Paraoxonase 2 is down-regulated by the Pseudomonas aeruginosa quorumsensing signal N-(3-oxododecanoyl)-L-homoserine lactone and attenuates oxidative stress induced by pyocyanin.
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.
Horke
Sven
S
Department of Pharmacology, University Medicine Mainz, Obere Zahlbacher Strasse 67, 55131 Mainz, Germany.
Witte
Ines
I
Altenhöfer
Sebastian
S
Wilgenbus
Petra
P
Goldeck
Marion
M
Förstermann
Ulrich
U
Xiao
Junhui
J
Kramer
Gerald L
GL
Haines
Donovan C
DC
Chowdhary
Puneet K
PK
Haley
Robert W
RW
Teiber
John F
JF
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D043303:Aryldialkylphosphatase / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000502:physiology; D002460:Cell Line; D002470:Cell Survival / Q000235:genetics / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects*; D004789:Enzyme Activation; D006714:Homoserine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects*; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011710:Pyocyanine / Q000494:pharmacology*; D053038:Quorum Sensing / Q000187:drug effects; D034741:RNA, Small Interfering / Q000235:genetics / Q000502:physiology; D014774:Virulence / Q000235:genetics
C109860:N-(3-oxododecanoyl)homoserine lactone; D034741:RNA, Small Interfering; D000001:Calcimycin; D006714:Homoserine; D011710:Pyocyanine; D043303:Aryldialkylphosphatase; D015107:4-Butyrolactone; D002118:Calcium