Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin

Abstract

Two virulence factors produced by Pseudomonas aeruginosa are pyocyanin and N-(3-oxododecanoyl)-L-homoserine lactone (3OC12). Pyocyanin damages host cells by generating ROS (reactive oxygen species). 3OC12 is a quorum-sensing signalling molecule which regulates bacterial gene expression and modulates host immune responses. PON2 (paraoxonase-2) is an esterase that inactivates 3OC12 and potentially attenuates Ps. aeruginosa virulence. Because increased intracellular Ca2+ initiates the degradation of PON2 mRNA and protein and 3OC12 causes increases in cytosolic Ca2+, we hypothesized that 3OC12 would also down-regulate PON2. 3OC12 and the Ca2+ ionophore A23187 caused a rapid cytosolic Ca2+ influx and down-regulated PON2 mRNA, protein and hydrolytic activity in A549 and EA.hy 926 cells. The decrease in PON2 hydrolytic activity was much more extensive and rapid than decreases in protein, suggesting a rapid post-translational mechanism which blocks PON2's hydrolytic activity. The Ca2+ chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid tetrakis(acetoxymethyl ester)] diminished the ability of 3OC12 to decrease PON2, demonstrating that the effects are mediated by Ca2+. PON2 also has antioxidative properties and we show that it protects cells from pyocyanin-induced oxidative stress. Knockdown of PON2 by transfecting cells with siRNA (small interfering RNA) rendered them more sensitive to, whereas overexpression of PON2 protected cells from, pyocyanin-induced ROS formation. Additionally, 3OC12 potentiated pyocyanin-induced ROS formation, presumably by inactivating PON2. These findings support a key role for PON2 in the defence against Ps. aeruginosa virulence, but also reveal a mechanism by which the bacterium may subvert the protection afforded by PON2.

One Enzyme, Two Functions

“…The PONs differ in localization as PON1 mainly associates with circulating serum high density lipoprotein particles, whereas the intracellular PON2 appears in two spliced isoforms that predominantly localize to the endoplasmic reticulum, mitochondria, and (peri)-nuclear region (7,9,15). Endogenous substrates of PON2 are largely unknown, but it has been shown that its lactonase activity dominantly hydrolyzes the Pseudomonas aeruginosa signaling lactone 3OC12, for which reason PON2 may add to the anti-bacterial defense (16,17), a mechanism apparently sensitive to major calcium disturbances (18,19). Further, studies with PON2-deficient mice indicated that PON2 suppresses atherosclerosis, and several studies described a significant ROS-diminishing function of PON2 in various cells (8,9,18,20,21).…”

“…ROS Detection-Unless stated otherwise, ROS was detected as previously published (9,18 PON2 Lactonase Activity-Deglycosylation and determination of PON2 lactonase activity were previously published (9,18). Importantly, in every experiment, PON2 mutant expression levels were analyzed by Western blotting using a ChemiDoc XRS imaging system (Bio-Rad) equipped with QuantityOne 4.6.7 software, normalized to ␣-tubulin, and subsumed comparing levels with PON2-WT.…”

“…Endogenous substrates of PON2 are largely unknown, but it has been shown that its lactonase activity dominantly hydrolyzes the Pseudomonas aeruginosa signaling lactone 3OC12, for which reason PON2 may add to the anti-bacterial defense (16,17), a mechanism apparently sensitive to major calcium disturbances (18,19). Further, studies with PON2-deficient mice indicated that PON2 suppresses atherosclerosis, and several studies described a significant ROS-diminishing function of PON2 in various cells (8,9,18,20,21). Although fundamental to understanding PON2 function, it is yet unanswered whether the lactonase activity and anti-oxidative function are interrelated.…”

Engineering Soluble Human Paraoxonase 2 for Quorum Quenching

“…The negative charge of the resulting intermediates is probably stabilized by the catalytic calcium. 142 . In addition, a polymorphism found in the PON2 coding sequence, with a substitution of a cysteine for a serine at position 311, alters glycosylation of the enzyme and decreases its lactonase activity, which could be of major importance for innate immunity in the lungs 143 .…”

Negative association between paraoxonase 2, anthropometric markers and metabolic syndrome

“…All three PONs are antioxidant enzymes and lactonases, but PON2 displays the highest activity hydrolyzing a number of acylhomoserine lactones [7,8].…”