Secreted Phosphatase and Deoxyribonuclease Are Required by Pseudomonas aeruginosa To Defend against Neutrophil Extracellular Traps

Abstract

Neutrophil extracellular traps (NETs) are produced by neutrophils as an innate immune defense mechanism to trap and kill microbial pathogens. NETs are comprised of ejected chromatin that forms a lattice structure enmeshed with numerous antimicrobial proteins. In addition to forming the structural backbone of NETs, extracellular DNA (eDNA) has membrane-disrupting antimicrobial activity that contributes to NET killing. Many pathogens produce secreted extracellular DNases to evade the antimicrobial activity of NETs. encodes an operon of two secreted enzymes, a predicted alkaline phosphatase and a DNase. The DNase () degrades eDNA to use as a nutrient source. Here we report that both eDNA and NETs are potent inducers of this DNase-phosphatase operon. Furthermore, the secreted DNase contributes to degrading NET DNA and defends against NET-mediated killing. We demonstrate that EddA has both alkaline phosphatase and phosphodiesterase (PDase) activities and also protects against the antimicrobial activity of NETs. Although the phosphatase does not cause DNA degradation similar to that of the DNase, its protective function is likely a result of removing the cation-chelating phosphates from the eDNA phosphodiester backbone. Therefore, both the DNase and PDase contribute to defense against NET killing of, highlighting the role of DNA-manipulating enzymes in targeting the eDNA in neutrophil extracellular traps.

In vivo evidence for extracellular DNA trap formation

“…Escaping NETs promotes spreading of pneumococci from the upper airways to the lungs and from the lungs into the blood stream during pneumonia 49 . Bacterial release of DNase and phosphatases contribute to defense against NETmediated killing of Pseudomonas aeruginosa, highlighting the role of manipulating enzymes in combating NETs by microorganisms 50 . In addition, the pneumococcal polysaccharide capsule protects from NET trapping, but is not required for resistance to NET-mediated killing ( Fig.…”

“…a Extracellular traps consisting of a DNA scaffold and granule proteins entrap and kill microorganisms6,7,14,152 . Certain pathogens have developed properties for escaping the physical entrapment by (b) encapsulation of bacteria to shield against traps 51 or (c) release of endonucleases to degrade the extracellular DNA scaffold leading to less efficient killing of bacteria49,50 .…”

Extracellular DNA controls expression of Pseudomonas aeruginosa genes involved in nutrient utilization, metal homeostasis, acid pH tolerance and virulence

“…Several groups have shown that DNA is an efficient nutrient source of carbon, nitrogen and phosphate [12][13][14]. eDNA and the DNA within NETs induce the expression of a two-gene operon encoding a secreted phosphatase and deoxyribonuclease in P. aeruginosa [14,15]. These secreted enzymes target DNA and protect P. aeruginosa from DNA killing during exposure to neutrophil extracellular traps [15].…”

Extracellular DNA controls expression ofPseudomonas aeruginosagenes involved in nutrient utilization, metal homeostasis, acid pH tolerance, and virulence

“…Several groups have shown that DNA is an efficient nutrient source of carbon, nitrogen and phosphate (12–14). Extracellular DNA and the DNA within neutrophil NETS induces the expression of a 2 gene operon encoding a secreted phosphatase and deoxyribonuclease in P. aerugino sa (14,15). These secreted enzymes target DNA and protect P. aeruginosa from DNA killing during exposure to neutrophil extracellular traps (15).…”

“…Extracellular DNA and the DNA within neutrophil NETS induces the expression of a 2 gene operon encoding a secreted phosphatase and deoxyribonuclease in P. aerugino sa (14,15). These secreted enzymes target DNA and protect P. aeruginosa from DNA killing during exposure to neutrophil extracellular traps (15). These genes are also induced under phosphate limiting conditions, and also contribute to DNA degradation and phosphate acquisition when using DNA as a nutrient (14).…”