Paraoxonase-2 deficiency enhances Pseudomonas aeruginosa quorum sensing in murine tracheal epithelia

Page 1 of 43 in PresS. Am J Physiol Lung Cell Mol Physiol (November 22, 2006). doi:10.1152/ajplung.00370.2006 Articles

Paraoxonase-2 Deficiency Enhances Pseudomonas aeruginosa Quorum Sensing in Murine Tracheal Epithelia

1*

1*

2,3

2

2,3

David A. Stoltz , Egon A. Ozer , Carey J. Ng , Janet M. Yu , Srinivasa T. Reddy , 2 3 4 1# Aldons J. Lusis , Noam Bourquard , Matthew R. Parsek , Joseph Zabner , Diana M. 2 Shih

* These authors contributed equally to this work. 1

Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA 2 Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA 3 Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA 4 Department of Microbiology, University of Washington, Seattle, 98195, USA #

To whom correspondence should be addressed at: Department of Internal Medicine Roy J. and Lucille A. Carver College of Medicine University of Iowa 440 EMRB Iowa City, IA 52242, USA Tel: 319-335-7608 Fax: 319-335-7623 E-mail: [email protected] Running Title: Paraoxonase and Murine Airway Epithelia

Copyright © 2006 by the American Physiological Society.

Page 2 of 43

1

ABSTRACT Pseudomonas aeruginosa is an important cause of nosocomial infections and is frequently present in the airways of cystic fibrosis patients. Quorum sensing mediates P. aeruginosa’s virulence and biofilm formation through density-dependent interbacterial signaling with autoinducers. N-3-oxododecanoyl homoserine lactone (3OC12-HSL) is the major autoinducer in P. aeruginosa. We have previously shown that human airway epithelia and paraoxonases (PONs) degrade 3OC12-HSL. This study investigated the role of PON1, PON2, and PON3 in airway epithelial cell inactivation of 3OC12-HSL. All three PONs were present in murine tracheal epithelial cells, with PON2 and PON3 expressed at the highest levels. Lysates of tracheal epithelial cells from PON2 knockout mice, but not PON1 or PON3 knockout mice, had impaired 3OC12-HSL inactivation compared to wild type mice. In contrast, PON1, PON2, or PON3 targeted deletions did not affect 3OC12-HSL degradation by intact epithelia. Overexpression of PON2 enhanced 3OC12-HSL degradation by human airway epithelial cell lysates, but not by intact epithelia. Finally, using a quorum-sensing reporter strain of P. aeruginosa, we found that quorum sensing was enhanced in PON2 deficient airway epithelia. In summary, these results show that loss of PON2 impairs 3OC12-HSL degradation by airway epithelial cells and suggests that diffusion of 3OC12-HSL into the airway cells can be the rate-limiting step for degradation of the molecule.

Keywords: N-3-oxododecanoyl homoserine lactone, 3OC12-HSL, cystic fibrosis, innate immunity, paraoxonase

Page 3 of 43

2

INTRODUCTION The lung is continuously exposed to a variety of infectious pathogens and foreign substances. Airway epithelial cells are uniquely designed for monitoring and clearing invading microbes through a number of mechanisms including: mucociliary clearance, proinflammatory cytokine and chemokine production, and release of antimicrobial peptides such as defensins, lactoferrin, and lysozyme (2). In the absence of impaired innate immune function or underlying comorbid medical conditions, these mechanisms are generally effective at preventing the development of pulmonary infections.

An uncommon, but serious, cause of respiratory tract infections is Pseudomonas aeruginosa, a Gram-negative bacterial pathogen that is ubiquitously present in the environment. Under suitable conditions and in a susceptible host this microorganism is a frequent cause of acute and chronic infections. P. aeruginosa is a common cause of hospital-acquired infections including cystitis, burn wound infections, and ventilator associated pneumonia. In addition, cystic fibrosis (CF) patients are frequently colonized with P. aeruginosa and pulmonary decline in CF is directly correlated to infection (22, 27). P. aeruginosa’s ability to produce chronic infections is, in part, explained by its capacity to produce and exist in biofilms. Biofilms are communities of bacteria that are characterized by a structured architecture consisting of bacteria contained within an extracellular matrix. This surrounding matrix is composed of both bacterial and host components. Biofilms typically display a marked resistance to antibiotic killing and therefore infections associated with biofilm-forming bacteria are difficult to eradicate (20).

Page 4 of 43

3

Many bacteria, including P. aeruginosa, utilize a cell-to-cell communication system termed quorum sensing to regulate biofilm formation, under certain conditions, and virulence factor production (12). Quorum sensing is dependent upon environmental concentrations of small quorum-sensing signaling molecules produced by bacteria and therefore bacterial cell density. Two well-studied quorum-sensing systems in P. aeruginosa, las and rhl, utilize molecules that are acylhomoserine lactones (AHLs). LasI is responsible for the production of N-3-oxododecanoyl homoserine lactone (3OC12HSL) and LasR is the transcription factor that responds to 3OC12-HSL. RhlI is an enzyme that synthesizes N-butanoyl homoserine lactone (C4-HSL) and RhlR is the transcription factor that responds to C4-HSL. Increasing concentrations of 3OC12-HSL lead to production of C4-HSL. These two systems constitute a regulatory cascade, with the las system controlling expression of the rhl system. Importantly, as many as 6% of the approximately 6,000 genes expressed by P. aeruginosa are influenced by the las or rhl quorum-sensing systems (10, 28, 33).

Paraoxonases (PONs) are enzymes originally described based upon their ability to hydrolyze organophosphates. Since the initial descriptions of PON’s enzymatic activity, three members of the PON family have been described: PON1, PON2, and PON3 (6). The three genes encoding the enzymes are located next to each other on the long arm of human chromosome 7. Human PON1 and PON3 are primarily produced in the liver and found circulating in serum bound to HDL particles. The majority of research related to PONs has been directed at the proposed protective role of PON1 in cardiovascular

Page 5 of 43

4 disease. PON2 is more ubiquitously expressed and is found in many tissues including placenta, brain, lung, and kidney (19). More recent studies have suggested that the native substrates for PONs are lactones (14). We have shown that human airway epithelial cells can inactivate AHLs, including 3OC12-HSL (4). This ability to interfere with quorum sensing may represent an additional function of airway epithelial cells in host defense. In support of this concept, PON1 is known to inhibit biofilm formation by P. aeruginosa (21). Our hypothesis is that PON activity in airway epithelial cells is important in quorum-sensing modulation and functions as part of the host defense response to P. aeruginosa. Here we describe the functional consequences of targeted deletion of PON1, PON2, and PON3 on quorum-sensing regulation by murine airway epithelial cells.

Page 6 of 43

5

MATERIALS AND METHODS Primary airway epithelial cell culture model. Primary human airway epithelial cells were isolated from trachea and bronchi of human donor lungs. The isolated cells were seeded onto collagen coated, semipermeable membranes (0.6 cm2 Millicell-HA; Millipore, Bedford, MA) and grown at the air-liquid interface as previously described (13, 40). All experiments were performed on well-differentiated human airway epithelia (HAE) (greater than 14 days after seeding of cells). Samples were collected with approval of the University of Iowa Institutional Review Board.

Murine tracheal epithelial cells were isolated and grown based upon the method previously described by You et al. (38). Mice tracheas were isolated following euthanasia. Tracheas were cut length-wise and immersed in dissociation media (15 mg Pronase/10 ml Ham’s F-12 media) at 4°C. Twenty-four hours later, epithelial cell clumps were harvested and resuspended in media containing a 50:50 ratio of DMEM:Ham’s F-12 supplemented with 4mM glutamine, 10 µg/ml insulin, 5 µg/ml transferrin, 62 µg/ml cholera toxin, 5 ng/ml epidermal growth factor, 0.03 mg/ml bovine pituitary extract, 10-8 M retinoic acid, 5% fetal bovine serum, and 20 U/µg/ml Pen-Strep. Harvested cells were pre-incubated in a culture dish at 37°C in 5% CO2 for 2-3 hours to remove fibroblasts. The epithelial cells were then seeded onto rat tail collagen pre-coated transwell membranes (0.33 cm2 insert membrane growth area, Co-Star; Corning, NY). Four days post-seeding, an apical air interface was attempted daily until a confluent culture epithelia

Page 7 of 43

6 was formed. Once formed, the polarized culture epithelia was maintained in media containing 50:50 DMEM:Ham’s F-12 supplemented with 4 mM glutamine, 10-8 M retinoic acid, 2% V/V NuSerum, and 20 U/µg/ml Pen-Strep. Culture epithelia were viable for 3-4 months.

Microscopic analysis of cultured epithelium. For light microscopy, epithelial cell cultures were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Samples were prepared for Transmission Electron Microscopy imaging using routine methods. Briefly, the intact filters were fixed in 2.5% gluteraldehyde in 0.1M cacodylate buffer pH 7.4, post fixed in 1% osmium tetroxide, en bloc stained in 2.5% uranyl acetate, and dehydrated in a graded series of ethanol. After infiltration with Eponate 12 (Ted Pella, Inc., Redding, CA) the samples were cured, sectioned at 70 nm and poststained with uranyl acetate and lead citrate. Sections on formvar coated and uncoated copper grids were imaged in a JEOL 1230 TEM (Peabody, MA) using a Gatan 2k x 2k UltraScan 1000 camera (Pleasanton, CA.).

3OC12-HSL degradation by intact epithelium and lysate preparations. For intact epithelium experiments, the apical and basolateral surfaces of cultured airway epithelium were washed twice with PBS containing calcium and magnesium (PBS +/+, pH 7.1). 3OC12-HSL in acidified ethyl acetate (EtAc) was dried under a nitrogen gas stream and then dissolved in PBS +/+ to achieve a final concentration of 10 µM 3OC12-HSL. Dissolved 3OC12-HSL was added to the apical (150 µl) and basolateral

Page 8 of 43

7 (250 µl) surface of human airway epithelia. For murine tracheal epithelial cultures, 75 µl and 250 µl were added to the apical and basolateral surfaces, respectively. Treated epithelia were incubated at 37°C in 5% CO2 and 6 µl samples of the apical media were collected at 0, 15, 30, and 60 minutes for determination of remaining 3OC12-HSL using a bioassay. These samples were added to 100 µl of EtAc and stored in airtight glass vials at -20°C.

For epithelial cell lysate experiments, epithelia were first washed with cold PBS +/+ and then 50 µl of lysis buffer (50 mM Tris·HCl, pH 6.9/150 mM NaCl/10 µM leupeptin/10 µM aprotinin/1 µM pepstatin A/1 mM phenylmethylsulfonyl fluoride/0.1 mg/ml benzamidine ) was added to the apical surface. After 20 min of rocking in lysis buffer at 4°C, the cells were scraped free from the membrane with a pipette tip and lysed by sonication (10 pulses) (Branson Sonifier 250, Danbury, CT). The cellular debris was cleared from the lysate by centrifugation (4500 g x 30 sec, 4°C). Relative protein concentrations were determined by a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA). Lysate preparations (10%) were diluted in PBS and incubated in the presence of 10 µM 3OC12-HSL (in PBS +/+) at 37°C. At various timepoints, 6 µl aliquots were collected and stored as described above.

3OC12-HSL and 3OC12-HSL quantitative assay. All AHLs were obtained from Sigma-Aldrich Chemicals (St. Louis, MO), except for 3OC12-HSL, which was obtained from Vertex Pharmaceuticals (Coralville, IA).

Page 9 of 43

8 3OC12-HSL was measured in a quantitative bioassay as previously described using Escherichia coli MG4 (pKDT17) (23).

CHO and epithelial cell culture gene transfer experiments. Chinese hamster ovary (CHO) cells were cultured as monolayers in 24-well plates. Recombinant adenovirus serotype 5 (Ad5) expressing hPON1, hPON2, hPON3, or GFP were produced by the University of Iowa Vector and Gene Targeting core (34). CHO cells were infected with recombinant Ad5 by CaPi transfection (8) and allowed to express for 48 hours. Synthetic AHLs were reconstituted in PBS +/+ to 20 µM and incubated for one hour on transfected CHO cells. 3OC12-HSL lactone ring hydrolysis was determined as described below.

Primary cultures of human airway epithelia were infected basolaterally with 100 MOI (multiplicity of infection) of Ad5 expressing hPON2 or GFP. The Millicells were returned to fresh media and incubated at 37°C for 48 hours and used for intact epithelia or lysate experiments as previously described above.

Western blot analysis of CHO cells. CHO cell lysates were made by incubation in lysis buffer (50 mM Tris·HCl, pH 7.5, 138 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM NaF, phenylmethylsulfonyl fluoride, leupeptin, pepstatin, and aprotinin) with 1% Triton X-100 for 20 min at 4°C on a rocker. Cells were scraped from the tissue culture plates and transferred to a microfuge tube. Sonication was performed for 10 sec and cell debris was cleared by centrifugation

Page 10 of 43

9 at 4,500 g for 30 sec, 4°C. The lysates were then combined with loading buffer and separated by SDS-PAGE. Protein was then transferred to polyvinylidene difluoride membranes (PVDF) (Immobilon-P; Millipore, Billerica, MA). PVDF membranes were blocked overnight at 4° C in 5% bovine serum albumin in PBS. Membranes were then incubated with the appropriate primary antibody for 2 h at room temperature and then washed 3 times in 1X TTBS (137 mM NaCl, 2.7 mM KCl, 2.5 mM Tris, 0.05% Tween 20). Primary antibodies were: PON1 – monoclonal, anti-human PON1 at 1:1000 (AbCAM, Cambridge, MA), PON2 – rabbit, polyclonal anti-human PON2 (Orbigen, San Diego, CA), and PON3 – rabbit, polyclonal anti-human PON3 (generated by Affinity BioReagents, Golden, CO). Secondary antibodies were conjugated to horseradish peroxidase (Amersham Biosciences, Buckingshire, England) and used at 1:20,000 for 1 h. Following 3 washes with 1X TTBS, immunoreactive bands were detected with SuperSignal solution (Pierce, Rockford, IL) and exposed to film.

Determination of unhydrolyzed 3OC12-HSL by HPLC. 3OC12-HSL was extracted from the culture media samples in acidified ethyl acetate, dried under sterile nitrogen gas, and concentrated 10-fold in methanol. Reconstituted samples were loaded onto a C18 HPLC column (Supelco, Bellefonte, PA) and eluted in a 60%-90% methanol:water gradient. Eluent from the HPLC column was ionized using positive electrospray and directed into a quadrupole ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) for analysis of lactone ring hydrolysis.

Page 11 of 43

10

P. aeruginosa quorum-sensing reporter experiments. Overnight cultures of a reporter strain of P. aeruginosa expressing lacZ under control of the qsc102 3OC12-HSL-responsive promoter (PAO1-qsc102-lacZ) were subcultured for 2 hrs at 37°C (100 µl in 5 ml LB containing gentamycin 100 µg/ml). The bacteria were washed twice with cold PBS +/+ and, following centrifugation, the bacterial pellet was resuspended in 0.5 ml PBS +/+. A 2 µl suspension of the bacteria (approximately 105 organisms) was applied to the apical surface of the epithelium and cultures were incubated at 37°C in 5% CO2. At various timepoints, the extracellular bacteria were collected from the apical surface by rinsing with 100 µl of PBS +/+. Quantitative bacterial counts were performed with standard plating methods and galactosidase expression was determined with Galacto-Light Plus (Applied Biosystems, Bedford, MA).

RT-PCR of murine tracheal epithelia. PON1, PON2, and PON3 mRNA levels in murine tracheal epithelial cells were determined as previously described (21).

Microarray analysis of HAE cultures. Total RNA was extracted from HAE cultures from eight separate donors using TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer’s recommendations. Following a 30 minute treatment with 1U/10µg RNA with DNase I (Invitrogen), an additional RNA cleanup step was performed using the Qiagen RNeasy

Page 12 of 43

11 total RNA isolation kit (Chatsworth, CA). The RNA was processed for expression array studies according to manufacturer’s recommended protocols and analyzed using the Affymetrix human U133A GeneChip (Santa Clara, CA) (26, 39).

PON knockout animal generation. PON1 and PON2 knockout animals were generated as previously described (18, 29). Generation of the PON3 knockout mouse will be described elsewhere (J. M. Yu, A. J. Lusis, and D. M. Shih, unpublished data). After germ line transmission, backcrossing was performed with C57BL/6J for 10, 6 and 4 generations for PON1, PON2, and PON3 knockout mice, respectively. Control mice were wild type littermates, for their respective experimental group. All experiments were reviewed and approved by the Animal Care and Use Committees at UCLA and the University of Iowa.

Statistical Analysis. Statistical analysis was performed using the Student’s t test. Statistical significance was set at p < 0.05.

Page 13 of 43

12

RESULTS Airway epithelial cells express PON1, PON2, and PON3 mRNA. We have previously shown that human airway epithelial cells are capable of degrading 3OC12-HSL (4) and that PONs are also able to inactivate 3OC12-HSL (21). Since a common site of P. aeruginosa infection is the lung, and PONs are capable of inactivating AHLs, we wanted to define the role of PON1, PON2, and PON3 in the airway epithelia response to quorum-sensing signals.

To investigate the expression profile of PON1, PON2, and PON3 in murine airway epithelium, we first cultured murine tracheal epithelial cells at the air-liquid interface. Figures 1A and 1B show hematoxylin and eosin as well as TEM images of the polarized epithelium grown at the air-liquid interface used for these studies. The cells become well differentiated and form a pseudostratified epithelia with ciliated cells present and an intact air-liquid interface. Transcripts for all three PONs were present. However, PON2 and PON3 were expressed at the highest level (Figure 2A). This is in contrast to human airway epithelia, that also express PON1, PON2, and PON3, but have much greater expression of PON2 mRNA compared to PON1 or PON3 (Figure 2B).

PON2 preferentially degrades 3OC12-HSL. PON is a promiscuous enzyme with multiple enzymatic activities including paraoxonase, aryl esterase, and lactonase activity (6). Recent studies have suggested that lactone molecules are likely to be PONs’ natural substrates (7, 14, 21). Since all three members of the PON family are expressed in airway epithelia, we first examined whether

Page 14 of 43

13 substrate specificity was different among the three PONs. CHO cells were transduced with adenoviruses encoding either PON1, PON2, or PON3 (Figure 3). Forty-eight hours later, the cells were exposed to AHLs of varying side chain lengths and substitution status. The active or closed-ring AHL forms in the cell media were determined by HPLC following a 60 minute incubation. Figure 3 shows that all three PONs have similar patterns of inactivating AHLs, with PON2 having the greatest lactonase activity specifically for 3OC12-HSL. Prior studies have shown that PON2 has much greater lactonase activity for AHLs, compared to PON1 or 3 (7, 21). PON2 specificity for 3OC12-HSL supports the hypothesis that this molecule may represent a natural substrate for PON2. We also found that only AHLs with acyl side chains 8 carbons in length or longer were susceptible to degradation by the PONs and the extent of lactonolysis following incubation with the PON-expressing cells was proportional to the side-chain length. Side-chain substitution also contributed to the amount of degradation in that 3OC12-HSL was more extensively degraded than C12-HSL, indicating that hydroxylation of the third carbon of the acyl side chain augments PON’s lactonase activity against the AHLs.

Murine tracheal epithelial cell lysates from PON2 knockout mice have impaired 3OC12-HSL inactivating ability. We next asked whether loss of individual PONs would impair the ability of murine tracheal epithelial cell lysates to inactivate 3OC12-HSL. PON1, PON2, and PON3 knockout mice were generated by targeted deletion. Tracheas were harvested

Page 15 of 43

14 from knockout mice, and their corresponding wild type littermates, and then cultures of murine tracheal epithelial cells were established.

Figure 4 shows that PON1, PON2, and PON3 knockout murine tracheal epithelia had little to no transcripts present for their respectively deleted PONs, compared to their corresponding wild type epithelia (Figure 2A).

To investigate the ability of murine tracheal epithelial cells, individually disrupted for each PON, to degrade 3OC12-HSL, we harvested cell lysates and exposed them to 3OC12-HSL. Wild type murine tracheal epithelial cell lysates degraded 3OC12-HSL over time with less than 25% of active 3OC12-HSL remaining after a 60 minute incubation. PON1 knockout and PON3 knockout tracheal epithelial cell lysates had preserved 3OC12-HSL degrading capacity compared to littermate controls. However, PON2 knockout epithelial cell lysates had significantly impaired ability to degrade 3OC12-HSL, with more than 75% of the unhydrolyzed 3OC12-HSL present at the end of a 60 minute incubation (Figure 5). In order to determine if this defect could be reversed, we added lysates from CHO cells transduced with AdPON2 to tracheal epithelial cell lysates from PON2 knockout mice. 3OC12-HSL inactivation was significantly improved with only 39.6 ± 1.7 % of unhydrolyzed 3OC12-HSL remaining after a 60 minute incubation, compared to PON2 knockout lysate alone (79.1 ± 5.1%). These data show that loss of PON1 or PON3 activity in murine tracheal epithelial cell lysates does not affect AHL degradation, but the absence of PON2 impairs this activity.

Page 16 of 43

15 Intact murine tracheal epithelial cell cultures have preserved capacity to degrade 3OC12-HSL despite loss of PON1, PON2, or PON3. In order to more closely simulate in vivo conditions, 3OC12-HSL hydrolysis by an intact epithelia was determined. We hypothesized that intact epithelia from PON2 knockout mice would display impaired 3OC12-HSL degradation based upon our results from the lysate experiments.

We first tested whether airway surface liquid (ASL) contained any AHL degrading activity. Following collection of ASL from 12 transwells (in a total volume of 100 µl), we incubated the collected fluid with 3OC12-HSL. There was no degradation by the ASL (data not shown), suggesting that PONs are either not secreted or not active in the apical compartment. This is consistent with lack of lactonase activity of human and mouse bronchoalveolar lavage fluid (4).

We then measured the hydrolysis of 10 µM 3OC12-HSL, following addition to the apical and basolateral compartments of the murine epithelial transwells. Figure 6 shows that intact epithelia from both PON1 and PON3 deficient mice had preserved capacity to degrade 3OC12-HSL. In contrast to our results from the PON2 knockout lysate data, intact epithelia from PON2 knockout mice also degraded 3OC12-HSL at a similar rate as control epithelia. This finding was surprising and raises several possible explanations. First, differing cellular localizations of mouse PON1, PON2, and PON3 may account for the observed differences. Second, the lysate experimental conditions may alter PON activity based upon yet undetermined factors including local calcium

Page 17 of 43

16 concentration, redox states, or endogenous inhibitors. Finally, diffusion of 3OC12-HSL across the cell membrane may be the rate-limiting step to degradation at the concentration tested.

Overexpression of PON2 in human airway epithelial cell cultures enhances 3OC12HSL degradation only in lysate preparations. In order to test if diffusion of 3OC12-HSL across the cell membrane was the ratelimiting step, we transduced HAEs with AdGFP or AdPON2. We predicted that if diffusion across the membrane were rate limiting, increasing PON2 levels would result in no change in 3OC12-HSL degradation, by intact epithelia, at the concentration tested. Moreover, increasing concentrations of 3OC12-HSL should result in enhanced degradation in the presence of higher PON2 concentrations. Alternatively, higher 3OC12-HSL concentrations in the setting of impaired lactonase activity (i.e, airway epithelia from PON2 deficient mice) would lead to decreased 3OC12-HSL degradation. We compared the rates of 3OC12-HSL degradation by hPON2 transduced epithelia in lysate and intact epithelia preparations. Figure 7 shows that PON2 transduction significantly enhanced inactivation of 3OC12-HSL in lysates. However, overexpression of PON2 did not influence the rate of 3OC12-HSL degradation by intact epithelium. These data suggest that at 10 µM 3OC12-HSL, diffusion across the cell membrane is rate-limiting for inactivation of 3OC12-HSL.

Intact tracheal epithelial cultures from PON2 knockout mice show enhanced quorumsensing reporting by P. aeruginosa.

Page 18 of 43

17 We next wanted to test the hypothesis that increased concentrations of 3OC12HSL in the setting of diminished PON activity would result in impaired degradation. However, concentrations of 3OC12-HSL greater than 10-20 µM are difficult to obtain under experimental conditions due to the molecule’s limited solubility in aqueous solutions. Therefore, to expose respiratory epithelium to greater 3OC12-HSL concentrations and more closely mimic in vivo conditions, we developed a P. aeruginosa -galactosidase ( -gal) quorum-sensing reporter assay. We first developed and tested this assay using serum from wild type and PON1 knockout mice. We used PON1 knockout sera because PON1 is the predominant PON present in sera. Figure 8A shows that over a 6 hour period, -gal expression increased in serum from both wild type and PON1 knockout mice. However, PON1 knockout mouse serum had significantly higher -gal expression, compared to control serum. This shows that in the absence of PON1, P. aeruginosa has enhanced quorum sensing as measured by increased -gal expression.

We next tested this quorum-sensing assay in intact respiratory epithelium. A 2 µl suspension of 105 cfu of PAO1-qsc-102-lacZ was applied to the apical surface of murine tracheal epithelial cell cultures. Following varying lengths of incubation, the apical fluid was collected for -gal measurements (quorum-sensing activity) and bacterial quantification. We hypothesized that PON2 knockout epithelia would show increased gal levels compared to controls. At 4 and 6 hours following addition of the bacteria, gal levels were increased with levels approximately doubled at the 6 hour timepoint, compared to control epithelia (Figure 8B). Interestingly, the bacterial counts were similar between the groups demonstrating that the observed differences in -gal expression were

Page 19 of 43

18 caused by changes in quorum sensing and not due to changes in bacterial replication.

Page 20 of 43

19

DISCUSSION The main results of this study show that murine tracheal epithelial cells are capable of degrading the P. aeruginosa quorum-sensing molecule, 3OC12-HSL. In addition, these cells express all three PON enzymes, with PON2 and PON3 being expressed at the highest levels. While targeted deletion of PON1 and PON3 had no effect on the ability of these cells to degrade 3OC12-HSL, loss of PON2 significantly impaired this ability in lysate preparations. Finally, the absence of PON2 enhanced P. aeruginosa quorum sensing in intact epithelia cultures. This study is the first to demonstrate the individual contributions of PON1, PON2, and PON3 in airway epithelial cell 3OC12HSL degradation.

We previously reported that human airway epithelial cells are capable of inactivating quorum-sensing molecules through an unknown mechanism (4). We, and others, have subsequently shown that PONs can hydrolyze the lactone ring of AHL quorum-sensing molecules, including 3OC12-HSL (7, 21, 37). This raised the question of whether or not PON functions as an inhibitor of quorum-sensing signaling and therefore as a regulator of P. aeruginosa virulence and, potentially, biofilm formation. In response to this question, we demonstrated that recombinant PON1 and wild type murine serum reduced biofilm formation, while biomass was enhanced in the presence of serum deficient in PON1 (from PON1 knockout mice). Paradoxically, PON1 knockout mice were protected in a P. aeruginosa peritonitis/sepsis model (21).

Page 21 of 43

20 The current study advances our knowledge regarding PON’s possible role in airway host defense. The respiratory epithelium plays a very important role in the host response to inhaled toxins and microbes that escape the upper airway clearance mechanisms. These cells are uniquely situated to prevent colonization, and ultimately infection, of the distal airways and alveoli. Airway epithelial cells can secrete a number of mediators that are important for mobilization of immune system cells, repair of injured epithelium, and direct pathogen killing with antimicrobial peptides such as -defensins, lactoferrin, lysozyme, LL-37, and secretory leukocyte proteinase inhibitor (2). We propose that PONs may function as a defense mechanism against bacterial pathogens that utilize lactone molecules to regulate their virulence. PONs are conserved from the nematode Caenorhabditis elegans to mammals, but the endogenous substrate(s) is unknown. Recently, we and others reported findings suggesting that the natural substrates for PONs are in fact lactone molecules (7, 14, 21). Consistent with this hypothesis is that while PON1, PON2, and PON3 all exhibit lactonase activity, there is greater individual variability for other substrates depending upon the PON enzyme examined (7, 32). Interestingly, in the human body, naturally occurring lactones are limited and mainly consist of metabolites of fatty acid oxidation. Therefore, the question remains whether exogenous lactones, possibly the AHL quorum-sensing molecules of P. aeruginosa, represent a natural substrate for PON.

We showed in this study that murine tracheal epithelial lysates from PON2 deficient mice had impaired 3OC12-HSL degrading activity, compared to PON1 or PON3 knockout cells. However, intact epithelium showed no differences. Several

Page 22 of 43

21 possible mechanisms may account for this discrepancy. First, although all three mouse PONs are membrane bound (15), they may exhibit different subcellular localization patterns. PON1 is bound to the extracellular membrane and transferred to HDL particles (5). In HeLa cells, PON2 localizes to cytosolic and cell membrane fractions, but is absent in the supernatant fraction (19). Human PON3 is also found on HDL, however, its subcellular localization is not well understood (25). A global survey study of organ and organelle protein expression in mouse provides evidence that mouse PON1, PON2, and PON3 are all membrane bound without further information on subcellular localization (website: http://tap.med.utoronto.ca/~mts/) (15). The method of lysate preparation (i.e., clearing centrifugation step) may affect which PONs are present in the reaction mixture and how they individually contribute to 3OC12-HSL degradation. Secondly, the chemical milieu that the PONs are exposed to may be different in the lysate versus intact epithelium preparations. A number of conditions have been shown to affect PON activity including: redox state, calcium concentration, lipid associations, and pH (1, 11, 17, 32). Finally, diffusion of 3OC12-HSL across the cell membrane may be the rate determining step for 3OC12-HSL inactivation. Findings from our intact epithelium studies, in which individual deletion of PON1, PON2, or PON3 did not affect 3OC12-HSL inactivation support the latter hypothesis. This suggests that diffusion of the molecule across the cell membrane limits the rate of quorum-sensing molecule inactivation by PON. Also supporting this hypothesis are the results of the experiments in which we over-expressed PON2 in human airway epithelial cells. In the PON2 deficiency model, the remaining two PONs, despite having relatively less lactonase activity compared to PON2, are still able to adequately degrade 3OC12-HSL in the intact epithelia studies. The importance of

Page 23 of 43

22 PONs in the regulation of quorum-sensing control will ultimately require a murine model with all three PONs deleted.

One advantage of this study is that with targeted deletion of the individual PONs, the specific contributions of PON1, PON2, and PON3 towards airway epithelial cell degradation of 3OC12-HSL could be examined. However, the true role of PON in airway host defense and regulation of quorum sensing remains to be determined. PON2 may not be required, but redundant, with PON1 and PON3 each contributing to lactonolysis of 3OC12-HSL. Alternatively, PON2 may be required but only for intracellular signaling events or against microbes that become intracellularly located (9). Finally, PON2 may be required, but only when extracellular concentrations of 3OC12HSL are markedly increased. AHL molecules are thought to be freely diffusible across cell membranes, although to varying degrees based upon the carbon side chain length. Very few studies have actually examined transport and kinetics of 3OC12-HSL movement across cell membranes. Using a chimeric sensor protein based on LasR, 3OC12-HSL was found to enter COS-1 cells and activate these reporter constructs (35). A study by Pearson et al. (24) reported that while C4-HSL reached steady state cell concentrations in cell suspensions of P. aeruginosa by 30 seconds, 3OC12-HSL required approximately 5 minutes to reach steady state levels. While these experiments were conducted using bacterial cells, it does suggest that 3OC12-HSL is delayed in crossing cell membranes.

Page 24 of 43

23 Airway epithelial cell paraoxonases may not only function as a component of the innate immune system directly acting at the bacterial level, but these enzymes also may be important for additional control of the inflammatory response by airway epithelial cells responding to these signals. Quorum-sensing molecules have been shown to affect many cells, including fibroblasts and epithelial cells. 3OC12-HSL induces an array of inflammatory molecules including: prostaglandin E2, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-alpha (30, 31). Recently, Kravchenko et al. reported that 3OC12-HSL treatment of epithelial cells and macrophages leads to phosphorylation of p38 and eukaryotic translation initiation factor 2 (eIF2a) (16), suggesting a possible pathway for 3OC12-HSL’s effects on the epithelial cell’s inflammatory response. P may regulate these responses through 3OC12-HSL inactivation and inhibit uncontrolled signaling of the inflammatory cascade.

If PON is a naturally occurring inhibitor of quorum sensing, then modulation of its activity or overexpression may represent a potential therapeutic target. P. aeruginosa infection of the lung is common and occurs in the acute setting, such as in nosocomial pneumonia and burn wound infections, as well as in chronic infections such as in cystic fibrosis patients. These infections are oftentimes difficult to treat and numerous therapeutic options, besides antibiotics, have been proposed. Quorum-sensing inhibitors have recently been investigated as a potential therapeutic agents with promising results. Experimental data regarding PON’s role in the response to an infection is very limited. Treatment of mice with a synthetic analogue of furanone, a naturally occurring quorumsensing inhibitor, improved bacterial clearance and mortality in a murine model of P.

Page 25 of 43

24 aeruginosa pulmonary infection (36). Similarly, mice treated with garlic extract, another recently described quorum-sensing inhibitor, enhanced bacterial clearance in a pneumonia model (3)

In summary, we have shown that murine tracheal epithelial cells express PON1, PON2, and PON3. These cells can degrade 3OC12-HSL, but in the absence of PON2 this activity is impaired. Since PON2 inactivates 3OC12-HSL to the greatest extent and is the most abundant PON in human airway epithelia, it is likely the primary enzyme responsible for quorum-sensing molecule inactivation. The PONs may play a key role in host defense to pathogens such as P. aeruginosa, but additional studies will be needed to further address this question.

Page 26 of 43

25

ACKNOWLEDGEMENTS We thank Lisa Jorgensen, Phil Karp, Thomas Moninger, Thomas Recker, Geri Traver, Peter Taft, Xu-Ping Wang, Yu-Rong Xia, and Yi-Shou Shi for excellent assistance and Michael Welsh, M.D. and Pete Greenberg, Ph.D. for insightful discussions. We appreciate the support of the Central Microscopy Research Facility, the Gene Transfer Vector Core, and the In Vitro Cell Models Core. These studies were supported in part by NHLBI and NIH grants (RO1 HL71776 to STR, PO1 HL30568 to AJL and DMS, and HL61234-08 to JZ).

Page 27 of 43

26

REFERENCES 1.

Aviram M, Rosenblat M, Billecke S, Erogul J, Sorenson R, Bisgaier CL, Newton RS, and La Du B. Human serum paraoxonase (PON 1) is inactivated by oxidized low density lipoprotein and preserved by antioxidants. Free Radic Biol Med 26: 892-904, 1999.

2.

Bals R and Hiemstra PS. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur Respir J 23: 327-333, 2004.

3.

Bjarnsholt T, Jensen PO, Rasmussen TB, Christophersen L, Calum H, Hentzer M, Hougen HP, Rygaard J, Moser C, Eberl L, Hoiby N, and Givskov M. Garlic blocks quorum sensing and promotes rapid clearing of pulmonary Pseudomonas aeruginosa infections. Microbiology 151: 3873-3880, 2005.

4.

Chun CK, Ozer EA, Welsh MJ, Zabner J, and Greenberg EP. Inactivation of a Pseudomonas aeruginosa quorum-sensing signal by human airway epithelia. Proc Natl Acad Sci U S A 101: 3587-3590, 2004.

5.

Deakin S, Leviev I, Gomaraschi M, Calabresi L, Franceschini G, and James RW. Enzymatically active paraoxonase-1 is located at the external membrane of producing cells and released by a high affinity, saturable, desorption mechanism. J Biol Chem 277: 4301-4308, 2002.

6.

Draganov DI and La Du BN. Pharmacogenetics of paraoxonase: a brief review. Naunyn Schmiedebergs Arch Pharmacol 369: 78-88, 2004.

7.

Draganov DI, Teiber JF, Speelman A, Osawa Y, Sunahara R, and La Du BN. Human paraoxonases (PON1, PON2, and PON3) are lactonases with overlapping and distinct substrate specificities. J Lipid Res 46: 1239-1247, 2005.

8.

Fasbender A, Lee JH, Walters RW, Moninger TO, Zabner J, and Welsh MJ. Incorporation of adenovirus in calcium phosphate precipitates enhances gene transfer to airway epithelia in vitro and in vivo. J Clin Invest 102: 184-193, 1998.

9.

Garcia-Medina R, Dunne WM, Singh PK, and Brody SL. Pseudomonas aeruginosa acquires biofilm-like properties within airway epithelial cells. Infect Immun 73: 8298-8305, 2005.

10.

Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N, Kumar N, Schembri MA, Song Z, Kristoffersen P, Manefield M, Costerton JW, Molin S, Eberl L, Steinberg P, Kjelleberg S, Hoiby N, and Givskov M. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. Embo J 22: 38033815, 2003.

Page 28 of 43

27

11.

James RW and Deakin SP. The importance of high-density lipoproteins for paraoxonase-1 secretion, stability, and activity. Free Radic Biol Med 37: 19861994, 2004.

12.

Juhas M, Eberl L, and Tummler B. Quorum sensing: the power of cooperation in the world of Pseudomonas. Environ Microbiol 7: 459-471, 2005.

13.

Karp PH, Moninger T, Weber SP, Nesselhauf TS, Launspach J, Zabner J, and Welsh MJ. An in vitro model of differentiated human airway epithelia: methods and evaluation of primary cultures. In: Epithelial Cell Culture Protocols, edited by Wise C. Totowa, NJ: Humana Press, Inc., 2002, p. 115-137.

14.

Khersonsky O and Tawfik DS. Structure-reactivity studies of serum paraoxonase PON1 suggest that its native activity is lactonase. Biochemistry 44: 6371-6382, 2005.

15.

Kislinger T, Cox B, Kannan A, Chung C, Hu P, Ignatchenko A, Scott MS, Gramolini AO, Morris Q, Hallett MT, Rossant J, Hughes TR, Frey B, and Emili A. Global survey of organ and organelle protein expression in mouse: combined proteomic and transcriptomic profiling. Cell 125: 173-186, 2006.

16.

Kravchenko VV, Kaufmann GF, Mathison JC, Scott DA, Katz AZ, Wood MR, Brogan AB, Lehmann M, Mee JM, Iwata K, Pan Q, Fearns C, Knaus UG, Meijler MM, Janda KD, and Ulevitch RJ. N-(3-OXO-ACYL) homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways. J Biol Chem 281: 28822-28830, 2006.

17.

Kuo CL and La Du BN. Calcium binding by human and rabbit serum paraoxonases. Structural stability and enzymatic activity. Drug Metab Dispos 26: 653-660, 1998.

18.

Ng CJ, Bourquard N, Grijalva V, Hama S, Shih DM, Navab M, Fogelman AM, Lusis AJ, Young S, and Reddy ST. Paraoxonase-2 deficiency aggravates atherosclerosis in mice despite lower apolipoprotein-B containing lipoproteins anti-atherogenic role for paraoxonase-2. J Biol Chem 281: 29491-29500, 2006.

19.

Ng CJ, Wadleigh DJ, Gangopadhyay A, Hama S, Grijalva VR, Navab M, Fogelman AM, and Reddy ST. Paraoxonase-2 is a ubiquitously expressed protein with antioxidant properties and is capable of preventing cell-mediated oxidative modification of low density lipoprotein. J Biol Chem 276: 44444-44449, 2001.

Page 29 of 43

28 20.

Nickel JC, Ruseska I, Wright JB, and Costerton JW. Tobramycin resistance of Pseudomonas aeruginosa cells growing as a biofilm on urinary catheter material. Antimicrob Agents Chemother 27: 619-624, 1985.

21.

Ozer EA, Pezzulo A, Shih DM, Chun C, Furlong C, Lusis AJ, Greenberg EP, and Zabner J. Human and murine paraoxonase 1 are host modulators of Pseudomonas aeruginosa quorum-sensing. FEMS Microbiol Lett 253: 29-37, 2005.

22.

Parad RB, Gerard CJ, Zurakowski D, Nichols DP, and Pier GB. Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype. Infect Immun 67: 4744-4750, 1999.

23.

Pearson JP, Gray KM, Passador L, Tucker KD, Eberhard A, Iglewski BH, and Greenberg EP. Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes. Proc Natl Acad Sci U S A 91: 197-201, 1994.

24.

Pearson JP, Van Delden C, and Iglewski BH. Active efflux and diffusion are involved in transport of Pseudomonas aeruginosa cell-to-cell signals. J Bacteriol 181: 1203-1210, 1999.

25.

Reddy ST, Wadleigh DJ, Grijalva V, Ng C, Hama S, Gangopadhyay A, Shih DM, Lusis AJ, Navab M, and Fogelman AM. Human paraoxonase-3 is an HDLassociated enzyme with biological activity similar to paraoxonase-1 protein but is not regulated by oxidized lipids. Arterioscler Thromb Vasc Biol 21: 542-547, 2001.

26.

Reese J, Das SK, Paria BC, Lim H, Song H, Matsumoto H, Knudtson KL, DuBois RN, and Dey SK. Global gene expression analysis to identify molecular markers of uterine receptivity and embryo implantation. J Biol Chem 276: 4413744145, 2001.

27.

Schaedel C, de Monestrol I, Hjelte L, Johannesson M, Kornfalt R, Lindblad A, Stradvik B, Wahlgren L, and Holmberg L. Predictors of deterioration of lung function in cystic fibrosis. Pediatr Pulmonol 33: 483-491, 2002.

28.

Schuster M, Lostroh CP, Ogi T, and Greenberg EP. Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185: 2066-2079, 2003.

29.

Shih DM, Gu L, Xia YR, Navab M, Li WF, Hama S, Castellani LW, Furlong CE, Costa LG, Fogelman AM, and Lusis AJ. Mice lacking serum paraoxonase are susceptible to organophosphate toxicity and atherosclerosis. Nature 394: 284287, 1998.

Page 30 of 43

29

30.

Smith RS, Harris SG, Phipps R, and Iglewski B. The Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)homoserine lactone contributes to virulence and induces inflammation in vivo. J Bacteriol 184: 1132-1139, 2002.

31.

Smith RS, Kelly R, Iglewski BH, and Phipps RP. The Pseudomonas autoinducer N-(3-oxododecanoyl) homoserine lactone induces cyclooxygenase-2 and prostaglandin E2 production in human lung fibroblasts: implications for inflammation. J Immunol 169: 2636-2642, 2002.

32.

Teiber JF, Draganov DI, and La Du BN. Lactonase and lactonizing activities of human serum paraoxonase (PON1) and rabbit serum PON3. Biochem Pharmacol 66: 887-896, 2003.

33.

Wagner VE, Bushnell D, Passador L, Brooks AI, and Iglewski BH. Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185: 2080-2095, 2003.

34.

Welsh MJ, Zabner J, Graham SM, Smith AE, Moscicki R, and Wadsworth SC. Adenovirus-mediated gene transfer for cystic fibrosis: Part A. Safety of dose and repeat administration in the nasal epithelium. Part B. Clinical efficacy in the maxillary sinus. Hum Gene Ther 6: 205-218, 1995.

35.

Williams SC, Patterson EK, Carty NL, Griswold JA, Hamood AN, and Rumbaugh KP. Pseudomonas aeruginosa autoinducer enters and functions in mammalian cells. J Bacteriol 186: 2281-2287, 2004.

36.

Wu H, Song Z, Hentzer M, Andersen JB, Molin S, Givskov M, and Hoiby N. Synthetic furanones inhibit quorum-sensing and enhance bacterial clearance in Pseudomonas aeruginosa lung infection in mice. J Antimicrob Chemother 53: 10541061, 2004.

37.

Yang F, Wang LH, Wang J, Dong YH, Hu JY, and Zhang LH. Quorum quenching enzyme activity is widely conserved in the sera of mammalian species. FEBS Lett 579: 3713-3717, 2005.

38.

You Y, Richer EJ, Huang T, and Brody SL. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am J Physiol Lung Cell Mol Physiol 283: L1315-1321, 2002.

39.

Zabner J, Scheetz TE, Almabrazi HG, Casavant TL, Huang J, Keshavjee S, and McCray PB, Jr. CFTR DeltaF508 mutation has minimal effect on the gene expression profile of differentiated human airway epithelia. Am J Physiol Lung Cell Mol Physiol 289: L545-553, 2005.

Page 31 of 43

30

40.

Zabner J, Wadsworth SC, Smith AE, and Welsh MJ. Adenovirus-mediated generation of cAMP-stimulated Cl- transport in cystic fibrosis airway epithelia in vitro: effect of promoter and administration method. Gene Ther 3: 458-465, 1996.

Page 32 of 43

31

FIGURE LEGENDS FIGURE 1: Imaging of murine tracheal epithelia. (A) Hematoxylin and eosin stained section of well-differentiated murine tracheal epithelial cells on transwell membranes (0.33 cm2) (bar = 10 µm); (B) Transmission electron microscopy of well-differentiated murine tracheal epithelial cells on transwell membranes (0.33 cm2) (bar = 2 µm).

FIGURE 2: Expression of PON1, 2, and 3 in murine tracheal and human airway epithelial cells. RT-PCR using primers specific for murine PON 1, 2, and 3 was performed on tracheal epithelial cell lysates. Microarray analysis, for PON1, 2, and 3 expression, was performed on RNA samples extracted from human airway epithelia samples. (A) Murine tracheal epithelia (B) human airway epithelia. Results are expressed as mRNA copies/1000 copies GAPDH and signal intensity, respectively.

FIGURE 3: Spectrum of inactivation of AHLs by PON1, PON2, and PON3. CHO cells were transfected with an adenovirus expressing (A) hPON1, (B) hPON2, or (C) hPON3. Unhydrolyzed AHLs were measured by HPLC following a 60 minute exposure. A representative experiment is shown. Three independent experiments yielded qualitatively similar results. Insets show Western blot analysis for PON1, PON2, or PON3 following transduction of CHO cells.

Page 33 of 43

32

FIGURE 4: Paraoxonase mRNA levels in murine tracheal epithelial cells following targeted deletion of PON1, PON2, or PON3. Total RNA was isolated from murine tracheal epithelial cells and subjected to RT-PCR using primers specific for murine paraoxonase 1, 2, and 3. Results are expressed as mRNA copies/1000 copies GAPDH.

FIGURE 5: Impaired 3OC12-HSL degradation by murine tracheal epithelial cell lysates from PON2 knockout mice. Following lysis of tracheal epithelial cell cultures from PON1, PON2, and PON3 knockout mice, lysates (10% volume) were incubated with 10 µM 3OC12-HSL. Following a 15, 30, or 60 minute incubation, lysate samples were obtained for measurement of inactivation of 3OC12-HSL. Data is expressed as the percentage of active 3OC12-HSL remaining compared to initial levels. Data is mean ± SEM. n=2-3 experiments/group performed in quadruplicates.

p < 0.05.

Page 34 of 43

33

FIGURE 6: Preserved 3OC12-HSL degradation by intact murine tracheal epithelial cell cultures from PON1, PON2, and PON3 knockout mice. Well-differentiated murine tracheal epithelial cell cultures were incubated in the presence of 3OC12-HSL (final concentration of 10 µM apically and basolaterally). Following a 15, 30, or 60 minute incubation, samples of apical media were obtained for measurement of inactivation of 3OC12-HSL. Data is expressed as the percentage of active 30C12-HSL remaining compared to initial levels. Data is mean ± SEM. n=2-3 experiments/group performed in quadruplicates.

FIGURE 7: Overexpression of PON2 in intact human airway epithelia cultures enhances 3OC12-HSL inactivation in lysate preparations, but not intact epithelia. Human airway epithelia cultures were transfected with an adenovirus expressing PON2 or GFP. 48 hours later, 3OC12-HSL was added to lysate samples (2% volume) or intact epithelia. 30 minutes later 3OC12-HSL inactivation was measured. Data is expressed as the percentage of active 3OC12-HSL remaining compared to initial levels. Data is mean ± SEM. Experiments were performed in quadruplicates.

Page 35 of 43

34 FIGURE 8: Loss of PON2 enhances P. aeruginosa quorum sensing. (A) PAO1-qsc-102-lacZ was incubated in the presence of wild type or PON1 deficient serum. 6 hours later bacterial counts and -gal levels were measured. (B) a 2 µl sample of PAO1-qsc-102 lacZ (105 cfu) was applied to the apical surface of wild type or PON2 deficient murine tracheal epithelia cultures. At 4 and 6 hours later bacterial counts and -gal levels were determined. Data is mean ± SEM. n=2-3 per group performed in triplicates. * p < 0.05.

Page 36 of 43

Page 37 of 43

mRNA copies/ 1000 copies GAPDH

FIGURE 2A 180 120 60 0

PON1

PON2

PON3

PON2

PON3

FIGURE 2B

Signal Intensity

5000 4000 3000 2000 1000 0

PON1

Page 38 of 43

AdGFP

100

AdPON1

150

PON1

50 0

3OC12 C12

C10

C8

C6

C4

C6

C4

C6

C4

AdGFP

100

AdPON2

150

PON2

50 0

3OC12 C12

C10

C8

100

AdGFP

150 AdPON3

Unhydrolyzed AHL (%)

Unhydrolyzed AHL (%)

Unhydrolyzed AHL (%)

FIGURE 3

PON3

50 0

3OC12 C12

C10

C8

Page 39 of 43

mRNA copies/ 1000 copies GAPDH

180

mRNA copies/ 1000 copies GAPDH

180

mRNA copies/ 1000 copies GAPDH

FIGURE 4

180

PON1KO PON1-KO

120 60 0 PON1

PON2

PON3

PON2KO PON2-KO

120 60 0 PON1

PON2

PON3

PON3KO PON3-KO

120 60 0 PON1

PON2

PON3

Page 40 of 43

3OC12-HSL Activity (%)

FIGURE 5

100 75 WT-PON1

50 25

PON1-KO

0

3OC12-HSL Activity (%)

0 100

*

*

75

40

60

PON2-KO

*

40

60

50 WT-PON2

25 0 0

3OC12HSL

20

20

100 75

WT-PON3

50 PON3-KO

25 0 0

20 40 Time (m)

60

Page 41 of 43

3OC12-HSL Activity (%)

FIGURE 6

100 75 50

WT-PON1

25 PON1-KO

0

3OC12HSL

0

20

40

60

100 75 PON2-KO

50 25

WT-PON2

0

3OC12HSL

0

20

40

60

100 75 PON3-KO

50 25

WT-PON3

0 0

20 40 Time (m)

60

Page 42 of 43

FIGURE 7 3OC12-HSL Activity (%)

100 80

*

AdGFP AdPON2

60 40 20 0 Lysate

Intact Epithelium

Page 43 of 43

FIG 8A

4

*

SERUM

3

PON1-KO

2 1

WT

0 0

2 4 Time(hrs) (h) TIME

6

FIG 8B 16

4

log10 CFU/well

12 8

*

EPITHELIUM

8

PON2-KO

6 4 2 0

WT KO

WT

0 0

2 4 Time(hrs) (h) TIME

6