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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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Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane.(A) Survival analysis of 1 × 107 CFU P. aeruginosa PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg2+. Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. (B) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg2+ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). (C) Visualization of the outer membrane integrity of P. aeruginosa PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [38] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. (D) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in (C) or DNA pretreated with DNase, PTase and Mg2+. Error bars represent SD from 6 fields of view. (E) Flow cytometry of DNA-exposed P. aeruginosa PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [28, 32]. 2.5 × 107 CFU P. aeruginosa PAO1 were exposed to 0.0125% DNA alone or pretreated as in (A) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. (F) Quantification of membrane-compromised, PI-stained P. aeruginosa PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in (E). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P<0.01 and P<0.05, respectively, between DNA alone sample and pretreated samples. Two-tailed student t-tests were performed to test for significant differences.
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ppat.1004593.g004: Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane.(A) Survival analysis of 1 × 107 CFU P. aeruginosa PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg2+. Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. (B) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg2+ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). (C) Visualization of the outer membrane integrity of P. aeruginosa PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [38] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. (D) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in (C) or DNA pretreated with DNase, PTase and Mg2+. Error bars represent SD from 6 fields of view. (E) Flow cytometry of DNA-exposed P. aeruginosa PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [28, 32]. 2.5 × 107 CFU P. aeruginosa PAO1 were exposed to 0.0125% DNA alone or pretreated as in (A) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. (F) Quantification of membrane-compromised, PI-stained P. aeruginosa PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in (E). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P<0.01 and P<0.05, respectively, between DNA alone sample and pretreated samples. Two-tailed student t-tests were performed to test for significant differences.

Mentions: To confirm the mechanism by which extracellular DNA kills bacteria, we monitored the loss of P. aeruginosa viability in the presence of dilute extracellular DNA (0.125% w/v; Fig. 4A). Bacterial survival was restored if DNA was pretreated with DNase or excess 5mM Mg2+, which degrades DNA or saturates its cation chelating ability, respectively, thus neutralizing the antibacterial activity (Fig. 4A). Pretreatment of extracellular DNA with calf intestinal alkaline phosphatase (PTase), which cleaves 5’-phosphates, also blocked the observed antibacterial activity (Fig. 4A). The addition of decreasing amounts of DNase, PTase or Mg2+, resulted in marked, dose-dependent, decreases in bacterial survival when challenged with 0.15% DNA (Fig. 4B). The DNA-mediated damage to the outer membrane leads to the formation of ChFP-enriched outer membrane vesicle-like structures (OMVs; Fig. 4C) [23]. However, incubation of P. aeruginosa with extracellular DNA pretreated with DNase, PTase and Mg2+ greatly diminished the number of ChFP-labeled OMVs relative to control conditions (Fig. 4D), confirming that the bactericidal mechanism of extracellular DNA is through disruption of the bacterial membrane.


DNA is an antimicrobial component of neutrophil extracellular traps.

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

Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane.(A) Survival analysis of 1 × 107 CFU P. aeruginosa PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg2+. Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. (B) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg2+ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). (C) Visualization of the outer membrane integrity of P. aeruginosa PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [38] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. (D) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in (C) or DNA pretreated with DNase, PTase and Mg2+. Error bars represent SD from 6 fields of view. (E) Flow cytometry of DNA-exposed P. aeruginosa PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [28, 32]. 2.5 × 107 CFU P. aeruginosa PAO1 were exposed to 0.0125% DNA alone or pretreated as in (A) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. (F) Quantification of membrane-compromised, PI-stained P. aeruginosa PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in (E). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P<0.01 and P<0.05, respectively, between DNA alone sample and pretreated samples. Two-tailed student t-tests were performed to test for significant differences.
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ppat.1004593.g004: Extracellular DNA exerts bactericidal activity through cation chelation-mediated disruption of the bacterial outer membrane.(A) Survival analysis of 1 × 107 CFU P. aeruginosa PAO1 coincubated with 0.125% (w/v) DNA or DNA pretreated with DNase I, PTase or 5 mM Mg2+. Bacterial counts were performed before (0) and after two hours treatment with DNA (2). Results are representative of three independent experiments. Error bars represent the standard deviation (SD) from eight replicates. (B) High, medium and low concentrations of DNase (430kU, 43kU, 4.3kU), PTase (50 U, 10 U, 1 U) or excess Mg2+ (5 mM, 500 μM, 5 μM) leads to increased levels of protection from killing with 0.15% DNA (w/v). (C) Visualization of the outer membrane integrity of P. aeruginosa PAO1::OM-lipoChFP expressing an outer membrane-localized mCherry fluorescent lipoprotein [38] immediately after 2% (w/v) DNA-exposure. Insets represent increased magnification of presented micrographs. Scale bar: 10 μM. (D) Quantification of ChFP-rich OMV generation in the field of view from 6 representative images generated from bacteria-DNA coincubation as described in (C) or DNA pretreated with DNase, PTase and Mg2+. Error bars represent SD from 6 fields of view. (E) Flow cytometry of DNA-exposed P. aeruginosa PAO1 using SYTO9-PI dual staining as a measure of membrane-compromised bacteria [28, 32]. 2.5 × 107 CFU P. aeruginosa PAO1 were exposed to 0.0125% DNA alone or pretreated as in (A) then immediately analyzed by the collection of positive events (N = 50 000) by BD LSRII. Numbers in corners represent the % of 50 000 events that fall into each quadrant gate. (F) Quantification of membrane-compromised, PI-stained P. aeruginosa PAO1 as measured by flow cytometry. Mean percent PI stained was derived from the average of three replicates (each with N = 50 000 for each plot) in each exposure condition as in (E). *** denotes a significant difference between the control and 0.125% DNA sample. ### and # denote a statistically significant difference, P<0.01 and P<0.05, respectively, between DNA alone sample and pretreated samples. Two-tailed student t-tests were performed to test for significant differences.
Mentions: To confirm the mechanism by which extracellular DNA kills bacteria, we monitored the loss of P. aeruginosa viability in the presence of dilute extracellular DNA (0.125% w/v; Fig. 4A). Bacterial survival was restored if DNA was pretreated with DNase or excess 5mM Mg2+, which degrades DNA or saturates its cation chelating ability, respectively, thus neutralizing the antibacterial activity (Fig. 4A). Pretreatment of extracellular DNA with calf intestinal alkaline phosphatase (PTase), which cleaves 5’-phosphates, also blocked the observed antibacterial activity (Fig. 4A). The addition of decreasing amounts of DNase, PTase or Mg2+, resulted in marked, dose-dependent, decreases in bacterial survival when challenged with 0.15% DNA (Fig. 4B). The DNA-mediated damage to the outer membrane leads to the formation of ChFP-enriched outer membrane vesicle-like structures (OMVs; Fig. 4C) [23]. However, incubation of P. aeruginosa with extracellular DNA pretreated with DNase, PTase and Mg2+ greatly diminished the number of ChFP-labeled OMVs relative to control conditions (Fig. 4D), confirming that the bactericidal mechanism of extracellular DNA is through disruption of the bacterial membrane.

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