Limits...
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.

Show MeSH

Related in: MedlinePlus

P. aeruginosa, E. coli and S. aureus differ in their ability to tolerate the bactericidal effects of NETs.(A) Survival analysis of 1 × 107 CFU P. aeruginosa, S. aureus and E. coli upon exposure to NETs (MOI 10:1). Bacterial viability was determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils and was normalized to bacterial counts in the absence of neutrophils. DNase I was added exogenously 0.5 hour prior to the end of the experiment to degrade NETs and ensure accurate counts of recoverable colonies. Results are representative of three independent replicates. ***P<0.001 versus P. aeruginosa PAO1. °°°P<0.001 versus non-DNase condition by one-way ANOVA with Bonferroni post tests. (B) Bacterial viability was determined by measuring luminescence from 1 × 107 CFU lux-tagged PAO1::p16Slux or E. coli DH5α/pσ70-lux in the absence or presence of PMA-induced NETs (MOI 10:1). Errors bars represent SEM from six replicates. All experiments were performed at least three times.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4295883&req=5

ppat.1004593.g003: P. aeruginosa, E. coli and S. aureus differ in their ability to tolerate the bactericidal effects of NETs.(A) Survival analysis of 1 × 107 CFU P. aeruginosa, S. aureus and E. coli upon exposure to NETs (MOI 10:1). Bacterial viability was determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils and was normalized to bacterial counts in the absence of neutrophils. DNase I was added exogenously 0.5 hour prior to the end of the experiment to degrade NETs and ensure accurate counts of recoverable colonies. Results are representative of three independent replicates. ***P<0.001 versus P. aeruginosa PAO1. °°°P<0.001 versus non-DNase condition by one-way ANOVA with Bonferroni post tests. (B) Bacterial viability was determined by measuring luminescence from 1 × 107 CFU lux-tagged PAO1::p16Slux or E. coli DH5α/pσ70-lux in the absence or presence of PMA-induced NETs (MOI 10:1). Errors bars represent SEM from six replicates. All experiments were performed at least three times.

Mentions: In order to characterize the bactericidal capacity of NETs, neutrophils were treated with PMA to stimulate maximal NETosis and cytochalasin D to block phagocytosis, thus restricting bacterial killing to extracellular NET function [1, 8, 21, 22]. Importantly, the addition of cytochalasin D had no effect on NETosis induced in PMA-treated neutrophils (S2 Fig.). We used the conventional method of direct bacterial counts to enumerate the number of bacteria before and after challenge with PMA-induced NETs. Direct counts of NET-exposed bacteria revealed that P. aeruginosa was most tolerant to NET killing, whereas S. aureus and E. coli were significantly more sensitive (Fig. 3A). The addition of deoxyribonuclease (DNase) restored bacterial survival of the NET-sensitive organisms E. coli and S. aureus, confirming that killing was mediated by extracellular NET function (Fig. 3A). Further, the kinetics of bacterial killing by PMA-generated NETs was determined by measuring the loss of luminescence from chromosomally-tagged luminescent P. aeruginosa strain, PAO1::p16Slux [23], and plasmid-borne luminescent E. coli / pσ70-lux [27]. This approach confirmed that P. aeruginosa was more tolerant to NET killing than E. coli, where luminescence rapidly decreased upon neutrophil challenge (Fig. 3B).


DNA is an antimicrobial component of neutrophil extracellular traps.

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

P. aeruginosa, E. coli and S. aureus differ in their ability to tolerate the bactericidal effects of NETs.(A) Survival analysis of 1 × 107 CFU P. aeruginosa, S. aureus and E. coli upon exposure to NETs (MOI 10:1). Bacterial viability was determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils and was normalized to bacterial counts in the absence of neutrophils. DNase I was added exogenously 0.5 hour prior to the end of the experiment to degrade NETs and ensure accurate counts of recoverable colonies. Results are representative of three independent replicates. ***P<0.001 versus P. aeruginosa PAO1. °°°P<0.001 versus non-DNase condition by one-way ANOVA with Bonferroni post tests. (B) Bacterial viability was determined by measuring luminescence from 1 × 107 CFU lux-tagged PAO1::p16Slux or E. coli DH5α/pσ70-lux in the absence or presence of PMA-induced NETs (MOI 10:1). Errors bars represent SEM from six replicates. All experiments were performed at least three times.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004593.g003: P. aeruginosa, E. coli and S. aureus differ in their ability to tolerate the bactericidal effects of NETs.(A) Survival analysis of 1 × 107 CFU P. aeruginosa, S. aureus and E. coli upon exposure to NETs (MOI 10:1). Bacterial viability was determined by direct plate counts (CFU/ml) before and after 4 hour incubation with PMA-activated neutrophils and was normalized to bacterial counts in the absence of neutrophils. DNase I was added exogenously 0.5 hour prior to the end of the experiment to degrade NETs and ensure accurate counts of recoverable colonies. Results are representative of three independent replicates. ***P<0.001 versus P. aeruginosa PAO1. °°°P<0.001 versus non-DNase condition by one-way ANOVA with Bonferroni post tests. (B) Bacterial viability was determined by measuring luminescence from 1 × 107 CFU lux-tagged PAO1::p16Slux or E. coli DH5α/pσ70-lux in the absence or presence of PMA-induced NETs (MOI 10:1). Errors bars represent SEM from six replicates. All experiments were performed at least three times.
Mentions: In order to characterize the bactericidal capacity of NETs, neutrophils were treated with PMA to stimulate maximal NETosis and cytochalasin D to block phagocytosis, thus restricting bacterial killing to extracellular NET function [1, 8, 21, 22]. Importantly, the addition of cytochalasin D had no effect on NETosis induced in PMA-treated neutrophils (S2 Fig.). We used the conventional method of direct bacterial counts to enumerate the number of bacteria before and after challenge with PMA-induced NETs. Direct counts of NET-exposed bacteria revealed that P. aeruginosa was most tolerant to NET killing, whereas S. aureus and E. coli were significantly more sensitive (Fig. 3A). The addition of deoxyribonuclease (DNase) restored bacterial survival of the NET-sensitive organisms E. coli and S. aureus, confirming that killing was mediated by extracellular NET function (Fig. 3A). Further, the kinetics of bacterial killing by PMA-generated NETs was determined by measuring the loss of luminescence from chromosomally-tagged luminescent P. aeruginosa strain, PAO1::p16Slux [23], and plasmid-borne luminescent E. coli / pσ70-lux [27]. This approach confirmed that P. aeruginosa was more tolerant to NET killing than E. coli, where luminescence rapidly decreased upon neutrophil challenge (Fig. 3B).

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