premature cellular senescence induced by pyocyanin, a redox-active pseudomonas aeruginosa toxin

Free Radical Biology & Medicine 41 (2006) 1670–1677www.elsevier.com/locate/freeradbiomed

Original Contribution

Premature cellular senescence induced by pyocyanin, a redox-activePseudomonas aeruginosa toxin

Michael Muller

Centre for Education and Research on Ageing, ANZAC Research Institute, University of Sydney, Concord RG Hospital, Sydney NSW 2139, Australia

Received 10 May 2006; revised 3 August 2006; accepted 1 September 2006Available online 8 September 2006

Abstract

Pseudomonas aeruginosa is an important nosocomial pathogen that can cause acute and chronic infection, particularly of the respiratorysystem. Pyocyanin is a major P. aeruginosa virulence factor that displays redox activity and induces oxidative stress in cellular systems. The effectof pyocyanin on replicating human pulmonary epithelial (A549) cells was investigated. Cells were exposed to pyocyanin for 24 h and theirsubsequent growth and development were followed for 7 days. Pyocyanin (5–10 μM) arrested cell growth and resulted in the development of amorphological phenotype consistent with cellular senescence, that is, an enlarged and flattened appearance. The senescent nature of these cells wassupported by positive staining for increased lysosomal content and senescence-associated β-galactosidase activity. All cells treated with pyocyanin(10 μM) converted to the senescent phenotype, which remained stable for up to 7 days. Exposure to pyocyanin at 25 μM or greater resulted in celldeath due to apoptosis. A549 cells exposed to pyocyanin generated hydrogen peroxide in a dose-dependent manner and the senescence-inducingeffect of pyocyanin was inhibited by the antioxidant, glutathione, suggesting the involvement of reactive oxygen species. The induction ofpremature cellular senescence by redox-active bacterial toxins may be a hitherto unrecognized aspect of infection pathology and a limiting factorin the tissue repair response to infection.© 2006 Elsevier Inc. All rights reserved.

Keywords: Premature cellular senescence; Pyocyanin; Cell replication; Tissue repair; Glutathione; Pseudomonas aeruginosa

Introduction

Pseudomonas aeruginosa is an important cause of acutenosocomial infection in immunocompromised patients, partic-ularly for those on mechanical ventilation. The organism is alsoa major cause of chronic infection for those with cystic fibrosisor bronchiectasis. The deterioration in lung function associatedwith chronic infection is strongly correlated with invasion andcolonization by P. aeruginosa [1]. Patients experience progres-sive loss of pulmonary function, which is secondary todestruction of lung tissues, particularly the epithelial lining,caused by pseudomonal colonization [2] and the host response

Abbreviations: CPD, cumulative population doublings; FBS, fetal bovineserum; PBS, phosphate-buffered saline; SA-β-gal, senescence-associated β-galactosidase.

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to chronic infection [3]. Consequently, epithelial cell replicationis a critical response in the process of tissue regeneration andthe restoration of lung function. Factors that inhibit cellreplication within this setting have the potential to contributeto lung injury.

Of the many virulence factors produced by P. aeruginosa,pyocyanin, a redox-active phenazine toxin, is emerging as aintegral component in the pathology of pseudomonal lungdisease and its presence is considered crucial to successfulinfection in mice [4,5]. The concentration of pyocyanin in thesol phase of sputa from infected patients has been reported to bein the range of 0.2 to 27.3 μg/ml (1 to 130 μM) with a medianvalue 1.7 μg/ml (8.1 μM) [6]. Due to its low molecular weight(210 Da) it is readily diffusible and permeates cell membranes.On entry to the cell it undergoes reduction by NAD(P)H and cansubsequently reduce molecular oxygen to superoxide [7], whichin turn dismutates to hydrogen peroxide. By this means,pyocyanin has been shown to induce oxidative stress inendothelial [8] and epithelial cells [9].

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1671M. Muller / Free Radical Biology & Medicine 41 (2006) 1670–1677

Pyocyanin modulates several cell functions includingreducing mucocillary clearance [10], neutrophil bactericidalfunction [11], airway IL-8 production [12], and eicosanoidproduction [13] and subsequent metabolism [14]. In additionto modulating cellular functions, studies have shown pyocya-nin to be inhibitory to the growth of bacterial [15], fungal [16],and mammalian cells [17]. The effect on bacterial growth wasfound to be due to the production of bactericidal reactiveoxygen species [18]. In contrast, inhibition of lymphocyteproliferation by pyocyanin [19] was due to down regulation ofIL-2 production and decreased expression of IL-2 receptors[20]. The growth of the human type II epithelial-like cell line,A549, was reported to be impaired by pyocyanin and the effectcould be partially inhibited by manganoporphyrin, a superox-ide scavenger, leading to the conclusion that pyocyanin-induced reactive oxygen species were responsible for growthinhibition [21].

However, studies have shown that exposure of mammaliancells to oxidants can produce diverse results with respect to cellgrowth that are dependent on oxidant concentration. Thus, lowlevels of reactive oxygen species can be mitogenic rather thangrowth inhibitory [22,23] while multiple sublethal exposures tooxidants such as hydrogen peroxide or tert-butyl hydroperoxidecan induce cells into a state of premature cellular senescence[24,25]. The senescent phenotype is characterized by an alteredmorphology that includes an enlarged and flattened cellularappearance, increased lysosomal content, and an inability torespond to mitogenic stimuli [26]. Exposure to higherconcentrations of oxidant species can result in cell death dueto apoptosis or necrosis.

As pyocyanin is considered to be a critical component in theestablishment of pseudomonal lung disease [4] and the toxin isknown to induce oxidative stress in mammalian cells [8,9] thisstudy was conducted to clarify the nature of the effect ofpyocyanin on lung epithelial cell growth. Here, evidence ispresented that replicating human type II epithelial-like cellsexposed to low concentrations of pyocyanin are induced into astate of persistent premature cellular senescence, while at higherconcentrations apoptosis predominates. It is further demon-strated that supplemental GSH is able to prevent pyocyanin-induced senescence. The implications for tissue regeneration inresponse to P. aeruginosa infection are discussed.

Materials and methods

All reagents were purchased from Sigma (Sydney, Australia)unless otherwise stated.

Cell culture

Human type II epithelial-like (A549) cells were obtainedfrom the ATCC and used from passages 42 to 61. Cells weregrown and maintained in complete RPMI 1640 (RPMI;Invitrogen, Sydney) containing 10% fetal bovine serum (FBS,Invitrogen Sydney), glutamine (2 mM), 100 U/ml penicillin,100 μg/ml streptomycin, and 2.5 μg/ml amphotericin B. Allexperiments were conducted in complete RPMI.

Preparation of pyocyanin

Pyocyanin was prepared by the photolysis of phenazinemethosulfate according to the method of Knight et al. [27] andpurified by thin-layer chromatography as described [14]. Thepurified compound was stored at −70°C in methanol andprotected from light at all times. Prior to use, the methanol wascompletely removed in a stream of nitrogen gas to leave thedried compound. When dry, the pyocyanin was reconstituted intissue culture medium and used immediately.

Treatment of A549 cells with pyocyanin

To determine the effect of pyocyanin on A549 cells, thefollowed plating procedure was followed for all assays exceptwhere noted. Cells were plated at 1 × 104 cell/well into 12-welltissue culture plates. At this low seeding density cells areisolated from one another and experience no or little contactinhibition during initial growth. After incubation for 24 h toallow for cell adherence, the medium was removed and freshcomplete medium containing the appropriate concentration ofpyocyanin was added. The cells were incubated for a further24 h in the presence of pyocyanin, the medium was removed,and cells were washed twice with fresh medium and finallyreplaced with pyocyanin-free complete RPMI. Changes of freshpyocyanin-free medium were made every 3 days thereafter.

Determination of cell proliferation

On Days 1, 3, and 7 postpyocyanin exposure the cells wereharvested by trypsinization, diluted in trypan blue and countedusing a hemocytometer. Cell population doublings weredetermined using the following formula:

PD ¼ log½Nt=N0�=log 2

where N0 and Nt are the number of seeded and harvested cells,respectively. Cumulative population doublings (CPD) weredetermined using the formula:

CPD ¼ APD

Cellular morphology

Cells were examined for morphological changes at 1, 3, and7 days postpyocyanin exposure by phase-contrast microscopyusing a Zeiss Axiovert 200 microscope. High-power fields werecaptured using a Zeiss Axiocam MRc digital camera and imageanalysis was performed with ImageJ software (NationalInstitute on Aging).

Detection of apoptosis

The presence of apoptotic cells was assessed by determiningcaspase activation using a Caspase-Glo 3/7 luminescence assaykit (Promega, Sydney). Cells were plated at 1 × 104 cell/ml(100 μl/well) into clear bottom, opaque wall 96-well tissue

Fig. 1. Effect of pyocyanin on A549 cell growth. Cells were plated at 1 × 104

cell/well and incubated for 24 h followed by a single 24-h exposure to pyocyanin(0–10 μM). Cell numbers were quantified on Days 0, 1, 3, and 7 (A) andcumulative population doublings (CPD) determined (B). The results representthe mean ± standard deviation (*P < 0.01; n = 9).

1672 M. Muller / Free Radical Biology & Medicine 41 (2006) 1670–1677

culture plates and incubated for 24 h. The medium was removedand the cells were then treated with pyocyanin for 24 h. Caspaseactivity was then assayed according to the instructionsaccompanying the kit. Plates were read with a Flurostar Optimaplate reader equipped with a luminescence probe (BMGLabtech, Australia).

Detection of cellular lysosomal content

An increase in senescence-associated lysosomal content wasdetected essentially as described by Kurtz et al. [28]. Onappropriate days, cells were washed three times with phosphate-buffered saline (PBS) followed by incubation for 10 min withacridine orange (20 μg/ml in Hanks' balanced salts solution) for3 min at 37°C. The cells were subsequently washed andexamined by fluorescence microscopy.

Senescence-associated β-galactosidase assay

The presence of senescence-associated β-galactosidase (SA-β-gal) activity was detected according to the method of Dimri etal. [29]. Briefly, cells were prepared as above and then washedtwice with PBS and fixed for 5 min in 2% formaldehyde/0.2%glutaraldehyde. The cells were washed once with PBS andincubated in a 37°C oven with freshly prepared SA-β-galstaining solution (final pH 6.0) containing citric acid (40 mM),sodium phosphate (40 mM), potassium ferrocyanide (5 mM),potassium ferricyanide (5 mM), sodium chloride (150 mM),magnesium chloride (2 mM), and 5-bromo-4-chloro-3-indolylβ-D-galactoside (X-Gal, 1 mg/ml). The cells were stained for16 h for full color development and subsequently examinedunder phase-contrast microscopy and the number of SA-β-gal-positive cells scored.

Determination of pyocyanin-generated hydrogen peroxide

Hydrogen peroxide production was determined by platingcells at 1 × 104 cell/ml into 96-well tissue culture plates (100 μl/well) for 24 h. The medium was removed and the cells werewashed twice with PBS. Hydrogen peroxide production wasthen assessed using the Amplex Red assay kit (MolecularProbes, USA) according to the manufactures' instructions. Allreagents were reconstituted in Hanks' balanced salts solution(without phenol red) containing pyocyanin (0 to 50 μM).Fluorescence was determined every 30 min using a FlurostarOptima plate reader (BMG Labtech, Australia).

Effect of the antioxidant GSH

A549 cells were plated at 1 × 104 cells/well into 12-welltissue culture plates. The cells were incubated for 24 h followedby 24 h in complete RPMI with or without pyocyanin (10 μM)and with or without GSH (0.1 to 10 mM). To compensate for theautoxidation of GSH in solution the half-life of GSH under theincubation conditions was determined and aliquots of additionalGSH were added to the culture medium at appropriate intervalsto maintain the designated concentration. The cells were harves-

ted after 24 h incubation by trypsinization, diluted in trypan blue,and counted using a hemocytometer.

Statistics

Data were analyzed by Student's t test or ANOVA withDunnett's or Tukey's posttest comparison using GraphPadPrism 2.01 (GraphPad Software Inc, San Diego, CA).

Results

Effect of pyocyanin on cell growth and viability

In the absence of pyocyanin, A549 cells exhibitedexponential growth (Fig. 1A) resulting in the cells becomingloosely confluent by Day 3 and tightly confluent by day 7.Treatment with pyocyanin at 1 μM did not affect cell growth atany time point. At 5 μM, pyocyanin inhibited replication (P <0.01) until Day 3 when cells began to proliferate. In contrast,pyocyanin at 10 μM resulted in complete growth arrest at alltime points. Determination of cumulative population doublingsover the 7 days revealed that control cells and cells exposed to1 μM pyocyanin underwent 13.7 ± 0.8 and 13.7 ± 1.0 CPD,respectively, while those exposed to 5 μM underwent 7.1 ± 2.0CPD. By Day 7 the CPD for treatment at 10 μM was 0.2 ± 1.8,indicating minimal replication (Fig. 1B). Exposure of A549cells to pyocyanin at 25 μM or greater for 24 h resulted in

Fig. 2. Pyocyanin activates the caspase cascade. A549 cells were treated for 24 hwith pyocyanin (1 to 50 μM) and caspase−3/−7 activities determined (RLU,relative light units). The results represent the mean ± standard deviation (*P <0.01; n = 4).

Fig. 4. Effect of pyocyanin on cell size. The mean areas of control cells (openbars) and cells treated with pyocyanin (10 μM; filled bars) were determined at 1,3, and 7 days. Areas are presented in square pixels and were obtained fromanalysis of three high-power fields per experiment (*P < 0.05; n = 9).


detachment of cells from their substrate, shrinkage of cells, andblebing of cell membranes, morphological characteristics ofapoptosis (data not shown). Determination of combinedcaspase-3 and -7 activities revealed a statistically significant(P < 0.01) increase, confirming activation of the apoptoticpathway (Fig. 2).

Treatment of A549 cells with pyocyanin results in a senescentmorphology

At all time points there was no discernable morphologicaldifference, as determined by phase-contrast microscopy,between control cells and cells treated with pyocyanin at1 μM. At 5 μM, pyocyanin caused an increase in cell size on

Fig. 3. Pyocyanin induces morphological changes in A549 cells. Control cells (Areplication and induces the formation of large cells (B,E). By Day 7 cells have prolif(C,F,I) and cells develop senescent morphology. Blue staining indicates SA-β-gexperiments. Scale bar = 10 μm.

Day 1 and by Day 3 a mixed population of normal size andenlarged cells developed (Fig. 3), although by Day 7 cell sizediminished to near normal dimensions. In contrast, pyocyaninat 10 μM caused all cells to exhibit an enlarged morphologythat persisted in a stable state for 7 days (Figs. 3 and 4). Inaddition to cellular enlargement, the nuclei of these cellsbecame altered, developing an enlarged and refractile appear-ance (Fig. 3).

Pyocyanin increases A549 lysosomal content

When examined by fluorescence microscopy followingstaining with acridine orange, control cells showed the

,D,F) grow and become confluent by Day 3. Pyocyanin (5 μM) inhibits cellerated and become confluent (H). Pyocyanin (10 μM) induces cell growth arrestalactosidase-positive cells. The images are representative of three separate

Fig. 5. Senescence-associated increase in cellular lysosomal content. Acridineorange was used to stain lysosomes in control (A) or pyocyanin (10 μM)-treatedcells (B) on Day 3. Green fluorescence is due to DNA and RNA labeling andyellow due to uptake of the fluorochrome by lysosomes (arrows). Eachexperiment was performed in triplicate; original magnification 100×.

Fig. 7. Hydrogen peroxide formation by A549 cells on exposure to pyocyanin.A549 cells were exposed to pyocyanin (0 to 50 μM) in complete growth mediumand hydrogen peroxide formation was monitored every 30 min. The resultsrepresent the mean of four individual experiments.


characteristic green fluorescence of DNA and RNA bindingonly. On treatment with pyocyanin (10 μM) yellow fluores-cence was observed, indicating lysosomal uptake of the dye(Fig. 5).

Fig. 6. Senescence-associated increase in β-galactosidase activity. Control andpyocyanin (10 μM)-treated cells were cultured for 1, 3, and 7 days and stainedfor SA-β-gal activity as described in the text. Five high-power fields wereexamined from each of three replicates per experiment and the number of totaland SA-β-gal-positive (blue staining) cells was determined. Results representthe mean ± standard deviation (n = 4).

Expression of SA-β-gal activity

Consistent with the development of an increased lysosomalcontent, exposure of A549 cells to pyocyanin resulted in theexpression of SA-β-gal activity (Fig. 6). Control cells expressedlow SA-β-gal activity, which did not exceed 5.5 ± 3.9% of totalcells counted (Fig. 6). Cells treated with pyocyanin (10 μM)resulted on Day 1 with 50.4 ± 14.4% of cells staining positivefor SA-β-gal activity. By Days 3 and 7 this had risen to greaterthan 95% of cells staining positive.

Generation of hydrogen peroxide by pyocyanin treatment

Exposure of A549 cells to pyocyanin resulted in theformation of H2O2 in a dose-dependent manner (Fig. 7).Basal production of H2O2 by untreated cells was determined tobe 1.6 pmol/h and this increased in the presence of pyocyanin toa maximum of 36.9 pmol/h at the highest concentration ofpyocyanin.

Fig. 8. GSH prevents pyocyanin-induced cellular senescence. Cells were platedat 1 × 104 cell/well and incubated for 24 h followed by a single 24-h exposure topyocyanin (10 μM) with or without the antioxidant, GSH (0.1 to 10 mM). Cellnumbers were quantified 24 h later. The results represent the mean ± standarddeviation (*P < 0.01; **P < 0.001; n = 6).


GSH prevents pyocyanin-induced cellular senescence

GSH is the most physiologically relevant antioxidant in theepithelial lining fluid of the lung therefore this antioxidant wasexamined for its ability to inhibit pyocyanin-induced senes-cence. The presence of GSH (0.1 to 10 mM) provided dose-dependent protection for A549 cells from the senescence-inducing effect of pyocyanin with a statistically significantresponse (P > 0.01) observed at 0.5 mM and above (Fig. 8).

Discussion

The present study has confirmed the previous observationthat pyocyanin causes the growth of A549 cells to be inhibited[21] and this observation has been extended to demonstrate thatthe mode of growth inhibition, at low concentrations, is by theinduction of cellular senescence, while at higher concentrationscell death due to apoptosis occurs. The senescent phenotypewas demonstrated by morphological criteria, that is, an enlargedand flattened appearance and the presence of increasedlysosomal content as determined by the lysosomotropicfluorochrome, acridine orange. Although there are fewbiochemical biomarkers for senescence, pyocyanin-inducedsenescent cells displayed enhanced expression of SA-β-galactivity, which is associated with an age-dependent increase inlysosomal mass [28] and is considered to be a marker ofsenescence [29]. Control cells exhibited only low level SA-β-gal activity and this was confined principally to confluentmonolayer populations, which is consistent with the findings ofothers [30]. A change in nuclear morphology was also observedfollowing pyocyanin treatment. Nuclei became enlarged andrefractile in appearance, a finding reported in other senescentcell populations [31].

Normal (nontumor) cells undergo a finite number of celldivisions before undergoing replicative senescence, when theybecome unable to further divide and unresponsive to mitogenicstimuli [26], a phenomenon referred to as the Hayflick limit.Replicative senescence is due to telomere erosion [32] and isconsidered to be irreversible. Recent studies have shown thatthe phenomenon of cellular senescence can be induced byoxidative stress due to frequent exposure of cells to sublethaldoses of hydrogen peroxide [33] or tert-butyl hydroperoxide[34], a condition generally referred to as stress-induced orpremature cellular senescence. The mechanism by whichpyocyanin induces premature cellular senescence appears tobe due to the chronic production of reactive oxygen species.Intracellular reduction of pyocyanin by NAD(P)H results in theproduction of superoxide and, by dismutation, hydrogenperoxide [8]. The ability of extracellular GSH to dosedependently inhibit the senescence-inducing effect of pyocya-nin supports this view.

Three separate mechanisms have been shown to promoteoxidant-induced cellular senescence. Persistent phosphorylationof p38MAPK following acute exposure to H2O2 was shown toresult in a senescent phenotype [35] while chronic exposure tooxidants has been demonstrated to induce telomere dysfunctionleading to senescence [36]. Alternatively, oxidative stress

resulting in nonspecific damage to DNA may impact on cellcycle progression leading to senescence [37]. In the case ofchronic exposure to pyocyanin it is likely that each of thesemechanisms are operative and further studies, includingterminal restriction analysis of telomere shortening rates, arein progress to clarify this point.

A vital host response to tissue injury is the ability ofremaining tissue to regenerate and restore normal physiologicalfunction to the injured site. Factors that inhibit cell replicationhave the potential to delay tissue repair and contribute tomorbidity and mortality. Epithelial cell replication, particularlythat of type II pneumocytes, is a critical step in the tissue repairprocess leading to restoration of lung function following injuryto the epithelial lining [38]. Hydrogen peroxide has beenreported to inhibit alveolar epithelial wound repair by theinduction of apoptosis in an in vitro wound repair assay [39].Data from the present study suggest that the presence ofpyocyanin at infection sites has the potential to compromise thetissue repair process by the induction of cellular senescence orapoptosis. Importantly, the senescence-inducing effect ofpyocyanin, in vitro, was inhibited, in a dose-dependent manner,by the presence of extracellular GSH. In vivo, the averageconcentration of GSH in the epithelial lining fluid of the lunghas been reported to be 257 μM (range 150 to 600 μM) innormal healthy subjects while the average concentration ofGSH for cystic fibrosis patients was found to be 78 μM (range<10 to 280 μM) [40]. The lower availability of GSH in thesepatients may compromise their ability to defend against thesenescence-inducing effect of pyocyanin. Recent clinical trialshave shown that GSH levels in epithelial lining fluid of patientscan be markedly increased (three- to fourfold) by the use ofaerosolized GSH with consequent improvement in lungfunction [41,42], a strategy that may provide some protectionagainst the effects of pyocyanin.

Although the present observations relate to human lungepithelial-like cells, they will also be relevant to other sites whereP. aeruginosa causes infections, in particular the cornea andskin. Pyocyanin has recently been implicated as an importantpseudomonal virulence factor for corneal infections [43] and thephenomenon of pyocyanin-induced cellular senescence may beparticularly relevant to burn patients infected with P. aeruginosa.Pyocyanin-induced cellular senescence may be a key factor inimpairing the regeneration of damaged skin tissue and thesuccessful establishment of tissue grafts. An early study onpyocyanin found that it inhibited the growth of epidermal cellsisolated from human skin explants [44] and preliminaryexperiments from this laboratory with other primary humancell isolates have shown the appearance of senescencecharacteristics on exposure to this toxin.

In conclusion, replicating human epithelial-like cells ex-posed to sublethal concentrations of pyocyanin underwentgrowth arrest and developed morphological and biochemicalcharacteristics consistent with that of cellular senescence. Whenexposed to 10 μM pyocyanin all cells developed a senescentphenotype that remained stable for several days, while at higherconcentrations pyocyanin caused replicating cells to becomeapoptotic. The ability of pyocyanin to induce senescence could


be completely inhibited by the presence of adequate levels ofGSH. Pyocyanin-induced cellular senescence and apoptosismay play an important role in limiting the tissue repair responseat pseudomonal infection sites.

Acknowledgments

This work was supported by a project grant (NHMRC352342) from the National Health and Medical ResearchCouncil of Australia.

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