paraoxonase-2 deficiency enhances pseudomonas aeruginosa quorum sensing in murine tracheal epithelia

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

David A. Stoltz1*, Egon A. Ozer1*

, Carey J. Ng2,3, Janet M. Yu2, Srinivasa T. Reddy2,3, Aldons J. Lusis2, Noam Bourquard3, Matthew R. Parsek4, Joseph Zabner1#, Diana M. Shih2

* These authors contributed equally to this work.

1Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine,

University of Iowa, Iowa City, IA 52242, USA2Department of Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA

90095, USA3Department of Molecular and Medical Pharmacology, David Geffen School of Medicine

at UCLA, Los Angeles, CA 90095, USA4Department of Microbiology, University of Washington, Seattle, 98195, USA

# To whom correspondence should be addressed at:

Department of Internal MedicineRoy J. and Lucille A. Carver College of MedicineUniversity of Iowa 440 EMRBIowa City, IA 52242, USATel: 319-335-7608Fax: 319-335-7623E-mail: [email protected]

Running Title:Paraoxonase and Murine Airway Epithelia

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

Copyright © 2006 by the American Physiological Society.

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

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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).

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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 (3OC12-

HSL) 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

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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.

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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, semi-

permeable 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

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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

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(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).

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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

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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.

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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 β-

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

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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.

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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

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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

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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.

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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

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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 3OC12-

HSL degradation only in lysate preparations.

In order to test if diffusion of 3OC12-HSL across the cell membrane was the rate-

limiting 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 quorum-

sensing reporting by P. aeruginosa.

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We next wanted to test the hypothesis that increased concentrations of 3OC12-

HSL 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

β-galactosidase (β-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, β-gal expression increased in serum from both wild type and

PON1 knockout mice. However, PON1 knockout mouse serum had significantly higher

β-gal expression, compared to control serum. This shows that in the absence of PON1,

P. aeruginosa has enhanced quorum sensing as measured by increased β-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 β-gal measurements (quorum-sensing activity) and bacterial

quantification. We hypothesized that PON2 knockout epithelia would show increased β-

gal levels compared to controls. At 4 and 6 hours following addition of the bacteria, β-

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 β-gal expression were

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

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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 3OC12-

HSL 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).

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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 β-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

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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

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http://tap.med.utoronto.ca/~mts/

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 3OC12-

HSL 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.

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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 2α (eIF2a) (16), suggesting a possible

pathway for 3OC12-HSL’s effects on the epithelial cell’s inflammatory response. PΟΝ

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 quorum-

sensing inhibitor, improved bacterial clearance and mortality in a murine model of P.

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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.

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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).

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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.

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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.

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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.

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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 β-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 β-gal levels were

determined. Data is mean ± SEM. n=2-3 per group performed in triplicates. * p < 0.05.

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FIGURE 2A

0

60

120

180

PON1 PON2 PON3

mR

NA

copi

es/

1000

copi

esG

AP

DH

010002000300040005000

PON1 PON2 PON3

Sig

nalI

nten

sity

FIGURE 2B

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0

50

100

150U

nhyd

roly

zed

AH

L(%

)

0

50

100

150

Unh

ydro

lyze

dA

HL

(%)

0

50

100

150

Unh

ydro

lyze

dA

HL

(%)

FIGURE 3

PON1

PON2

PON3

3OC12 C12 C10 C6C8 C4

3OC12 C12 C10 C6C8 C4

3OC12 C12 C10 C6C8 C4

AdPO

N1

AdG

FP

AdPO

N2

AdG

FP

AdPO

N3

AdG

FP

of 43

0

60

120

180

PON1 PON2 PON3

mR

NA

copi

es/

1000

copi

esG

AP

DH

PON2KO

0

60

120

180

PON1 PON2 PON3

mR

NA

copi

es/

1000

copi

esG

AP

DH

PON3KO

FIGURE 4

0

60

120

180

PON1 PON2 PON3

mR

NA

copi

es/

1000

copi

esG

AP

DH

PON1KOPON1-KO

PON3-KO

PON2-KO

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0255075

100

0 20 40 60

3OC

12-H

SL

Act

ivity

(%)

0255075

100

0 20 40 60

3OC

12-H

SL

Act

ivity

(%)

FIGURE 5

WT-PON1

PON1-KO

WT-PON2

PON2-KO

WT-PON3

PON3-KO

* * *

0255075

100

0 20 40 60

3OC

12-

HS

L

Time (m)

of 43

0255075

100

0 20 40 60

3OC

12-

HS

L

0255075

100

0 20 40 60

3OC

12-

HS

L

0255075

100

0 20 40 60

3OC

12-H

SL

Act

ivity

(%)

FIGURE 6

Time (m)

WT-PON1

PON1-KO

WT-PON2

PON2-KO

WT-PON3

PON3-KO

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0

20

40

60

80

100

3OC

12-H

SL

Act

ivity

(%) AdGFP

AdPON2

FIGURE 7

IntactEpithelium

Lysate

*

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0

4

8

12

16

0 2 4 6TIME (hrs)

0

1

2

3

4

0 2 4 6TIME (hrs)

FIG 8A

02468

log1

0C

FU/w

ell

PON1-KO

WT

PON2-KO

WT

FIG 8B

WT KO

SERUM

EPITHELIUM

Time (h)

Time (h)

*

*

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paraoxonase-2 deficiency enhances pseudomonas aeruginosa quorum sensing in murine tracheal epithelia

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