Paraoxonases-2 and -3 Are Important Defense Enzymes against Pseudomonas aeruginosa Virulence Factors due to Their Anti-Oxidative and Anti-Inflammatory Properties

Abstract

The pathogen Pseudomonas aeruginosa causes serious damage in immunocompromised patients by secretion of various virulence factors, among them the quorum sensing N-(3-oxododecanoyl)-L-homoserine lactone (3OC12) and the redox-active pyocyanin (PCN). Paraoxonase-2 (PON2) may protect against P. aeruginosa infections, as it efficiently inactivates 3OC12 and diminishes PCN-induced oxidative stress. This defense could be circumvented because 3OC12 mediates intracellular Ca(2+)-rise in host cells, which causes rapid inactivation and degradation of PON2. Importantly, we recently found that the PON2 paralogue PON3 prevents mitochondrial radical formation. Here we investigated its role as additional potential defense mechanism against P. aeruginosa infections. Our studies demonstrate that PON3 diminished PCN-induced oxidative stress. Moreover, it showed clear anti-inflammatory potential by protecting against NF-κB activation and IL-8 release. The latter similarly applied to PON2. Furthermore, we observed a Ca(2+)-mediated inactivation and degradation of PON3, again in accordance with previous findings for PON2. Our results suggest that the anti-oxidative and anti-inflammatory functions of PON2 and PON3 are an important part of our innate defense system against P. aeruginosa infections. Furthermore, we conclude that P. aeruginosa circumvents PON3 protection by the same pathway as for PON2. This may help identifying underlying mechanisms in order to sustain the protection afforded by these enzymes.

Figure 1

PON2 or PON3 overexpression diminishes ROS production induced by P. aeruginosa signaling molecule pyocyanin (PCN). (a) Naïve, PON2-GFP, or PON3-GFP overexpressing HEK293 cells were loaded with carboxy-H₂DCFDA and stimulated with PCN (2.4 μM). Carboxy-H₂DCFDA fluorescence as means of ROS was recorded over several hours. (b) Similar to A. Naïve, PON2-GFP, PON3-GFP, or PON3-dsRed overexpressing EA.hy 926 cells were loaded with L-012 and stimulated with PCN (2.4 μM). Curve maxima calculated by nonlinear regression showed statistically significant differences (P < 0.001) between naïve and PON2 or PON3 overexpressing cells.

Figure 2

PON2 or PON3 overexpression diminishes NF-κB activation induced by P. aeruginosa signaling molecule PCN. A549 cells transiently overexpressing HA or PON2-HA (a) and GFP or PON3-GFP (b) were stimulated with PCN (100 μM, 4 h) and analyzed for NF-κB activation. Symbols represent ± S.E.M. n = 6–9; ***P < 0.001.

Figure 3

PCN induces IL-8 secretion, which can be lowered by PON2 or PON3 overexpression. (a) Naïve, PON2-GFP or PON3-GFP overexpressing EA.hy 926 cells were treated with PCN (10 μM, 16 h). Cell supernatants were analyzed for the secretion of the listed cytokines and chemokines by ELISA. (b) Quantitative evaluation of results from panel (a) Fold induction of IL-8 release was calculated between untreated and PCN-treated samples.

Figure 4

PON2 and PON3 activity decrease after 3OC12 treatment. (a) PON2-GFP overexpressing HEK293 cells were treated with 3OC12 (100 μM) for the indicated durations and tested for 3OC12-HSL hydrolytic activity. (b) PON3-GFP overexpressing HEK293 cells were treated with 3OC12 (100 μM) for the indicated durations and tested for lovastatinase hydrolytic activity. Symbols represent ± S.E.M. n = 3; ***P < 0.001.

Figure 5

PON3 mRNA is not actively degraded in response to 3OC12 treatment. (a) A549 cells were treated with 3OC12 (100 μM) for the indicated durations and analyzed for PON3 mRNA levels by qRT-PCR. (b) A549 cells were treated with 3OC12 (100 μM, 24 h) or with DRB (100 μM, 24 h) or combinations thereof. There was no statistically significant difference in PON3 mRNA levels after DRB or DRB/3OC12 treatment. Symbols represent ± S.E.M. n = 3; ***P < 0.001 versus control.

Figure 6

Both PON2 and PON3 are degraded after 3OC12 treatment. (a) A549 cells were treated with 3OC12 (100 μM) for the indicated durations or (b) with thapsigargin (24 h) with the indicated concentrations. Lysates (50 μg of protein) were analyzed by Western blotting using anti-PON2, anti-PON3, or anti-α-tubulin antibodies. One representative blot is shown. Results (right) are the means ± S.E.M. of three replicate analyses; **P < 0.01; ***P < 0.001.