of P. aeruginosa PAO1 (2 × 107 CFU) coincubated for four hours with PMA-stimulated neutrophils alone (1 × 106; MOI: 10) or with the addition of DNase I, PTase and 5 mM Mg2+. N = 50 000 for each plot. Numbers in each corner represent the % of 50 000 events that fall into each quadrant gate.
Mentions: The bactericidal activity of neutrophil extracellular traps is attributed to direct contact and exposure of bacteria to the antimicrobial proteins embedded in the DNA scaffold of NETs [1, 2]. Given the antimicrobial activity of DNA, we propose that the DNA backbone of the NET itself is antibacterial. Therefore, if DNA contributes to bacterial killing, treatments that quench the cation chelation potential of the DNA backbone will block bactericidal activity of NETs. To address this possibility, PMA-activated neutrophils were treated with the addition of DNase, PTase or excess Mg2+ and bacterial viability was monitored. The DNA-targeted treatments completely protected P. aeruginosa and E. coli from killing by neutrophil extracellular traps (Fig. 5A). To confirm these results, we monitored the luminescence of P. aeruginosa PAO1::p16Slux co-incubated with PMA-activated neutrophils and observed that the antibacterial effects of NETs were neutralized by treatment with exogenous DNase, Mg2+ cations and PTase (Fig. 5B).

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