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Infection and Immunity, June 2008, p. 2512-2519, Vol. 76, No. 6
0019-9567/08/$08.00+0     doi:10.1128/IAI.01606-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Dominant Role of Paraoxonases in Inactivation of the Pseudomonas aeruginosa Quorum-Sensing Signal N-(3-Oxododecanoyl)-L-Homoserine Lactone {triangledown}

John F. Teiber,1* Sven Horke,2 Donovan C. Haines,3 Puneet K. Chowdhary,3 Junhui Xiao,1 Gerald L. Kramer,1 Robert W. Haley,1 and Dragomir I. Draganov4

Department of Internal Medicine, Division of Epidemiology, The University of Texas Southwestern Medical Center, Dallas, Texas 75390,1 Department of Pharmacology, Johannes Gutenberg University, Mainz, Germany,2 Department of Chemistry, University of Texas at Dallas, Richardson, Texas 75083,3 Department of Metabolism, WIL Research Laboratories, LLC, Ashland, Ohio 448054

Received 5 December 2007/ Returned for modification 8 January 2008/ Accepted 5 March 2008


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ABSTRACT
 
The pathogenic bacterium Pseudomonas aeruginosa causes serious infections in immunocompromised patients. N-(3-Oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key component of P. aeruginosa's quorum-sensing system and regulates the expression of many virulence factors. 3OC12-HSL was previously shown to be hydrolytically inactivated by the paraoxonase (PON) family of calcium-dependent esterases, consisting of PON1, PON2, and PON3. Here we determined the specific activities of purified human PONs for 3OC12-HSL hydrolysis, including the common PON1 polymorphic forms, and found they were in the following order: PON2 >> PON1192R > PON1192Q > PON3. PON2 exhibited a high specific activity of 7.6 ± 0.4 µmols/min/mg at 10 µM 3OC12-HSL, making it the best PON2 substrate identified to date. By use of class-specific inhibitors, approximately 85 and 95% of the 3OC12-HSL lactonase activity were attributable to PON1 in mouse and human sera, respectively. In mouse liver homogenates, the activity was metal dependent, with magnesium- and manganese-dependent lactonase activities comprising 10 to 15% of the calcium-dependent activity. In mouse lung homogenates, all of the activity was calcium dependent. The calcium-dependent activities were irreversibly inhibited by extended EDTA treatment, implicating PONs as the major enzymes inactivating 3OC12-HSL. In human HepG2 and EA.hy 926 cell lysates, the 3OC12-HSL lactonase activity closely paralleled the PON2 protein levels after PON2 knockdown by small interfering RNA treatment of the cells. These findings suggest that PONs, particularly PON2, could be an important mechanism by which 3OC12-HSL is inactivated in mammals.


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INTRODUCTION
 
Pseudomonas aeruginosa is an opportunistic bacterium which causes serious infections in immunocompromised and cystic fibrosis patients (10). As with many gram-negative bacteria, P. aeruginosa produces acyl-homoserine lactone (AHL) quorum-sensing (QS) signaling molecules termed autoinducers which allow the single-celled organisms to coordinate their actions (36). N-(3-Oxododecanoyl)-L-homoserine lactone (3OC12-HSL) is a key autoinducer synthesized by P. aeruginosa which regulates the expression of extracellular virulence factors and biofilm formation (5, 36). Rats and mice experimentally infected with P. aeruginosa mutants deficient in the ability to produce or respond to 3OC12-HSL exhibited significantly diminished lung pathology, bacterial dissemination, and morbidity and accelerated bacterial clearance compared to animals infected with wild-type bacteria, demonstrating the importance of 3OC12-HSL for P. aeruginosa pathogenicity (14, 21, 27, 31, 40). 3OC12-HSL also has an array of immunomodulatory effects on eukaryotic cells, including the induction of apoptosis, inhibition of leukocyte proliferation, activation of neutrophils and macrophages, and induction of proinflammatory mediators (7, 15, 34, 37, 39, 43). Recently, it was shown that a number of mammalian cell lines were able to inactive 3OC12-HSL (5), providing a possible mechanism for reduction of bacterial virulence.

Mammalian paraoxonases (PONs) are a unique, highly conserved family of calcium-dependent esterases consisting of PON1, PON2, and PON3 (8). Human PON1 and PON3 are synthesized predominantly in the liver from where PON1 and some PON3 are secreted into the blood and associated with high-density lipoproteins (HDL) (29). PON2 is not in serum but is expressed in many tissues and cell types (8, 25). PONs exhibit antioxidative properties and afford protection from atherosclerosis in mouse models; however, the mechanisms by which they mediate these properties are not yet established (1, 16, 24, 32, 33). PONs hydrolyze a broad range of esters, including phosphotriesters, arylesters, and lactones, and have overlapping, but also distinct, substrate specificities (9). Although the physiological function(s) and natural substrates for the PONs are uncertain, accumulating evidence indicates that the lactonase activity of the PONs may be its natural function (9, 17). Serum PON1 hydrolyzes the lactone ring of 3OC12-HSL (26) and the lactonase activity of the PONs extends over a number of AHL QS compounds with various acyl chain lengths (9, 38).

Sera from PON1 knockout (PON1–/–) mice are deficient in 3OC12-HSL hydrolytic activity, but surprisingly, PON1–/– mice had a higher rate of survival than wild-type mice after intraperitoneal injection of the bacterium P. aeruginosa (26). PON2 and PON3 were shown to be up-regulated in the PON1–/– mouse airway epithelium, and it was suggested that this up-regulation may lead to increased inactivation of 3OC12-HSL. However, the possible up-regulation and contribution of other enzymes toward 3OC12-HSL inactivation in this model were not investigated.

For murine tracheal epithelial cells, PON2 was shown to be important for the inactivation of 3OC12-HSL by demonstrating that the cell lysates from PON2-deficient mice had an impaired ability to hydrolyze 3OC12-HSL and that P. aeruginosa QS was enhanced in these epithelial cell cultures (38). Interestingly, in intact epithelial cells there was no difference in the rates of 3OC12-HSL inactivation between the wild-type and PON2-deficient cells. Thus, the relative importance of the role that PON2 plays in the intact cells is not clear, and other inactivation pathways, PON or non-PON, may be important for 3OC12-HSL inactivation in these cells.

Mammals express a broad range of enzymes, such as carboxylesterases, amidases, acylases, proteases, oxidases, and reductases, which could potentially inactive 3OC12-HSL, as well as other AHLs, and the importance of PON-mediated inactivation of 3OC12-HSL relative to other enzymatic pathways is not known. Therefore, the aim of this study was to determine if there are other mammalian enzymes which may inactivate 3OC12-HSL and to evaluate the contribution of the PONs to 3OC12-HSL metabolism. We found that the human PONs, particularly PON2, could efficiently hydrolyze 3OC12-HSL. PONs were the major enzymes inactivating this lactone in human and mouse serum, mouse lung and liver homogenates, and cultured human cell lysates. Thus, our study suggests that the PONs, especially PON2, may represent a key defense mechanism against the P. aeruginosa QS autoinducer 3OC12-HSL.


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MATERIALS AND METHODS
 
Human serum was obtained from the pathology laboratory (University of Texas Southwestern Medical Center, Dallas, TX) and frozen and stored at –80°C. Recombinant human PONs were expressed in Trichoplusia ni High Five insect cells and purified as previously described (9). PON2 has a common coding region polymorphism at position 311 (serine or cysteine); the cysteine 311 variant was used for this study. Human hepatoma (HepG2) cells were from the American Type Culture Collection (Rockville, MD). All chemicals used were of analytical grade or better and purchased from Sigma-Aldrich (St. Louis, MO) or Fisher Scientific (Pittsburg, PA).

Synthesis of 3OC12-HSL. 3OC12-HSL was synthesized by acylating Meldrum's acid with decanoyl chloride and then reacting the resulting adduct with D-HSL as reported previously (4). The reaction was carried out in 10 mM HCl at room temperature for 3 h. The 3OC12-HSL product was purified by chromatography over silica gel. Purity and identity were confirmed by thin-layer chromatography and 1H nuclear magnetic resonance.

Tissue homogenates. ICR mice were injected with 0.5 ml of 100 U/ml heparin solution in saline and livers and lungs were perfused through the portal vein and heart, respectively, with phosphate-buffered saline (PBS). Organs were harvested and rinsed with PBS and stored at –80°C until use. Thawed organs were homogenized on ice in 1 volume of 25 mM Tris, pH 7.4, containing 1 mM CaCl2 with 10 passes on a Polytron homogenizer followed by 10 passes on a Potter-Elvehjem homogenizer. Samples were frozen at –80°C until use.

Cell culture. HepG2 cells were cultured with minimal essential medium containing L-glutamine (Mediatech, Inc., Herndon, VA) supplemented with 10% fetal bovine serum. Human endothelial (EA.hy 926) cells were cultured as previously described (16). Cells were grown in 75-cm2 flasks at 37°C in a humidified atmosphere at 5% CO2. At 70 to 90% confluence, cells were detached with trypsin and pelleted. Pellets were washed with PBS and stored at –80°C until use. Frozen cell pellets were thawed and briefly sonicated on ice in 25 mM Tris buffer (pH 7.4) containing 0.05% n-dodecyl-β-D-maltoside and 1 mM of CaCl2 and analyzed for 3OC12-HSL activity as described below. Protein concentrations of the cell lysates and tissue homogenates were determined by the bicinchoninic acid method (Pierce, Rockford, IL).

Enzymatic assays. For 3OC12-HSL hydrolysis reactions, 5 mM Tris buffer, pH 7.4, containing 1 mM of CaCl2 was added to microcentrifuge tubes containing the samples, or sample buffer for controls, and preincubated at 37°C for 1 min. Reactions with 50- or 100-µl final mixture volumes were initiated by adding a 1% volume of the 3OC12-HSL stock solution (in methanol) and incubated at 37°C. At substrate concentrations of over 100 µM, the solution started to cloud, indicating a lack of solubility. Therefore, substrate concentrations were kept below 100 µM. Reactions were stopped with an equal volume of ice-cold acetonitrile, and the mixtures were vortex mixed and centrifuged to pellet the precipitated protein. Supernatants (30 µl) were chromatographed on a Shimadzu high-performance liquid chromatography (HPLC) system equipped with a UV/visible detector set at 205 nm by use of a Restek Pinnacle II C18 column (250 by 4.6 mm; 5-µm particles). Samples were eluted isocratically with water:acetonitrile:acetic acid (32:68:0.2 [vol/vol/vol]) at 1 ml/min. Retention times for 3OC12-HSL and the 3OC12-HSL acid product were 6.3 and 3.8 min, respectively. The 3OC12-HSL acid product was made by incubating 3OC12-HSL in 5 mM NaOH at room temperature for 4 h, and the ratios of peak areas for equivalent concentrations of 3OC12-HSL and 3OC12-HSL acid were experimentally determined to be 1:1.2. Initial rates were estimated under conditions in which less than 10% of the substrate was hydrolyzed and in which product formation was linear with incubation times and protein concentrations.

For the inhibition assays, serum and tissue homogenates were incubated overnight at 4°C in 25 mM Tris (pH 7.4) containing either 1 mM CaCl2, 10 mM EDTA, or 2 mM di-isopropyl fluorphosphonate (DFP) with 1 mM CaCl2. For determination of the metal ion requirements for 3OC12-HSL hydrolysis, lung and liver homogenates were chelated with 10 mM EDTA (in 5 mM Tris, pH 7.4) for 2 min at room temperature, and the reactions were carried out as described above in buffer containing 10 mM of either CaCl2, MgCl2 or MnCl2, or 1 mM ZnCl2. To irreversibly inhibit the PONs in the tissue homogenates, 100 µl of the homogenate was dialyzed overnight in microdialysis units (Pierce, Rockford, IL) at 4°C in 25 mM Tris (pH 7.4) containing 10 mM EDTA. Cell lysates were obtained by brief sonication of cell pellets on ice in 25 mM Tris buffer (pH 7.4) containing 0.05% n-dodecyl-β-D-maltoside and either 1 or 10 mM EDTA or 1 mM CaCl2. Lysates treated with 1 mM EDTA were quickly diluted into buffer containing 10 mM of either CaCl2, MgCl2, MnCl2, or 1 mM ZnCl2 and then analyzed for 3OC12-HSL activity. Lysates treated with 10 mM EDTA or 1 mM CaCl2 were kept overnight at 4°C and then analyzed for 3OC12-HSL activity in the presence of 10 mM CaCl2.

p-Nitrophenyl acetate (pNPA) hydrolysis was determined on a Multiskan Spectrum microplate reader (Thermo Electron Co., Vantaa, Finland). Serum or tissue homogenate was added to the 96-well plate and reactions (0.2-ml final mixture volume) were initiated by adding 1 mM of pNPA in 50 mM Tris, pH 8, with or without 1 mM CaCl2. The increase in absorbance at 412 nm, due to the release of p-nitrophenol, was monitored, and initial rates were calculated from the amount of p-nitrophenol released using a molar extinction coefficient of 17,100 M–1 cm–1.

For chlorpyrifos-oxon (CPO) hydrolysis assays, 50 mM Tris buffer, pH 8.5, containing 1 mM of CaCl2 was added to microcentrifuge tubes containing diluted purified enzyme, serum, or homogenates and preincubated at 37°C for 1 min. Reactions, 50-µl final mixture volume, were initiated by adding 0.5 µl of CPO stock solution (in methanol) so the final concentration was 0.32 mM, and mixtures were incubated at 37°C for 30 min. Reactions were stopped and reaction mixtures processed as described above for 3OC12-HSL. Supernatants (40 µl) were chromatographed on a BAS HPLC system equipped with a UV/visible detector set at 230 nm and a Beckman C18 column (250 by 4.6 mm; 5-µm particles). Samples were eluted at 1 ml/min in 70% acetonitrile containing 0.2% acetic acid. Retention times for CPO and the 3,5,6-trichloro-2-pyridinol product were 4.2 and 5.2 min, respectively. The ratios of peak areas for equivalent concentrations of CPO and 3,5,6-trichloro-2-pyridinol were experimentally determined to be 1:0.9. Initial rates were estimated under conditions in which less than 10% of the substrate was hydrolyzed. Hydrolysis of lovastatin and (±)5-hydroxy-6E,Z,11Z,14Z-eicosateterenoic acid, 1,5-lactone (5-HL), was determined as described previously (9).

RNA interference. RNA interference experiments were performed as described previously (16). To estimate PON2 knockdown, lysates were analyzed by Western blotting using PON2 and alpha-tubulin antibodies and enhanced chemiluminescence-based detection methods. X-ray films were scanned and densitometrically evaluated using Quantity One V4.6.2 software (Bio-Rad, Hercules, CA). PON2 protein levels were normalized to alpha-tubulin.

Western blotting. Westerns were performed on lysates as described previously (9) with minor modifications. Gels (12% Tris-HCl) were run under denaturing conditions and transferred to polyvinylidene difluoride membranes and membranes were blocked overnight at 4°C with StartingBlock PBS (Pierce, Rockford, IL) containing 0.05% Tween 20. The PON1 and PON2 standards were purified as previously described (9).

Statistical analysis. Data were analyzed using a two-tailed Student t test.


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RESULTS
 
3OC12-HSL hydrolysis by purified proteins. The specific activities of purified recombinant human PONs were determined, allowing a direct comparison of the enzymes' efficiencies for 3OC12-HSL (Table 1). The specific activities of the PONs with the proposed natural PON substrate, 5-HL, is also shown in Table 1 for comparison. All three PONs hydrolyzed 3OC12-HSL, with PON2 being the most efficient. Notably, the specific activity of PON2 with 3OC12-HSL was higher than with 5-HL, which was previously the best substrate known for PON2 (9). Increasing the substrate concentration from 10 to 25 µM resulted in increases in rates of approximately twofold or more (Table 1), indicating that a concentration of 10 µM is well below saturating for all the PONs. Human PON1 has a common coding region polymorphism, Q or R at position 192, which has been shown to significantly affect its hydrolytic rates with some substrates (8, 12). To determine if the PON1 192Q/R polymorphism influences 3OC12-HSL hydrolysis, the two isoforms were analyzed separately. The specific activity of PON192R was approximately twice that of PON192Q at the substrate concentrations tested (Table 1).


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  		TABLE 1. Specific activities for 5-HL and 3OC12-HSL hydrolysis by purified proteins

With some substrates, the lactonase activity of PON1 has been shown to be up to 20 times higher when the enzyme is present on HDL, its natural state in blood, than what is seen for the purified protein (13). Therefore, we sought to determine if HDL-associated PON1 was more active with 3OC12-AHL than it was with the purified recombinant enzyme. The rates of 3OC12-AHL (25 µM) hydrolysis in two human serum samples homozygous for PON192Q and PON192R were 630 and 1,740 nmol/min/mg of PON1, respectively. These values are similar to the corresponding rates with the purified recombinant PON192Q and PON192R isoforms (Table 1), suggesting that association with HDL has a limited effect on PON1 3OC12-AHL lactonase activity.

Two other commercially available purified esterases were also tested for their abilities to inactivate 3OC12-HSL. The lactonase activities of porcine liver carboxyl esterase and kidney N-acylase were 3 to 5 orders of magnitude lower than that of the PONs (Table 1). Interestingly, we did not detect any 3OC12-HSL N-acylase activity with the porcine kidney N-acylase (data not shown), which has been shown to cleave the acyl group off the open acid form of AHLs (41).

3OC12-HSL hydrolysis in serum and tissue and cellular homogenates. To identify the potential contribution of other mammalian enzymes with esterase or acylase activity in 3OC12-HSL metabolism, we tested the abilities of mouse serum and lung and liver homogenates and human serum and HepG2 and EA.hy 926 cell lysates to hydrolyze 3OC12-HSL. All of the samples converted 3OC12-HSL to the 3OC12-HSL acid product. Figure 1 shows representative HPLC chromatograms of the analysis of 3OC12-HSL metabolism by mouse serum and liver homogenates. No other peaks were apparent in the chromatograms, and all of the lactone that was hydrolyzed could be accounted for by the formation of the 3OC12-HSL acid product. This indicates that under the assay conditions, only lactonase, and no acylase, activity was present in the samples. The hydrolytic rates for the serum and tissue and cell homogenates are presented in Table 2.


Figure 1
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  		FIG. 1. HPLC chromatograms showing substrate and product peaks of 3OC12-HSL incubated in the presence of serum and liver homogenates. 3OC12-HSL was incubated at a concentration of 25 µM in the presence of diluted mouse serum (A and B) or 0.1 mg/ml of liver homogenate (C and D) for 0 min (A and C) or 20 min (B and D). Proteins were precipitated and supernatants were analyzed by HPLC as described in Materials and Methods.


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  		TABLE 2. Rates of 3OC12-HSL hydrolysis by serum, tissue homogenates, and cell lysates

Inhibition of 3OC12-HSL hydrolysis in serum. Mouse serum contains significant levels of carboxylesterases in addition to PON1, while human serum contains butyrylcholinesterase, PON1, and low levels of PON3 but no carboxylesterases (22). The abilities of these enzymes to hydrolyze 3OC12-HSL were determined by treating the serum with the class-specific esterase inhibitors DFP, an irreversible inhibitor of serine esterases, and EDTA, which inhibits metal-dependent esterases, including PONs (9, 19), which require calcium for activity. After treatment of the serum with the inhibitors, inhibition was verified by determining the residual activity with pNPA, which is hydrolyzed by carboxlyesterases, butyrylcholinesterase, and PONs. In the serum samples, the percentage of pNPA activity that remained after treatment with DFP corresponded exactly to the percentage of activity inhibited by EDTA (Fig. 2A), demonstrating that all the activity in the samples could be accounted for by DFP- and EDTA-sensitive esterases.


Figure 2
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  		FIG. 2. Inhibition of pNPA and 3OC12-HSL hydrolysis in mouse and pooled human sera by class-specific esterase inhibitors. Diluted serum was treated with DFP (1 mM, gray bars) or EDTA (10 mM, hatched bars) or left untreated (white bars) and incubated overnight at 4°C. Samples were then analyzed for pNPA (A) and 3OC12-HSL (B) hydrolysis as described in Materials and Methods. Data are the means of at least three replicates and error bars represent the standard deviations. *, Significantly different from the control P value of <0.005.

DFP did not inhibit 3OC12-HSL hydrolysis in human serum, but it did inhibit this activity in mouse serum by about 10% (Fig. 2B), indicating a minor contribution by a serine esterase(s) in the mouse serum. The extensive inhibition by EDTA in mouse and human serum samples demonstrates that 3OC12-HSL hydrolysis is almost completely mediated by PON, the only metal-dependent esterase present in the serum (22). The residual activity remaining after EDTA treatment in the human serum, and the roughly 5% in the mouse serum that was not attributable to serine esterase(s) may be due to albumin. Albumin does have esterase activity (22), and we found that purified human albumin hydrolyzed 3OC12-HSL (50 µM) at a rate of 0.017 ± 0.001 nmol/min/mg, which is about 5 orders of magnitude lower than the specific activity of purified human PON1.

Inhibition of 3OC12-HSL hydrolysis in mouse tissue homogenates. As an important metabolic organ, the liver contains many enzymes that could potentially metabolize 3OC12-HSL. Because the lung is a common target of P. aeruginosa infection in compromised patients, we also examined mouse lung homogenate. Pretreatment of the homogenates with DFP resulted in 91.3% and 89.1% inhibitions of pNPA activity in lung and liver, respectively (data not shown). In contrast, the hydrolysis of 3OC12-HSL in either of the tissue homogenates was not inhibited by DFP (Fig. 3).


Figure 3
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  		FIG. 3. Inhibition and metal requirements for the hydrolysis of 3OC12-HSL in mouse lung (A) and liver (B) homogenates. To determine the metal requirements, diluted homogenates were incubated for 2 min in the presence of 10 mM EDTA and then assayed for 3OC12-HSL hydrolysis in the presence of no metal or the indicated metal at a substrate concentration of 50 µM as described in Materials and Methods. The controls were not treated with EDTA and were assayed in the presence of 1 mM CaCl2 as described in Materials and Methods. For inhibition studies with DFP, the homogenates were treated with 1 mM of the inhibitor or, for the controls, were left untreated, incubated overnight at 4°C, and then analyzed for 3OC12-HSL hydrolysis at a substrate concentration of 50 µM as described in Materials and Methods. Data are the means of at least three replicates and error bars represent the standard deviations. *, Significantly different from the control P value of <0.005.

PONs contain two calcium ions, a low-affinity calcium required for catalytic activity and a higher-affinity calcium required for structural maintenance (20). The structural integrity of the PONs can be maintained by other metals, but only calcium can support the hydrolytic activity. Therefore, to identify the metal requirements for the hydrolysis, samples were briefly treated with EDTA to remove the catalytic calcium from PON and then tested for 3OC12-AHL hydrolysis in the absence of any metal or the presence of different metal ions. In the lung and liver homogenates, 3OC12-AHL activity was completely inhibited by EDTA but could be restored to the non-EDTA-treated levels in the presence of calcium (Fig. 3). In the lung homogenate, no activity was detected in the presence of magnesium, manganese, or zinc (Fig. 3A). In the liver homogenate, there was some magnesium- and manganese-dependent activity, but this was only about 10 to 15% of the calcium-dependent activity (Fig. 3B). This indicates a minor contribution by an esterase(s) which could be supported by magnesium and/or manganese.

There are other mammalian esterases, such as secretory phospholipases and the calpains, which are also calcium dependent (11, 18). Unlike these esterases, however, PONs are irreversibly inactivated when stripped of their structural calcium. To distinguish between hydrolysis of 3OC12-AHL by PONs and other potential calcium-dependent esterases, the homogenates were dialyzed overnight in the presence of 10 mM EDTA to strip PONs of their structural calcium and then analyzed for 3OC12-AHL activity in the presence of calcium. No activity was detected (data not shown), indicating that the calcium-dependent 3OC12-AHL activity in the homogenates is mediated by an esterase(s) which is irreversibly inactivated after the removal of metal ions for an extended period. These properties are consistent with PON-mediated activity.

Inhibition of 3OC12-HSL hydrolysis in HepG2 and EA.hy 926 cell lysates. To investigate further the potential of human enzymes to hydrolyze 3OC12-HSL, we characterized the ability of HepG2 and EA.hy 926 cell homogenates to hydrolyze this lactone. HepG2 cells are of human liver origin and express a wide range of metabolic enzymes, including phase I and phase II enzyme systems (23). EA.hy 926 cells are a well-characterized endothelium-derived cell line that has been shown to express PON2 (16). As shown in Fig. 4, the activity in the lysates was calcium dependent, with the residual activity detected in the presence of other metals being near our detection limit. When cells were lysed in the presence of calcium or EDTA, kept overnight at 4°C, and then analyzed in the presence of calcium, no activity was lost in the calcium-treated cells; however, no activity was detected in the EDTA-treated cells (data not shown). As with the mouse lung and liver homogenates, this calcium-dependent activity, which can be irreversibly inactivated by EDTA, implicates PON(s) as the main contributor to 3OC12-HSL hydrolysis in the lysates.


Figure 4
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  		FIG. 4. Metal requirements for the hydrolysis of 3OC12-HSL by HepG2 (A) and EA.hy 926 (B) cell lysates. Cells were lysed in the presence of 1 mM EDTA and then immediately diluted into buffer containing the metal shown. Lysates were tested for 3OC12-HSL hydrolytic activity at a substrate concentration of 50 µM in buffer containing the metals indicated in the figure as described in Materials and Methods. Data are the means of at least three replicates and error bars represent the standard deviations. *, Significantly different from the control P value of <0.005.

Western blot analysis revealed that PON2 is expressed in both cell lines, but PON1 was not detected (Fig. 5). Furthermore, the cell lysates had no detectable CPO hydrolytic activity (detection limit, 50 pg PON1/µg of lysate), which is specific for PON1, or lovastatinase activity (detection limit, 50 pg PON3/µg of lysate), which is specific for PON3, indicating that these PON proteins are not present in the cells or are present at very low levels (data not shown). Based on the specific activity of PON2 for 3OC12-HSL (Table 1) and the 3OC12-HSL hydrolytic activity in the cell lysates (Table 2), the amounts of PON2 in the HepG2 and EA.hy 926 cells were calculated to be 0.38 and 0.08 ng/mg of cell lysate, respectively. By use of these values, the amounts of PON2 loaded on the Westerns (Fig. 5B) were calculated to be approximately 5.7 ng and 1.3 ng of PON2 for HepG2 and EA.hy 926 cells, respectively. As seen in Fig. 5B, the intensity of 5 ng of the recombinant PON2 band is similar to the intensity of the upper bands for the HepG2 cells (an estimated 5.7 ng of PON2) but significantly higher than the upper bands for the EA.hy 926 cells (an estimated 1.3 ng of PON2). This suggests that the 3OC12-HSL-specific activity of the purified recombinant PON2 is in the same range as the specific activity of the PON2 in the cell lysates.


Figure 5
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  		FIG. 5. Western blot analysis of PON1 and PON2 in HepG2 and EA.hy 926 cell lysates. Lysates from preparations of two different pellets of HepG2 and EA.hy 926 cells (15 µg of protein), purified human serum PON1 and purified recombinant PON2, were analyzed by Western blotting with anti-PON1 4C.10 (A) or anti-PON2 (B) antibodies as described in Materials and Methods. (A) Lane 1, 10 ng of PON1; lanes 2 to 5, HepG2 and EA.hy 926 cell lysates; lane 6, 1 ng of PON1. (B) Lanes 1 and 3, EA.hy 926 cell lysates; lanes 2 and 4, HepG2 cell lysates; lane 6, 5 ng of PON2. The lower band in panel B is the mRNA splice variant of PON2 designated PON2-isoform 2 (16). The recombinant PON2 migrates faster than the endogenous cellular PON2 due to differences in the extents of glycosylation.

Finally, to estimate the relative importance of PON2 toward 3OC12-HSL hydrolytic activity, PON2 expression was reduced in the cells by transfection with PON2-specific small interfering RNAs (siRNAs). Western blotting demonstrated that PON2 protein levels were efficiently reduced by PON2-specific siRNA treatment (Fig. 6A). Figure 6B and C are representative of several different experiments and demonstrate that the decrease in PON2 mass very closely paralleled the decrease in the 3OC12-HSL lactonase activity in both HepG2 and EA.hy 926 cell lysates. As controls, closely comparable results were obtained with two different PON2-specific siRNAs (Fig. 6C and data not shown).


Figure 6
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  		FIG. 6. 3OC12-HSL hydrolysis and PON2 mass in PON2 siRNA-treated HepG2 and EA.hy 926 cell lysates. Cells were either left untreated, treated with PON2-specific siRNAs, or treated with a nonspecific scrambled siRNA as described in Materials and Methods. (A) Western analysis of PON2 protein in cell lysates of treated or untreated cells. Lysates from the pellets of the HepG2 (B) and EA.hy 926 (C) cells were prepared and assayed for PON2 mass (dark bars) by Western analysis and 3OC12-HSL hydrolytic activity (light bars) as described in Materials and Methods. For 3OC12-HSL hydrolytic activities, the data are the means of at least three replicates and error bars represent the standard deviations. * Significantly different from the control siRNA P value of <0.01.


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DISCUSSION
 
In this study, the contributions of PONs and other enzymatic pathways toward the inactivation of the P. aeruginosa virulence factor 3OC12-HSL were investigated in serum, mouse lung and liver homogenates, and in human HepG2 and EA.hy 296 cell lysates. These tissue sources were chosen because they are known to express a wide range of metabolic enzymes, including PONs, carboxylesterases, proteases, acylases, and amidases, with the potential to metabolize 3OC12-HSL. Whereas all the sources had significant 3OC12-HSL lactonase activity, the PONs were identified as the main, or only, contributors to this activity in the tested samples. That no 3OC12-HSL N-acylase activity was detected indicates the absence of a 3OC12-HSL N-acylase in the mammalian tissues tested.

Previous studies have shown that PONs can hydrolyze 3OC12-HSL (26, 42); however, these studies were performed using cells transfected with human PONs, which allows only a semiquantitative assessment of their activity. Here we determined the 3OC12-HSL lactonase-specific activities of purified human PONs for 3OC12-HSL hydrolysis, allowing a direct comparison of their efficiencies for 3OC12-HSL. The rate of 3OC12-HSL hydrolysis by PON2 was very high and about 22 and 75 times greater than the rates exhibited by PON1 and PON3, respectively, at substrate concentrations from 10 µM to 25 µM (Table 1). Although the local concentrations of 3OC12-HSL during P. aeruginosa infection in vivo are not known, the concentrations used in this study are considered to be within the physiological range. 3OC12-HSL concentrations of 1 to 10 µM are achieved in P. aeruginosa laboratory cultures, and concentrations from 300 to 600 µM have been detected in biofilms in vitro (3, 5).

For the inhibition studies, we took advantage of the PONs' calcium requirements for both catalytic activity and structural integrity to estimate the PONs' contribution to 3OC12-HSL hydrolysis. Nearly 100% of the 3OC12-HSL lactonase activity in the human serum, mouse lung, and human cell homogenates was calcium dependent and irreversibly inhibited by extended incubation in the absence of divalent metal ions. In human serum, the inactivation of 3OC12-HSL is mediated by PON1, because PON2 is not present in the serum (25) and PON3 is much less abundant than PON1 (29) and has a low 3OC12-HSL activity (Table 1). In mouse serum, an enzyme(s) other than PON1 contributes to the inactivation of 3OC12-HSL, as previously shown (26). Our findings confirm this and demonstrate that about 10% of the activity is due to a serine (DFP-sensitive) esterase. All three PONs are expressed in the mouse lung and liver (24, 28, 33); however, the levels of each PON paralog in these tissues are uncertain. We found that mouse lung homogenates have very low organophosphatase (PON1-specific) hydrolytic activity (unpublished observation). This finding, together with the relatively low 3OC12-HSL lactonase activity of PON3, suggests that PON2 is the key 3OC12-HSL lactonase in the mouse lung.

The 3OC12-HSL hydrolytic activities of a serine esterase that we identified in mouse serum and a magnesium- and manganese-dependent esterase that we identified in mouse liver were very low compared with the calcium-dependent (PON) activity. Despite the considerably lower activities of these esterases, in tissues or cells in which they are expressed at significant levels and in which PONs are deficient, they may play a role in inactivation of this QS signal. Metabolic inactivation of 3OC12-HSL through oxidative and reductive pathways may also occur. We have found that cytochrome P450s oxidize 3OC12-HSL, although our initial studies with human liver microsomes suggest that the rates are much lower than those exhibited by the PONs (data not shown).

The specificity of 3OC12-HSL hydrolysis for the PONs in the tissues examined and the very high specific activity of PON2 for 3OC12-HSL suggest that PON2 may be a primary mechanism by which mammals inactivate 3OC12-HSL. PON2 is expressed in a very broad range of tissues and cell types, including cells likely to come in contact with P. aeruginosa, such as airway epithelial cells, macrophages, endothelial cells, and fibroblasts (16, 30, 38). Indeed, in the two human cell lines tested in this study, hepatoma HepG2 and endothelial EA.hy 926 cells, we demonstrated that PON2 exclusively mediated the hydrolysis of 3OC12-HSL in the cell lysates. Our findings concur with a previous study by Stoltz et al. which demonstrated that murine tracheal epithelial cell lysates from PON2-deficient mice had an impaired ability to inactivate 3OC12-HSL compared to what was seen for the wild-type cells (38). However, paradoxically, Stoltz et al. also observed that the rates of inactivation of 3OC12-HSL in intact PON2-deficient and wild-type cells were the same (38). Recent studies have demonstrated the localization of PON2 to the endoplasmic reticulum and nuclear envelope, but not the plasma membrane, in murine macrophages and a number of human cell lines (16, 35), which suggests that PON2 3OC12-HSL inactivation likely occurs intracellularly. This is consistent with the suggestion by Stoltz et al. that transport of 3OC12-HSL into the cells may be rate limiting for inactivation and that PON1 and/or PON3 are present at levels high enough to rapidly inactivate the 3OC12-HSL that enters the PON2-deficient cells. It is important to note, however, that Stoltz et al. also demonstrated that PON2-deficient epithelial cultures are less able to attenuate QS by P. aeruginosa than are wild-type cultures after infection of the cells with the bacteria (38). It is thus possible that cell permeability for 3OC12-HSL and/or PON2 activity or cellular localization is altered during infection/inflammation so that under conditions of infection, PON2 inactivates 3OC12-HSL. Further studies are necessary to clarify the role that PON2, and the other PONs, may play in inactivating 3OC12-HSL and attenuating P. aeruginosa QS in cell cultures and in vivo.

PONs are known to hydrolyze a of number of AHLs, including those produced in other pathogenic genera, such as Burkholderia, Yersinia, Serratia, and Aeromonas (2, 9, 38). Thus, in addition to 3OC12-HSL, PONs may also be predominant mammalian enzymes inactivating a number of AHLs. Brucella melitensis is an intracellular pathogen which has been shown to utilize N-dodecanoyl-L-HSL to control virulence in an in vivo model (6). N-Dodecanoyl-L-C12-HSL is hydrolytically inactivated by the PONs (9, 38) and PON2 may be more relevant for the inactivation of AHLs utilized by B. melitensis and other intracellular pathogens, whereas PON1, which is highly expressed in the liver and associated with HDL, may be more important for extracellular inactivation and clearance of 3OC12-HSL from the blood. Continued research on the AHL lactonase activities of the PONs will improve our understanding of the mechanisms by which the host defends against gram-negative bacteria and may result in the identification of PONs as an important therapeutic target.


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ACKNOWLEDGMENTS
 
This study was supported in part by the U.S. Army Medical Research and Material Command under cooperative agreement no. DAMD17-01-1-074.

The content of this article does not necessarily reflect the position or policy of the U.S. government, and no official endorsement should be inferred.


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FOOTNOTES
 
* Corresponding author. Mailing address: The University of Texas Southwestern Medical Center, Department of Internal Medicine, Division of Epidemiology, 5323 Harry Hines Blvd., Dallas, TX 75390-8874. Phone: (214) 648-2454. Fax: (214) 648-7893. E-mail: john.teiber{at}utsouthwestern.edu Back

{triangledown} Published ahead of print on 17 March 2008. Back

Editor: B. A. McCormick


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Infection and Immunity, June 2008, p. 2512-2519, Vol. 76, No. 6
0019-9567/08/$08.00+0     doi:10.1128/IAI.01606-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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