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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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Pseudomonas aeruginosa responds to DNA in neutrophil extracellular traps and induces protective bacterial surface modifications.(A) Survival analysis of 1 × 107 CFU wild-type P. aeruginosa PAO1 or mutants with defects in the aminoarabinose-LPS modification (PA3553::lux) or in spermidine synthesis (PA4774::lux) after coincubation with 0.125% DNA. *** denotes a statistically significant difference between time 0 and 2 hours post coincubation with 0.125% DNA. ### denotes a statistically significant difference of P<0.001 between wild-type and mutant P. aeruginosa when exposed to 0.125% DNA. Two-tailed student t-tests were performed to test for significant differences. Error bars represent SD from eight replicates. (B) 2 × 107 CFU P. aeruginosa PAO1 spermidine synthesis (PA4774::lux) and aminoarabinose modification (PA3553::lux) transcriptional reporter strains were incubated with PMA-activated neutrophils (MOI 10:1) and gene expression (luminescence as quantified by CPS) was measured every 20 minutes in the absence (empty squares) and presence (solid circles) of NETs. Error bars represent SEM from six replicates. (C) The effect of DNase or 2 mM Mg2+ treatment on NET-mediated gene induction of 2 × 107 CFU PA4774::lux or PA3553::lux after four hours of coincubation (MOI 10:1). *P<0.05, ***P<0.001 versus bacteria alone (white bar), #P<0.05, ###P<0.001 versus NET exposure (black bar) as determined by one-way ANOVA with Bonferroni post tests. (D) Bacterial survival analysis of 2 × 107 CFU NET-exposed P. aeruginosa PAO1 wild-type, aminoarabinose modification mutant PA3553::lux, or the spermidine synthesis mutants PA47743/4::lux, PA4774::lux (MOI 10:1) Error bars represent the SEM from 6 replicates. *** denotes a statistically significant difference of P<0.001 versus wild-type survival as determined by one-way ANOVA with Bonferroni post tests. All assays were conducted at least three times and representative data is presented.
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ppat.1004593.g007: Pseudomonas aeruginosa responds to DNA in neutrophil extracellular traps and induces protective bacterial surface modifications.(A) Survival analysis of 1 × 107 CFU wild-type P. aeruginosa PAO1 or mutants with defects in the aminoarabinose-LPS modification (PA3553::lux) or in spermidine synthesis (PA4774::lux) after coincubation with 0.125% DNA. *** denotes a statistically significant difference between time 0 and 2 hours post coincubation with 0.125% DNA. ### denotes a statistically significant difference of P<0.001 between wild-type and mutant P. aeruginosa when exposed to 0.125% DNA. Two-tailed student t-tests were performed to test for significant differences. Error bars represent SD from eight replicates. (B) 2 × 107 CFU P. aeruginosa PAO1 spermidine synthesis (PA4774::lux) and aminoarabinose modification (PA3553::lux) transcriptional reporter strains were incubated with PMA-activated neutrophils (MOI 10:1) and gene expression (luminescence as quantified by CPS) was measured every 20 minutes in the absence (empty squares) and presence (solid circles) of NETs. Error bars represent SEM from six replicates. (C) The effect of DNase or 2 mM Mg2+ treatment on NET-mediated gene induction of 2 × 107 CFU PA4774::lux or PA3553::lux after four hours of coincubation (MOI 10:1). *P<0.05, ***P<0.001 versus bacteria alone (white bar), #P<0.05, ###P<0.001 versus NET exposure (black bar) as determined by one-way ANOVA with Bonferroni post tests. (D) Bacterial survival analysis of 2 × 107 CFU NET-exposed P. aeruginosa PAO1 wild-type, aminoarabinose modification mutant PA3553::lux, or the spermidine synthesis mutants PA47743/4::lux, PA4774::lux (MOI 10:1) Error bars represent the SEM from 6 replicates. *** denotes a statistically significant difference of P<0.001 versus wild-type survival as determined by one-way ANOVA with Bonferroni post tests. All assays were conducted at least three times and representative data is presented.

Mentions: Subinhibitory concentrations of extracellular DNA sequester Mg2+ and trigger the expression of multiple surface modifications that are known to protect the bacterial outer membrane from antimicrobial peptide (AP) damage and killing [23–25]. The arn operon (PA3552-PA3559) is required for the covalent addition of aminoarabinose to the phosphates of lipid A and the spermidine synthesis genes (PA4773-PA4774; speDE homologs) are required for production of the polycation spermidine on the outer surface [23, 24]. Both modifications substitute for divalent metal cations, mask the negative charges of the outer surface and thus contribute to AP resistance [23, 24, 29–31]. Given that these modifications stabilize the bacterial envelope, we sought to determine whether these surface modification pathways provided a more general mechanism to resist bacterial membrane damage. We noted that P. aeruginosa strains with mutations in the arn or spermidine biosynthetic pathways were significantly less capable of surviving exposure to DNA (Fig. 7A).


DNA is an antimicrobial component of neutrophil extracellular traps.

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

Pseudomonas aeruginosa responds to DNA in neutrophil extracellular traps and induces protective bacterial surface modifications.(A) Survival analysis of 1 × 107 CFU wild-type P. aeruginosa PAO1 or mutants with defects in the aminoarabinose-LPS modification (PA3553::lux) or in spermidine synthesis (PA4774::lux) after coincubation with 0.125% DNA. *** denotes a statistically significant difference between time 0 and 2 hours post coincubation with 0.125% DNA. ### denotes a statistically significant difference of P<0.001 between wild-type and mutant P. aeruginosa when exposed to 0.125% DNA. Two-tailed student t-tests were performed to test for significant differences. Error bars represent SD from eight replicates. (B) 2 × 107 CFU P. aeruginosa PAO1 spermidine synthesis (PA4774::lux) and aminoarabinose modification (PA3553::lux) transcriptional reporter strains were incubated with PMA-activated neutrophils (MOI 10:1) and gene expression (luminescence as quantified by CPS) was measured every 20 minutes in the absence (empty squares) and presence (solid circles) of NETs. Error bars represent SEM from six replicates. (C) The effect of DNase or 2 mM Mg2+ treatment on NET-mediated gene induction of 2 × 107 CFU PA4774::lux or PA3553::lux after four hours of coincubation (MOI 10:1). *P<0.05, ***P<0.001 versus bacteria alone (white bar), #P<0.05, ###P<0.001 versus NET exposure (black bar) as determined by one-way ANOVA with Bonferroni post tests. (D) Bacterial survival analysis of 2 × 107 CFU NET-exposed P. aeruginosa PAO1 wild-type, aminoarabinose modification mutant PA3553::lux, or the spermidine synthesis mutants PA47743/4::lux, PA4774::lux (MOI 10:1) Error bars represent the SEM from 6 replicates. *** denotes a statistically significant difference of P<0.001 versus wild-type survival as determined by one-way ANOVA with Bonferroni post tests. All assays were conducted at least three times and representative data is presented.
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ppat.1004593.g007: Pseudomonas aeruginosa responds to DNA in neutrophil extracellular traps and induces protective bacterial surface modifications.(A) Survival analysis of 1 × 107 CFU wild-type P. aeruginosa PAO1 or mutants with defects in the aminoarabinose-LPS modification (PA3553::lux) or in spermidine synthesis (PA4774::lux) after coincubation with 0.125% DNA. *** denotes a statistically significant difference between time 0 and 2 hours post coincubation with 0.125% DNA. ### denotes a statistically significant difference of P<0.001 between wild-type and mutant P. aeruginosa when exposed to 0.125% DNA. Two-tailed student t-tests were performed to test for significant differences. Error bars represent SD from eight replicates. (B) 2 × 107 CFU P. aeruginosa PAO1 spermidine synthesis (PA4774::lux) and aminoarabinose modification (PA3553::lux) transcriptional reporter strains were incubated with PMA-activated neutrophils (MOI 10:1) and gene expression (luminescence as quantified by CPS) was measured every 20 minutes in the absence (empty squares) and presence (solid circles) of NETs. Error bars represent SEM from six replicates. (C) The effect of DNase or 2 mM Mg2+ treatment on NET-mediated gene induction of 2 × 107 CFU PA4774::lux or PA3553::lux after four hours of coincubation (MOI 10:1). *P<0.05, ***P<0.001 versus bacteria alone (white bar), #P<0.05, ###P<0.001 versus NET exposure (black bar) as determined by one-way ANOVA with Bonferroni post tests. (D) Bacterial survival analysis of 2 × 107 CFU NET-exposed P. aeruginosa PAO1 wild-type, aminoarabinose modification mutant PA3553::lux, or the spermidine synthesis mutants PA47743/4::lux, PA4774::lux (MOI 10:1) Error bars represent the SEM from 6 replicates. *** denotes a statistically significant difference of P<0.001 versus wild-type survival as determined by one-way ANOVA with Bonferroni post tests. All assays were conducted at least three times and representative data is presented.
Mentions: Subinhibitory concentrations of extracellular DNA sequester Mg2+ and trigger the expression of multiple surface modifications that are known to protect the bacterial outer membrane from antimicrobial peptide (AP) damage and killing [23–25]. The arn operon (PA3552-PA3559) is required for the covalent addition of aminoarabinose to the phosphates of lipid A and the spermidine synthesis genes (PA4773-PA4774; speDE homologs) are required for production of the polycation spermidine on the outer surface [23, 24]. Both modifications substitute for divalent metal cations, mask the negative charges of the outer surface and thus contribute to AP resistance [23, 24, 29–31]. Given that these modifications stabilize the bacterial envelope, we sought to determine whether these surface modification pathways provided a more general mechanism to resist bacterial membrane damage. We noted that P. aeruginosa strains with mutations in the arn or spermidine biosynthetic pathways were significantly less capable of surviving exposure to DNA (Fig. 7A).

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