Modulation of Pseudomonas aeruginosa gene expression by host

Molecular Microbiology (2003)

50

(5) 1477ndash1491 doi101046j1365-2958200303803x

copy 2003 Blackwell Publishing Ltd

Blackwell Science LtdOxford UKMMIMolecular Microbiology1365-2958Blackwell Publishing Ltd 200350

514771491

Original Article

Modulation of P aeruginosa by oropharyngeal floraK Duan et al

Accepted 28 August 2003 For correspondence E-mailsuretteucalgaryca Tel (

+

1) 403 220 2744 Fax (

+

1) 403 270 2772

Modulation of

Pseudomonas aeruginosa

gene expression by host microflora through interspecies communication

Kangmin Duan

1

Carol Dammel

2

Jeffrey Stein

2

Harvey Rabin

1

and Michael G Surette

13

1

Department of Microbiology and Infectious Diseases University of Calgary Calgary AB Canada T2N 4N1

2

Quorex Pharmaceuticals Inc Carlsbad CA 92008-7326 USA

3

Department of Biochemistry and Molecular Biology University of Calgary Calgary AB Canada T2N 4N1

Summary

The change in gene expression patterns in responseto host environments is a prerequisite for bacterialinfection Bacterial diseases often occur as an out-come of the complex interactions between pathogensand the host The indigenous usually non-pathogenicmicroflora is a ubiquitous constituent of the host Inorder to understand the interactions between patho-gens and the resident microflora and how they affectthe gene expression patterns of the pathogens andcontribute to bacterial diseases the interactionsbetween pathogenic


andavirulent oropharyngeal flora (OF) strains isolatedfrom sputum samples of cystic fibrosis (CF) patientswere investigated Animal experiments using a ratlung infection model indicate that the presence of OFbacteria enhanced lung damage caused by

P aerugi-nosa

Genome-wide transcriptional analysis with a

lux

reporter-based promoter library demonstrated that

ordfordfordfordf

4 of genes in the genome responded to the pres-ence of OF strains using an

in vitro

system Charac-terization of a subset of the regulated genes indicatesthat they fall into seven functional classes and largeportions of the upregulated genes are genes impor-tant for

P aeruginosa

pathogenesis Autoinducer-2(AI-2)-mediated quorum sensing a proposed inter-species signalling system accounted for some butnot all of the gene regulation A substantial amountof AI-2 was detected directly in sputum samplesfrom CF patients and in cultures of most non-pseudomonad bacteria isolated from the sputa Tran-scriptional profiling of a set of defined

P aeruginosa

virulence factor promoters revealed that OF and exog-enous AI-2 could upregulate overlapping subsets ofthese genes These results suggest important contri-butions of the host microflora to

P aeruginosa

infec-tion by modulating gene expression via interspeciescommunications

Introduction

The occurrence of a bacterial infection relies on the patho-genrsquos ability to regulate its gene expression in responseto the host environment and is also directed by the hostresponse Bacterial diseases are often an outcome of thecomplex interactions between the pathogens and the hostIn humans bacterial pathogens not only interact with thehost but also inevitably with the indigenous generallyavirulent microflora that is often a constituent of the hostsThe interactions between pathogenic microbes and thehosts have been the focus of intensive studies and detailedmolecular mechanisms have been revealed The relation-ship between bacterial pathogenicity and the interactionsof a pathogen with the resident microflora however ismuch less understood with the exception of the protectiveroles of the resident flora against invasion of pathogens(Falk

et al

1998 Guarner and Malagelada 2003)


is a major opportunisticpathogen in humans capable of causing serious infec-tions In patients with cystic fibrosis (CF)

P aeruginosa

chronic infection ultimately causes pulmonary failureresulting in premature mortality (Stover

et al

2000) Thealtered ion transport associated with CF patients givesrise to a more viscous pulmonary mucous layer therebyimpairing ciliary clearance compromising the hostimmune response and permitting microbial colonization ofthe lungs (Bals

et al

1999) Chronic colonization andinfection by

P aeruginosa

occurs in

ordf

80 of CF patientsby 18 years of age (Rajan and Saiman 2002) andpatients commonly experience periodic infections by otherpathogens such as

Staphylococcus aureus

and

Haemo-philus influenzae

Burkholderia cepacia

has also emergedas an important pathogen frequently associated with highmortality in CF patients (Hart and Winstanley 2002 Soni

et al

2002) In addition to

P aeruginosa

B cepacia

andsecondary pathogens a variety of other microorganismsare also present in CF lungs (Coenye

et al

2002) includ-

1478

K Duan

et al


Molecular Microbiology

50

1477ndash1491

ing oropharyngeal flora (OF) bacteria such as viridansgroup streptococci and coagulase-negative staphylococciwhich only colonize the upper respiratory tracts in healthyadults To understand the pathogenesis of CF infection itis necessary to learn the molecular interactions and cellndashcell communications in this complex microbial communityin the lungs of CF patients

Quorum sensing is a cell density-dependent cellndashcellsignalling system that uses small extracellular moleculestermed autoinducers to co-ordinate gene expression in abacterial community In Gram-negative bacteria autoin-ducers are typically acyl-homoserine lactone (HSL)molecules and in Gram-positive bacteria typically oli-gopeptides (Miller and Bassler 2001)

P aeruginosa

pos-sesses two acyl-homoserine lactone quorum-sensingsystems

lasRlasI

(Passador

et al

1993) and

rhlRrhlI

(Brint and Ohman 1995 Latifi

et al

1995 Ochsner andReiser 1995) and one 2-heptyl-3-hydroxy-4-quinolone-based signalling system (denoted PQS for

Pseudomonas

quinolone signal) (Pesci

et al

1999) Quorum sensing in

P aeruginosa

affects a broad range of genes and is centralto its pathogenesis Up to 11 of genes in the genome arecontrolled by quorum sensing (Schuster

et al

2003 Wag-ner

et al

2003) Quorum sensing regulates a range ofimportant cellular and secreted virulence factors and playsimportant roles in the development of biofilms (Fuqua

et al

2001 Miller and Bassler 2001 Whitehead

et al

2001Smith and Iglewski 2003) Recent data suggest that

Paeruginosa

lives in biofilms in the lungs of CF patients(Singh

et al

2000) and biofilms may be primarily anaer-obic (Yoon

et al

2002)

P aeruginosa

autoinducers aredetectable in the lungs suggesting that quorum sensingcontrols

in vivo

pathogenicity (Singh

et al

2000 Erickson

et al

2002 Favre-Bonte

et al

2002 Middleton

et al

2002)

The LuxS-dependent quorum-sensing system is aunique system the signalling molecule of which termedautoinducer-2 (AI-2) results from spontaneous cyclizationof the LuxS product (45-dihydroxy-23-pentanedione)(Surette

et al

1999 Schauder

et al

2001) The

luxS

family of genes is spread widely among Gram-positiveand Gram-negative bacteria and AI-2 has been proposedto be an interspecies signalling molecule harbouring theinformation of both density and fitness of a bacterial com-munity (Bassler 1999 Schauder

et al

2001 Xavier andBassler 2003) Recent studies indicate that AI-2 is ableto regulate a range of genes and cellular processes

(DeLisa

et al

2001 Stevenson and Babb 2002) It hasbeen linked to virulence in a number of bacteria including

Neisseria meningitidis

(Winzer

et al

2002)

Streptococ-cus pyogenes

(Lyon

et al

2001)

Vibrio cholerae

(Miller

et al

2002 Zhu

et al

2002)

Clostridium perfringens

(Ohtani

et al

2002) and

Actinobacillus actinomycetem-comitans

(Fong

et al

2001) Evidence has shown that AI-2 is involved in mixed-species biofilm formation (McNab

et al

2003) and interspecies gene regulation (Fong

et al

2001) The

P aeruginosa

PAO1 genome however lacksan identifiable

luxS

gene homologue (Stover

et al

2000)and therefore does not produce AI-2

To understand the contribution of microbial interactionsto

P aeruginosa

pathogenesis and host diseases animalstudies have been carried out to analyse the effect of OFon

P aeruginosa

virulence The molecular interactionsbetween pathogenic

P aeruginosa

and avirulent strainsisolated from sputum samples of CF patients have beeninvestigated with an

in vitro

system

P aeruginosa

genesresponding to OF bacteria have been identified bygenome-wide gene expression profiling in the presenceof OF strains The role of AI-2-mediated quorum sensingin the bacterial interactions has also been assessed Evi-dence is presented that suggests important contributionsof host microflora to

P aeruginosa

infection via modulationof its gene expression

Results

Mixed microbial species in CF lungs shown by clinical microbiology data

Present in the lungs of CF patients is a complex microbialcommunity dominated by chronic infecting pathogensmost frequently

P aeruginosa

The constitution of thismicrobial community is influenced by the physiologicalstatus of the patients and antibiotic treatments Becausenon-pathogenic species are often neglected in clinicallaboratories (Shreve

et al

1999) many other speciesmay be present in the CF lungs This view is supportedby recent findings that uncommon bacterial species livein the lungs of CF patients (Coenye

et al

2002)The microbial dynamics in the lungs of CF patients over

an extended period is reflected in the microbiological datagathered from sputum samples of CF patients This isdemonstrated by the data from the Adult CF Clinic inFoothills Hospital Calgary Alberta Canada Figure 1A

Fig 1

Microbiological analysis of sputum samples from CF patientsA Longitudinal microbiology data from a single CF patient colonized primarily by

P aeruginosa

showing the transient infections by secondary pathogens and the persistent colonization of

Pseudomonas

and normal oropharyngeal flora Bacterial levels are in colony-forming units (cfu)ml

-

1

sputumB Concentration of

Pseudomonas

(solid bars) and normal oropharyngeal flora (open bars) from CF patients The average cfu ml

-

1

sputum was calculated for eight patients (with a minimum of 40 sputum samples per patient) collected over a 10-year span The error bars represent standard deviation in cfu

Modulation of

P aeruginosa

by oropharyngeal flora

1479



50

1477ndash1491

1480

K Duan

et al



50

1477ndash1491

shows the data collected on a single patient chronicallyinfected with

P aeruginosa

during the period from May1990 to April 2001 Representatives of normal respiratorytract flora or OF strains such as viridans group

Strepto-coccus

and

Staphylococcus

spp were consistentlypresent as a significant fraction of the microflora in thesputa Periodical colonization by

H influenzae

S aureus

and

Candida albicans

was also observed The patientdata presented in Fig 1A are typical of most patients inthis collection Similar patterns were obtained in the sputaof patients that were not infected with

P aeruginosa

ieOF strains were consistently present at significant con-centrations (data not shown) To generate a more com-prehensive illustration of prevalence of these mixedcommunities in CF lungs the average concentrations of

Pseudomonas

and normal oropharyngeal flora from cysticfibrosis patients with chronic

Pseudomonas

infections arepresented in Fig 1B The average cfu ml

-

1

sputum wascalculated for eight patients who had a minimum of 40sputum samples during this time The concentration of OFwas regularly equivalent to or higher than that of

Pseudomonas

In vivo

evaluation of the effect of OF bacteria on

P aeruginosa virulence

Association of P aeruginosa and other bacteria in thelungs of CF patients suggests that interactions occurbetween different bacteria To investigate the potentialcontributions of the associated bacteria to the pathoge-nicity of P aeruginosa the virulence of P aeruginosa inthe presence of an OF bacterium was tested in vivo usingthe agar bead rat lung infection model (Cash et al 1979)Three groups of eight rats were inoculated intratracheally

with P aeruginosa PAO1 alone PAO1 plus CF004 andCF004 alone CF004 is a Streptococcus strain isolatedfrom a CF sputum sample Seven days after infection therats were sacrificed and quantitative bacteriology andlung pathology were performed The percentage of con-solidation in the lung tissue was measured Consolidatedareas are tissues where accumulation of pulmonaryoedema fluid andor infiltration of inflammatory cells haveoccurred therefore more consolidation indicates moresevere lung damage As shown in Fig 2A significantenhancement of P aeruginosa virulence in the presenceof the OF strain was observed as indicated by increasedlung damage (P lt 00001 t-test) No significant change inP aeruginosa loads was observed in the co-infected groupcompared with the group infected with P aeruginosaalone (P = 04) (Fig 2B) Similarly OF loads remainedunchanged in the co-infection group and OF alone group(P = 09)

Screening of P aeruginosa genes modulated by OF bacteria

In order to understand the contribution of OF bacteria toP aeruginosa virulence and to investigate the molecularinteractions between P aeruginosa and OF bacteria anin vitro method was developed to screen for P aeruginosagenes that are modulated in the presence of OF bacteriaThe system uses a P aeruginosa random promoter libraryconstructed with the luxCDABE reporter carried on a low-copy-number plasmid pMS402 The activity of any individ-ual promoter is thus represented by the amount of lightgenerated by the clone containing the construct By mea-suring luminescence in a multilabel plate counter the Paeruginosa library can be screened temporally under dif-

B Fig 2 Quantitative pathology and bacteriology of the rat lungs 7 days after infection using the agar beads modelA The rat lungs co-infected with P aeruginosa and OF strain CF004 (group 2) showed signifi-cantly more consolidation than that infected with P aeruginosa alone (group 1) or OF alone (group 3) (P lt 00001 unpaired t-test) Consol-idation indicates accumulation of pulmonary oedema fluid andor infiltration of inflammatory cells More consolidation denotes more severe lung damageB P aeruginosa (solid bars) and CF004 (grey bars) bacterial loads (cfu) in the lungs of the three groups

Modulation of P aeruginosa by oropharyngeal flora 1481

copy 2003 Blackwell Publishing Ltd Molecular Microbiology 50 1477ndash1491

ferent conditions to identify differentially regulated promot-ers hence differentially regulated genes transcribed fromthese promoters

Using this method we screened 3456 P aeruginosa(ATCC27853) clones for differentially expressed promot-ers in the presence of two Gram-positive OF bacteriaStreptococcus strain CF004 and Staphylococcus strainCF018 Both strains were isolated from a single sputumsample from a CF patient The luminescence from eachP aeruginosa promoter clone in both a monoculture of Paeruginosa and a co-culture with CF004 or CF018 wasmeasured The luminescence data of the P aeruginosapromoter clones at the 75 h time point in the initial screenare shown in Fig 3A (CF004) and B (CF018) The initialscreening identified 280 promoters potentially regulatedby CF004 and 252 by CF018 Rescreening of thesepotential positives by temporally resolved gene expres-sion profiling combined with growth evaluation confirmed214 promoters affected by CF004 and 171 by CF018representing ordf6 and 5 of the P aeruginosa clones

respectively (ordf 4 of P aeruginosa operons assumingthat the library is a random library) Among these promot-ers 152 were common to both strains The regulated Paeruginosa promoters can be clustered into three classesthose regulated by both strains (class I) CF004 only(class II) or CF018 only (class III) (Fig 3C) The differentregulation of P aeruginosa promoters by these two strainssuggests that there are common as well as unique signalsor pathways in the interactions between P aeruginosa andthese Gram-positive bacteria Screening a subset of reg-ulated promoter clones against other OF isolates alsorevealed varied levels of gene regulation by different iso-lates (data not shown) This may point to a more promi-nent role for specific OF strains in affecting P aeruginosagene expression but may also simply be a reflection ofthe co-culture conditions

Characterization of the modulated P aeruginosa genes

A subset of the affected promoters was sequenced and

Fig 3 Scatter plot of the initial screen data at the 75 h time point P aeruginosa promoter clones were screened for differential expression in response to the presence of CF004 (A) or CF018 (B) Each point in the plots represents the activity of one individual promoter in the library The promoters unaffected by the co-culture conditions (blue) fall along the diagonal (ie their expression is the same in both the monoculture and co-culture) Points in red distributed above and below the diagonal indicate promoters with a minimum 25-fold downregulation and upregulation respectively in the presence of CF004 or CF018C Representatives of regulated promoters identified in initial screening are clustered by expression profiles Red to green colour gradient indicates the levels of expression from high to low data are normalized by the maximal value of each promoter Three classes of regulated promoters are marked those regulated by both strains (class I) CF004 only (class II) or CF018 only (class III)

1482 K Duan et al


compared with the annotated P aeruginosa PAO1genome (Stover et al 2000) and GenBank data (httpwwwncbinlmnihgov) to identify the genes regulatedTable 1 lists the 48 operons with known or putative genefunctions that are expressed from the regulated promot-ers These genes can be classified into seven groups by

function An additional 33 characterized promoters areassociated with genes encoding proteins with unknownfunction (data not shown) Seven promoters that arelocated at the 5cent end of the annotated genes but orientatedin the opposite direction were also identified and desig-nated orphan promoters (data not shown) These orphan

Table 1 P aeruginosa genes with known or putative functions expressed by OF-regulated promoters

GroupaGene orPA no Description

Maximum fold regulationb Reference

(1) Protein relevant to virulence

xcpP Secretion protein XcpP type II protein secretion apparatus

+39 Akrim et al (1993)

PA1882 Probable efflux transporter SugE family 67 similar to SugE protein (E coli ) shown to have cationic drug export function

+36 Stover et al (2000) Chung and Saier (2002)

cbpD Chitin-binding protein possibly involved in biofilm formation or pathogenicity

+30 Folders et al (2000)

PA4381 Probable two-component response regulator 91 similar to ColR (P fluorescens) which is crucial for colonization

+11 Stover et al (2000)

lasB Elastase one of the major virulence factors

+7 Bever and Iglewski (1988)

orf5c P aeruginosa pathogenicity island PAGI-1 gene a homologue of RpoN-dependent transcriptional activators similar to PrpR (E coli )

+5 Liang et al (2001)

wbpT c Putative a-D-alactosyltransferases involved in O-antigen biosynthesis of P aeruginosa serotype O6

+4 Belanger et al (1999)

ndk Nucleoside diphosphate kinase producing GTP from ATP involved in alginate synthesis via GDP-mannose production

+4 Sundin et al (1996)

phnBd Anthranilate synthase component II involved in phenazine biosynthesis

+2 Mavrodi et al (2001)

PA1282e Probable MFS transporter similar to multidrug efflux protein in Saureus and M smegmatis


phzM Phenazine-specific methyltransferase -29 Essar et al (1990) Mavrodi et al (2001)

(2) Transporter ormembraneprotein

PA4500 Probable binding protein component of ABC transporter


PA4037 Probable ATP-binding component of ABC transporter


PA3038 Probable porin +7 Stover et al (2000)PA2114 Probable MFS transporter -5 Stover et al (2000)lppL Lipopeptide LppL precursor -460 Jann et al (1990)

(3) Metabolism PA1551 Probable ferridoxin +19 Stover et al (2000)PA0182e Probable short-chain dehydrogenase +18 Stover et al (2000)PA5400 Probable electron transfer flavoprotein

alpha subunit+14 Stover et al (2000)

purD Phosphoribosylglycineamide synthetase purine metabolism


dnaE DNA polymerase III alpha chain +5 Stover et al (2000)lysS Lysyl-tRNA synthetase +5 Stover et al (2000)PA2953e Electron transfer flavoprotein-ubiquinone

oxidoreductase+5 Stover et al (2000)

PA1919 Probable radical-activating enzyme 62 similar to NrdG (Ralstonia eutropha)


argFd Ornithine carbamoyltransferase arginine biosynthesis


PA5127 Probable rRNA methylase +2 Stover et al (2000)proS Prolyl-tRNA synthetase +2 Stover et al (2000)thiG Probable thiamine biosynthesis protein

thiazole moiety+2 Stover et al (2000)

metG Methionyl-tRNA synthetase -152 Stover et al (2000)



promoters could potentially transcribe small open readingframes (ORFs) or small regulatory RNA molecules suchas antisense RNA thus having the potential of regulatinggene expression on the other strand In addition to thethree with putative functions included in Table 1 another14 promoters share no sequence homology with the PAO1genome neither do they share homology with any otherbacterial sequences in the GenBank They representunique sequences in this clinical P aeruginosa isolateThe identification of these unique clones probably reflectsthe plasticity of the Pseudomonas genome and suggeststhat a fraction of strain-specific genes may be associatedwith microbial interactions

Among the genes affected by OF are a relatively largenumber of well-characterized P aeruginosa virulence fac-tor genes or genes relevant to P aeruginosa pathogenicity(Table 1 the first group) lasB which encodes elastase amajor virulence factor that contributes to inflammatorydamage of the respiratory epithelia and interferes with

host immunological defences (Bever and Iglewski 1988)was upregulated to a maximum of sevenfold during a 24 htime course The most activated gene in this group wasxcpP which encodes a type II protein secretion apparatusresponsible for secreting and chaperoning a number ofvirulence factors including elastase and exotoxin A (Akrimet al 1993) Two probable multidrug efflux genes PA1282and PA1882 were also upregulated PA1282 encodes amajor facilitator superfamily (MFS) transporter proteinsimilar to multidrug efflux proteins QacA and QacB in Saureus and LfrA in Mycobacterium smegmatis (Stoveret al 2000) PA1882 encodes a small protein belongingto the SugE subfamily of the small multidrug resistancefamily (SMR) SugE has recently been shown to havecationic drug export function in Escherichia coli (Chungand Saier 2002) and the same function has been pro-posed for the SMR family Efflux pumps are not onlyimportant for bacterial antibiotic resistance but can alsocontribute to a pathogenrsquos invasiveness by assisting

(4) Iron uptakeor storage

PA0112 Membrane protein 49 similar to a region of putative haem O oxygenase in Aquifex aeolicus


PA4515 Conserved hypothetical protein iron uptake factor PiuC


hemE Uroporphyrinogen decarboxylase haem biosynthesis


(5) Transcriptionalregulators

lrp Global transcriptional regulator leucine-responsive regulatory protein


PA1136 Probable transcriptional regulator 42 similar to transcriptional activator LasR


PA2489 Probable transcriptional regulator +2 Stover et al (2000)PA2766e Probable transcriptional regulator TetR

family+2 Stover et al (2000)

PA3898 Probable AraC family transcriptional regulator

-9 Stover et al (2000)

PA2879 Probable transcriptional regulator LysR family




PA3604 Probable two-component response regulator LuxR family


PA1226 Probable transcriptional regulator TetR family


(6) Secretedprotein

PA3309 Conserved hypothetical protein with universal stress protein motif


pra Protein activator extracellular protein +8 Hardegger et al (1994)(7) Other functional merAc Mercuric ion reductase +2 Brown et al (1983)

protein rpsT e 30S ribosomal protein S20 -16 Stover et al (2000)rpsQe 30S ribosomal protein S17 -23 Stover et al (2000)pctA Chemotactic transducer -74 Kuroda et al (1995)PA2561 Probable chemotaxis transducer -86 Stover et al (2000)



Table 1 cont

a Although multiple genes are often controlled by one promoter only the first gene in each operon is listed Promoters not listed include 33promoters expressing genes with unknown functions seven orphan promoters and 14 promoters not found in PAO or other sequenced bacteriab Approximate maximum fold regulation in the presence of CF004 or CF018 at the 48 time points (+) upregulation (ndash) downregulationc Promoters not present in the PAO1 genomed Promoters represented more than once by non-identical clones or two identical clones (argF )e Promoters regulated by only one OF strain

1484 K Duan et al


export of virulence determinants (Hirakata et al 2002)CbpD encoding a chitin-binding protein has only beenfound in clinical isolates of P aeruginosa but not in soilpseudomonads It has been suggested that CbpD mayhave a role as an adhesin mediating P aeruginosa colo-nization of eukaryotic cells (Folders et al 2000) ndkencodes a nucleoside diphosphate kinase involved in alg-inate production (Sundin et al 1996) a process criticalfor P aeruginosa pathogenicity PA4381 encodes a prob-able two-component response regulator similar to ColR inPseudomonas fluorescens which is crucial for the bacte-riumrsquos ability to colonize plants although the mechanismby which ColR functions remains unclear (Dekkers et al1998) Two genes wbpT and orf5 are present in P aerug-inosa ATCC27853 but not in PAO1 wbpT encodes aputative a-D-galactosyltransferase involved in O-antigenbiosynthesis of P aeruginosa serotype O6 (Belangeret al 1999) orf5 is a P aeruginosa pathogenicity islandPAGI-1 gene encoding a homologue of RpoN-dependenttranscriptional activator (Liang et al 2001) Identificationof these upregulated genes that are relevant to pathoge-nicity might explain the observation in the animal studiesthat P aeruginosa virulence was enhanced in the pres-ence of OF

The only negatively affected gene in this group wasphzM It encodes a phenazine-specific methyltransferaseinvolved in the conversion of phenazine-1-carboxylic acid(PCA) to pyocyanin (Mavrodi et al 2001) As phenazinecompounds are not only virulence factors but alsoinvolved in bacterial competition (ie inhibiting or killingother bacteria in the vicinity) the downregulation of phzMin response to the presence of OF bacteria seems tocontradict the antagonistic competition function of pyocy-anin However as PCA itself is an active phenazine com-pound downregulation of phzM may simply indicate aratio change in the phenazine compounds In contrastphnB a related promoter identified in our assay wasupregulated about twofold A previous study showed thatphnA and phnB encode an anthranilate synthase influenc-ing pyocyanin production (Essar et al 1990) but the pre-cise role of the synthase in pyocyanin or PCA biosynthesisis not clear (Mavrodi et al 2001)

The second group of genes affected by OF encodesfive membrane proteins lppL encodes a lipopeptide andthe other four encode probable transporters The 13genes in the third group are largely involved in the cellularprocesses of protein and DNA and electron transferThree iron utilization-related genes were identified andclassed in the fourth group

Ten of the regulated genes (including the PA4391 in thefirst group) encode known or putative transcriptional reg-ulators This number represents 97 of the 103 charac-terized promoters close to the ratio of 94 in the PAO1genome for genes encoding either transcription regulators

or two-component regulatory proteins (Stover et al2000) Except for the lrp gene encoding a global transcrip-tional regulator (Stover et al 2000) the functions ortargets of the remaining regulators in this group areunknown

AI-2 is produced in sputum cultures and by most bacterial isolates from CF sputum

One possible contributor in the interactions between OFbacteria and P aeruginosa is the AI-2-mediated signallingthat is proposed as an interspecies communication path-way (Xavier and Bassler 2003) In an effort to probe therole of AI-2 in the microbial community in the lungs of CFpatients the first question addressed was whether AI-2was produced in this community Thirty independent spu-tum collections were used to inoculate brainndashheart infu-sion (BHI) and THY media These cultures were grown for16 h and AI-2 activity in cell-free supernatants was mea-sured using the Vibrio harveyi reporter system All thesamples tested exhibited AI-2 activity at different levelsFigure 4A shows the data from 10 independent patientsamples

To verify these results AI-2 production by individualstrains was tested A sputum sample from a single patientwas diluted and isolated on different solid media Repre-sentative isolates were grown in BHI and AI-2 activity wasmeasured As illustrated in Fig 4B the results indicatethat the AI-2 signalling molecule was produced by mostisolates confirming that the bacteria present in the lungsof CF patients can produce AI-2 P aeruginosa isolateswere the only strains negative for AI-2 production Con-sistent with this observation the P aeruginosa PAO1genome does not contain luxS a gene that is required forAI-2 production Despite the possible difference in geneticcomposition morphologically varied P aeruginosa iso-lates from several sputum samples were also tested andnone produced detectable AI-2

AI-2 activity can be detected directly in sputa and in BAL of rats co-infected with P aeruginosa and a sputum isolate

To assess whether AI-2 activity is present in the lungs ofCF patients AI-2 was measured directly in the sputumsamples The insoluble materials were precipitated bycentrifugation and the macromolecule substances werereduced by methanol precipitation As shown in Fig 5 asubstantial amount of AI-2 activity could be detecteddirectly in the cleared sputum samples of CF patientssuggesting that AI-2 is produced in the lungs of CFpatients It is noteworthy that the AI-2 activity was detect-able in the sample from the patient who was not colonizedby P aeruginosa (sample CFY in Fig 5) The amount ofAI-2 in sputum samples was comparable to that in the



culture supernatant of Salmonella typhimurium LT2 Incomparison acyl-homoserine lactone autoinducers havealso been detected in sputum samples from CF patientsbut the concentrations are relatively lower compared withculture supernatants ie 1ndash22 nM for 3-oxo-C12-HSL and1ndash5 nM for C4-HSL (Erickson et al 2002)

One limitation of the above experiments is that sputumsamples could potentially be contaminated with upperrespiratory tract material during excretion To address thisissue and to determine whether AI-2 could accumulate inthe lung AI-2 activity was measured in rat bronchoalveo-lar lavage fluids As exhibited in Fig 5 a significantamount of AI-2 was detected in the bronchoalveolar lav-age fluid of the animals co-infected with P aeruginosa andGram-positive isolate CF004 but not in those of the ani-mals infected with P aeruginosa alone These data indi-cate that AI-2 can be produced and accumulate to a

substantial level in the lung supporting the involvementof AI-2 in the interactions in the microbial community Thehigh concentration of AI-2 detected in sputum sampleswould suggest that this could not all be accounted for bycontamination of the sample during expelling This sameargument applies to the presence of normal oropharyn-geal flora in sputum samples

Pseudomonas aeruginosa virulence factor genes are modulated by OF bacteria and AI-2

To investigate the effect of AI-2 on P aeruginosagene expression and pathogenicity a group of 21 well-characterized P aeruginosa genes that are related topathogenicity (Table 2) was tested for differential expres-sion in the presence of AI-2 Their response to OF strainCF004 was tested in parallel experiments The promoterregions of these genes or their accommodating operonswere polymerase chain reaction (PCR) amplified clonedin pMS402 and transferred back into P aeruginosa Thetemporal expression of these genes was tested over aperiod of 24 h in the presence of exogenous AI-2 orCF004 As listed in Table 2 six genes were regulated byboth AI-2 and the OF strain and three were regulated onlyby the OF strain These upregulated virulence factorsinclude rhamnosyltransferase gene rhlA involved in rham-nolipid synthesis (Ochsner et al 1994) elastase genelasB (Bever and Iglewski 1988) exotoxin genes exoT(Yahr et al 1996) exoS (Kulich et al 1994) and exoY(Yahr et al 1998) and the phenazine synthesis genesphzA1 and phzA2 (Mavrodi et al 2001) The stationaryphase sigma factor rpoS (Tanaka and Takahashi 1994)and flagellin type B gene fliC (Brimer and Montie 1998

Fig 4 AI-2 detectionA AI-2 activities in the culture supernatants grown from sputum samples from 10 CF patients AI-2 activity is represented as fold induction of luminescence above the medium control using the V harveyi reporter system The cultures were grown for 16 h in BHI and culture supernatants were added at 5 in the assays S typhimurium LT2 was used as a positive controlB AI-2 production of the bacterial strains isolated from a single sputum sample Nos 1 7 and 9 Staphylococcus spp nos 2 and 12 Klebsiella spp nos 4 5 and 11 Streptococcus spp no 8 coagu-lase-negative Staphylococcus spp nos 3 6 and 10 Pseudomonas spp 5 cell-free overnight culture supernatants were added to the AI-2 assays

Fig 5 AI-2 activity in sputum samples and in BAL of rats co-infected with P aeruginosa and sputum isolate CFX1 AI-2 activity in four independent sputum samples was detected directly using sputum extracts at 5 Sample CFY was from a CF patient who was not colonized by P aeruginosa at the time of sample collection Rat BAL 1 and BAL 2 represent BAL samples from rats infected with P aerug-inosa alone and those co-infected with P aeruginosa and CF004 respectively

1486 K Duan et al


Feldman et al 1998) both of which are relevant to Paeruginosa virulence were also upregulated The resultsindicate that AI-2 was able to modulate P aeruginosagene expression patterns and pathogenicity AI-2-mediated signalling represents one of the pathways thatOF bacteria use to interact with P aeruginosa

Discussion

The usually non-pathogenic indigenous microflora orlsquonormal florarsquo is a ubiquitous constituent of the animal orhuman host In healthy individuals the upper respiratorytracts are colonized by a variety of microorganisms com-prising the normal flora or OF and the lower tractsalthough constantly inoculated normally remain sterile Incontrast a diverse OF is present along with the opportu-nistic pathogen P aeruginosa in the lower respiratorytracts of adult CF patients Analysis of clinical microbiol-ogy data from CF patients over a long period revealed thatS aureus and H influenzae are periodically present in thelungs of CF patients The normally avirulent oropharyn-geal flora strains such as coagulase-negative Staphylo-coccus spp and viridans Streptococcus spp are alsopresent in the sputum samples in substantial concentra-tions (106-108 cfu ml-1) In typical clinical microbiologypractice the types of bacteria monitored are limited Bac-teria characterized in this study inevitably under-representthe actual bacterial diversity in the CF lungs consistentwith the recent findings that a large number of lsquounusualrsquo

species are present in the lungs of CF patients (Coenyeet al 2002)

The animal experiments using the agar beads infectionmodel showed that P aeruginosa pathogenicity was sig-nificantly enhanced by the presence of OF whereas theOF strain itself showed little virulence The increasedlung damage was not the result of changes in the levelsof P aeruginosa in the co-infections as there was nosignificant increase in bacterial load This is reminiscentof some clinical situations in which no significant changein bacterial load is observed during periods of exacerba-tion and recovery (Jaffar-Bandjee et al 1995 Wolteret al 1999) Although resident flora can play an impor-tant protective role against the invasion of pathogens(Tancrede 1992 Falk et al 1998) in our experimentalconditions OF bacteria seem to contribute to diseaseprogression

Broad response in P aeruginosa seems to be inducedby the presence of OF Although competition for nutrientswas expected to be one of the factors the observedresponse appears to be a result of more complex interac-tions between P aeruginosa and OF bacteria Only arelatively small number of genes involved in metabolismwere affected (Table 1) As indicated by cfu counting inthe co-culture condition the OF strains grew poorly andwere unlikely to be able to compete effectively with the Paeruginosa for nutritional resources We expected compe-tition for iron to be a factor in a mixed microbial commu-nity however the limited number of affected iron

Table 2 Regulation of P aeruginosa virulence-associated genes by OF strain CF004 and AI-2

Gene PA no Function description

Fold inductiona

CF004 AI-2

Regulated virulence factorsphzA2 PA1899 Phenazine synthesis cluster 2 first gene 71 43lasB PA3724 Elastase 60 24phzA1 PA4210 Phenazine synthesis cluster 1 first gene 30 32rhlA PA3479 Rhaminolipid (rhamnosyltransferase chain A) 20 22exoT PA0044 Exoenzyme T (99 similar to ADP-ribosyltransferase) 19 19fliC PA1092 Flagellin type B 18 36exoS PA3841 Exoenzyme S (ADP-ribosyltransferase) 23 nexoY PA2191 Belongs to the exoenzyme S regulon adenylate cyclase 19 nrpoS PA3622 Stationary phase sigma factor 18 n

Unaffected virulence factorslasA PA1871 Protease (staphylolytic protease preproenzyme LasA)aprA PA1249 Alkaline protease (alkaline metalloproteinase precursor)adh PA2407 Putative adhesion proteinhem PA0041 Putative haemagglutininpilG PA0408 Type IV fimbrial (part of the pilGHIJKL gene cluster)algD PA3540 Alginate synthesis (GDP-mannose 6-dehydrogenase AlgD)plcH PA0844 Haemolytic phospholipase C (haemolysin) precursortoxA PA1148 Exotoxin ApvcA PA2254 Pyoverdine chromophore biosynthesis protein PvcA first of four ORF clusterrnr PA4937 Exoribonuclease RNase R (virulence protein VacB)migA PA0705 Probable glycosyl transferase (mucin-inducible gene)oprH PA1178 Outer membrane protein H1 precursor and PhoPQ operon

a Approximate maximum fold induction in the presence of CF004 and AI-2 respectively



utilization genes suggests that iron competition if any inthe co-cultures did not result in significant changes in Paeruginosa gene expression Consistent with this obser-vation a parallel screen for iron-regulated genes in Paeruginosa identified a predominately non-overlappingset of promoters (data not shown) The results from theanalysis of defined virulence factor genes also indicatethat the pvcA (Stintzi et al 1999) and toxA (Gray et al1984) genes which are strongly affected by iron were notaffected by the presence of OF strains

The upregulation of a significant number of virulencegenes in P aeruginosa by OF strains is intriguing andprobably explains the increased lung damage observed inthe presence of the OF strain in the animal experimentsThe upregulation of a pathogenrsquos virulence factors bymicroflora underscores the importance of bacterial inter-actions in pathogenicity The interactions between bacte-ria and their host are believed to determine the evolutionof many of the virulence factors that pathogens possess(Cotter and DiRita 2000) The large number of P aerug-inosa virulence-related genes affected by other bacteriain this study suggests that the interactions amongmicrobes in microflora may also contribute to the evolutionof these virulence factors Bacterial pathogens especiallythose that inhabit both environmental niches as well asanimal hosts may rely on these interactions to maintaintheir virulence when out of the hosts On the other handthese virulence factors may play a role in the developmentof microbial communities

The regulation of P aeruginosa gene expression andvirulence involves its own repertoire of cellndashcell signallingmolecules (Fuqua et al 2001 Miller and Bassler 2001Whitehead et al 2001 Smith and Iglewski 2003) Thepathways through which the OF strains affect P aerugi-nosa gene expression are expected to be multifactorialand complex One of the signals seems to be autoinducer-2 (AI-2) produced by non-pseudomonad strains It seemsthat P aeruginosa is modulating its behaviour by monitor-ing the environmental conditions and by eavesdroppingon the other bacteria via AI-2 and probably other signalsAI-2 in the sputum samples could reach a substantialamount which is readily detectable by the V harveyiassay The samples were processed to remove insolubleand macromolecule substances but were not concen-trated The levels of AI-2 activity measured are unavoid-ably lower than actual levels because of the lost activityduring sample preparation A caveat to the use of sputumsamples is that they may potentially be contaminated withupper respiratory tract material during production Giventhe high concentration of OF bacteria and AI-2 present itseems unlikely that this activity is exclusively caused bycontamination This is further supported by the presenceof AI-2 in bronchoalveolar lavage fluid from chronicallyinfected rat lungs

The regulation of a number of virulence factor genes byAI-2 indicates that this signal produced by OF strainscontributed at least in part to the observed modulationof P aeruginosa gene expression and changes in itspathogenicity The AI-2-regulated P aeruginosa virulencefactor genes partially overlap those modulated by OFstrains suggesting that in the co-culture experiments AI-2 was one of the signals but not the only signal producedby the OF that regulates P aeruginosa gene expressionIn the lungs of CF patients this AI-2-mediated effectcould also originate from secondary pathogens suggest-ing that the virulence of secondary pathogens could alsobe delivered through enhancing the virulence of the pri-mary pathogen P aeruginosa It is also possible that Paeruginosa could influence virulence factor genes of sec-ondary pathogens or those potentially present in the OFstrains

Our evidence indicates that OF bacteria that are notnormally thought of as serious problems in CF may con-tribute to lung pathology by influencing the gene expres-sion of P aeruginosa Part of the influence of OF on Paeruginosa may result from interspecies communicationincluding AI-2-mediated signalling The clinical efficacy ofa course of antibiotic therapy does not always correlatewith proven inhibition of replication of P aeruginosa Paeruginosa bacterial density in sputum can remain highdespite clinically effective treatment (Jaffar-Bandjee et al1995) The data presented here suggest that one contrib-uting mode of action of these drugs may be througheffects on OF strains Similar effects on OF strains mayalso explain part of the efficacy of macrolide antibioticssuch as azithromycin that have little antipseudomonalactivity but have been proven to be of clinical benefit inCF (Pechersquore 2001 Saiman 2002 Schoumlni 2003) Proto-cols that take into consideration the potential significanceof OFndashP aeruginosa interactions in CF may lead to moreefficacious therapeutic interventions and improved clinicaloutcome

Experimental procedures

Bacterial strains plasmids and culture conditions

Pseudomonas aeruginosa PAO1 (Holloway 1955) and theclinical isolate ATCC27853 that was used to construct thepromoter library were grown on LuriandashBertani (LB) plates orLB broth at 37infinC Salmonella typhimurium LT2 (McClellandet al 2001) was also grown in LB Vibrio harveyi BB170(Bassler et al 1994) was grown on LB plates or in autoin-ducer bioassay (AB) medium (Greenberg et al 1979) at30infinC The culture conditions for bacteria in different assaysare described separately

The lux-based promoter reporter plasmid pMS402 wasconstructed by joining the PacI fragment of pCS26Pac(Bjarnason et al 2003) with the EcoRIndashBamHI fragment ofpUCP28T (West et al 1994) which was blunt-ended by fill-

1488 K Duan et al


ing in recessed termini and was attached with a blunt-endPacI linker

Bacteria in sputum samples from CF patients were isolatedby first diluting the sputum sample in THY (ToddndashHewitt brothsupplemented with 05 yeast extract) and then grown onagar plates including blood agar BHI agar ToddndashHewitt agarand Pseudomonas isolation agar After growth in a CO2 incu-bator or anaerobic jars colonies were counted and cell andcolony morphology was examined Distinct isolates weregrown on blood agar plates and collected Microbial specieswere characterized using conventional microbiological andbiochemical procedures (Murray 1995) The OF strainsCF004 and CF018 were isolated from a single sputumsample from a CF patient and classified as viridans groupStreptococcus spp and Staphylococcus spp respectivelyby microbiological and biochemical tests

Pseudomonas aeruginosa was grown at 37infinC on LB platesor in M9 minimal medium supplemented with casamino acid(01) glucose (05) and trimethoprim at 200 mg ml-1

where appropriate CF004 and CF018 were grown at 37infinCin BHI medium or on blood agar plates Pseudomonas isola-tion agar (PIA) and BactoTM Columbia CNA agar were usedas selection media for P aeruginosa and Gram-positivestrains respectively Both monoculture and co-culture assayswere set up in multiwell plates (384-well or 96-well) filled withthe supplemented M9 medium (150 ml per well) The monoc-ultures were inoculated with P aeruginosa (1500 dilution)and co-cultures were inoculated with P aeruginosa (1500dilution) together with CF004 or CF018 (1200 dilution)Under these co-culture conditions P aeruginosa grew nor-mally and reached 2 yen 109 cfu ml-1 during the 24 h of growthThe OF bacteria retained a stable but low growth in thiscondition and had 1 yen 104-2 yen 105 cfu ml-1 after 24 h growthThese co-culture conditions were selected to minimize com-petition for nutrients in order not to select for biosyntheticgenes and presumably favour the detection of genes regu-lated by cellndashcell signalling

Animal infection test

Animal studies were carried out by Pathoprobe R and D usingan established rat lung infection model (Cash et al 1979)Three groups of male SpraguendashDawley rats (eight in eachgroup) weighing between 180 g and 200 g were inoculatedintratracheally with 005 ml of bacterium suspensions of 106

cfu ml-1 embedded in agar beads (P aeruginosa alone OFstrain alone and a mixture of equal amounts of both) Therats were sacrificed for quantitative bacteriology and histopa-thology at the end of 7 days after infection The pathogenicityof P aeruginosa was assessed by the lung damage indicatedas percentage of consolidation

Random promoter library construction

The promoter library was constructed and validated accord-ing to procedures developed in our laboratory (Bjarnasonet al 2003) Briefly Sau3A partially digested P aeruginosachromosomal DNA fragments with the sizes of 1ndash2 kb wereselected by sucrose gradient centrifugation and ligated intothe BamHI restriction site upstream of the promoterless

luxCDABE operon in the low-copy-number plasmid pMS402The ligated DNA was transformed into P aeruginosa by elec-troporation The transformants were picked into 384-wellplates and promoter-containing clones were selected bymeasuring luminescence production in LB and M9 media atseveral time points during growth A library of 3456 promoterclones was constructed and used for screening CF004- andCF018-regulated promoters Using the 626 Mbp P aerugi-nosa PAO1 genome size (Stover et al 2000) as referenceand the estimation that every 2ndash25 kb of DNA may containone transcription unit ordf2500ndash3130 promoter regions are pre-dicted in the P aeruginosa genome

Promoter screening and clustering

In the initial screening the luminescence of each promoterclone in a monoculture of P aeruginosa or co-culture withCF004 or CF018 was measured in a Wallac Victor2 model1450 multilabel counter (Perkin-Elmer Life Sciences) at 15 hintervals for 105 h and then at 24 h The luminescence wasmeasured as counts per second (cps) After rearraying thepotential regulated clones in new multiwell plates rescreen-ing was carried out in a similar manner However the lumi-nescence of each well was measured every 30 min for 24 hand bacterial growth in the wells was monitored at the sametime by measuring optical density (OD600) The growth of thebacteria was also confirmed by plating selected cultures onselective media and cfu counting

The promoter clustering was performed according to thesimilarity in their expression profiles using the CLUSTER pro-gram and visualized using TREEVIEW (Eisen et al 1998)

Sequence analysis of the modulated promoters

A subset of OF-regulated promoters was PCR amplifiedusing pZE05 (CCAGCTGGCAATTCCGA) and pZE06(AATCATCACTTTCGGGAA) primers which flank the BamHIsite of pMS402 The PCR products were sequenced usingan automatic sequencer and DNA sequences were analysedusing the BLAST program from the National Center for Bio-technology Information (httpwwwncbinlmnihgovBLAST)and VECTOR NTI 71 (Informax) programs

Measurement of AI-2 activity

AI-2 activity was measured using the V harveyi reporterassay as described previously (Surette and Bassler 1998)Briefly an overnight culture of V harveyi was diluted 15000in AB medium (Greenberg et al 1979) on a 96-well microtitreplate and 10 ml of samples (or diluted samples) to be testedwas added to 90 ml of the diluted culture The plates wereincubated at 30infinC with shaking at 200 rpm The lumines-cence values of individual cultures (wells) were measuredevery hour for a total of 12 h in a Victor2 Multilabel counterand AI-2 activity was reported as fold induction of lumines-cence by the reporter strain above the medium control

AI-2 activity in sputa was measured after the sputum sam-ples were processed by the following procedures The sputawere passed through an 18-gauge needle 20 times and a 21-gauge needle five times Then the samples were cleared by



centrifugation at 10 000 g for 15 min Macromolecules in thecleared fluid were reduced via precipitation by an equal vol-ume of methanol and filtration through a 022-mm pore sizefilter Methanol in the extracts was removed by evaporationin a SpeedVac and samples were adjusted to the originalvolume by adding sterile water AI-2 in culture supernatant orbronchoalveolar lavage (BAL) fluid was measured directlyafter the samples were subjected to centrifugation at15 000 g for 10 min and sterilization by passing through a022-mm pore size filter

Construction of a P aeruginosa virulence factor gene set

The promoter regions of selected P aeruginosa virulencefactors were amplified by PCR and cloned at the XhoIndashBamHI sites of pMS402 The expression of the virulencefactors in a monoculture of P aeruginosa alone or co-culturewith CF004 was measured as luminescence productiontaken at 30 min intervals for 24 h using a Wallac Victor2 Mul-tilabel counter

In vitro synthesis of AI-2

AI-2 was synthesized as described previously (Schauderet al 2001) The reaction was carried out for 1 h at 37infinC in10 mM sodium phosphate buffer (pH 75) containing 1 mMsubstrate S-adenosylhomocysteine (SAH) and 1 mg ml-1

purified E coli LuxSndash and PfxndashGST fusion proteins Afterincubation the reaction mixture was filtered through Biomax-5 Centricon Plus-20 centrifugation filters (Millipore) to removeproteins The amount of AI-2 in the preparation was esti-mated by fold induction of luminescence using the V harveyisystem described above

Acknowledgements

We thank our colleagues and members of the Surette labo-ratory for helpful discussions and critical reading of the manu-script MGS is supported by a Canada Research Chair inMicrobial Gene Expression and a Senior Scholar Award fromthe Alberta Heritage Foundation for Medical Research(AHFMR) KD is a recipient of an AHFMR postdoctoralfellowship This research was supported by the CanadianInstitutes of Health Research and Quorex Pharmaceuticals

References

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Bassler BL (1999) How bacteria talk to each other regula-tion of gene expression by quorum sensing Curr OpinMicrobiol 2 582ndash587

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Bever RA and Iglewski BH (1988) Molecular character-ization and nucleotide sequence of the Pseudomonasaeruginosa elastase structural gene J Bacteriol 1704309ndash4314

Bjarnason J Southward C and Surette MG (2003)Genomic profiling of iron-responsive genes in Salmonellaby high throughput screening of a random promoter libraryJ Bacteriol 185 4973ndash4982

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Brint JM and Ohman DE (1995) Synthesis of multipleexoproducts in Pseudomonas aeruginosa is under the con-trol of RhlR-RhlI another set of regulators in strain PAO1with homology to the autoinducer-responsive LuxR-LuxIfamily J Bacteriol 177 7155ndash7163

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Coenye T Goris J Spilker T Vandamme P andLiPuma JJ (2002) Characterization of unusual bacteriaisolated from respiratory secretions of cystic fibrosispatients and description of Inquilinus limosus gen nov spnov J Clin Microbiol 40 2062ndash2069

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DeLisa MP Wu CF Wang L Valdes JJ and BentleyWE (2001) DNA microarray-based identification of genescontrolled by autoinducer 2-stimulated quorum sensing inEscherichia coli J Bacteriol 183 5239ndash5247

Eisen MB Spellman PT Brown PO and Botstein D(1998) Cluster analysis and display of genome-wideexpression patterns Proc Natl Acad Sci USA 95 14863ndash14868

Erickson DL Endersby R Kirkham A Stuber K Voll-man DD Rabin HR et al (2002) Pseudomonas aerug-

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Falk PG Hooper LV Midtvedt T and Gordon JI (1998)Creating and maintaining the gastrointestinal ecosystemwhat we know and need to know from gnotobiology Micro-biol Mol Biol Rev 62 1157ndash1170

Favre-Bonte S Pache JC Robert J Blanc D PechereJC and van Delden C (2002) Detection of Pseudomo-nas aeruginosa cell-to-cell signals in lung tissue of cysticfibrosis patients Microb Pathog 32 143ndash147

Feldman M Bryan R Rajan S Scheffler L Brunnert STang H and Prince A (1998) Role of flagella in patho-genesis of Pseudomonas aeruginosa pulmonary infectionInfect Immun 66 43ndash51

Folders J Tommassen J van Loon LC and Bitter W(2000) Identification of a chitin-binding protein secreted byPseudomonas aeruginosa J Bacteriol 182 1257ndash1263

Fong KP Chung WO Lamont RJ and Demuth DR(2001) Intra- and interspecies regulation of gene expres-sion by Actinobacillus actinomycetemcomitans LuxS InfectImmun 69 7625ndash7634

Fuqua C Parsek MR and Greenberg EP (2001) Regu-lation of gene expression by cell-to-cell communicationacyl-homoserine lactone quorum sensing Annu RevGenet 35 439ndash468

Gray GL Smith DH Baldridge JS Harkins RN VasilML Chen EY and Heyneker HL (1984) Cloningnucleotide sequence and expression in Escherichia coli ofthe exotoxin A structural gene of Pseudomonas aerugi-nosa Proc Natl Acad Sci USA 81 2645ndash2649

Greenberg EP Hastings JW and Ulitzer S (1979)Induction of luciferase synthesis in Beneckea harveyi byother marine bacteria Arch Microbiol 120 87ndash91

Guarner F and Malagelada JR (2003) Gut flora in healthand disease Lancet 361 512ndash519

Hardegger M Koch AK Ochsner UA Fiechter A andReiser J (1994) Cloning and heterologous expression ofa gene encoding an alkane-induced extracellular proteininvolved in alkane assimilation from Pseudomonas aerug-inosa Appl Environ Microbiol 60 3679ndash3687

Hart CA and Winstanley C (2002) Persistent and aggres-sive bacteria in the lungs of cystic fibrosis children Br MedBull 61 81ndash96

Hirakata Y Srikumar R Poole K Gotoh N SuematsuT Kohno S et al (2002) Multidrug efflux systems playan important role in the invasiveness of Pseudomonasaeruginosa J Exp Med 196 109ndash118

Holloway BW (1955) Genetic recombination in Pseudomo-nas aeruginosa J Gen Microbiol 13 572ndash581

Jaffar-Bandjee MC Lazdunski A Bally M Carrere JChazalette JP and Galabert C (1995) Production ofelastase exotoxin A and alkaline protease in sputa duringpulmonary exacerbation of cystic fibrosis in patients chron-ically infected by Pseudomonas aeruginosa J Clin Micro-biol 33 924ndash929

Jann A Cavard D Martin C Cami B and Patte JC(1990) A lipopeptide-encoding sequence upstream fromthe lysA gene of Pseudomonas aeruginosa Mol Microbiol4 677ndash682

Kulich SM Yahr TL Mende-Mueller LM Barbieri JTand Frank DW (1994) Cloning the structural gene for the49-kDa form of exoenzyme S (exoS) from Pseudomonasaeruginosa strain 388 J Biol Chem 269 10431ndash10437

Kuroda A Kumano T Taguchi K Nikata T Kato J andOhtake H (1995) Molecular cloning and characterizationof a chemotactic transducer gene in Pseudomonas aerug-inosa J Bacteriol 177 7019ndash7025

Latifi A Winson MK Foglino M Bycroft BW StewartGS Lazdunski A and Williams P (1995) Multiplehomologues of LuxR and LuxI control expression of viru-lence determinants and secondary metabolites throughquorum sensing in Pseudomonas aeruginosa PAO1 MolMicrobiol 17 333ndash343

Liang X Pham XQ Olson MV and Lory S (2001)Identification of a genomic island present in the majority ofpathogenic isolates of Pseudomonas aeruginosa J Bacte-riol 183 843ndash853

Lyon WR Madden JC Levin JC Stein JL and Cap-aron MG (2001) Mutation of luxS affects growth andvirulence factor expression in Streptococcus pyogenesMol Microbiol 42 145ndash157

McClelland M Sanderson KE Spieth J Clifton SWLatreille P Courtney L et al (2001) Complete genomesequence of Salmonella enterica serovar TyphimuriumLT2 Nature 413 852ndash856

McNab R Ford SK El-Sabaeny A Barbieri B CookGS and Lamont RJ (2003) LuxS-based signaling inStreptococcus gordonii autoinducer 2 controls carbohy-drate metabolism and biofilm formation with Porphyromo-nas gingivalis J Bacteriol 185 274ndash284

Mavrodi DV Bonsall RF Delaney SM Soule MJPhillips G and Thomashow LS (2001) Functional anal-ysis of genes for biosynthesis of pyocyanin and phenazine-1-carboxamide from Pseudomonas aeruginosa PAO1 JBacteriol 183 6454ndash6465

Middleton B Rodgers HC Camara M Knox AJ Will-iams P and Hardman A (2002) Direct detection of N-acylhomoserine lactones in cystic fibrosis sputum FEMSMicrobiol Lett 207 1ndash7

Miller MB and Bassler BL (2001) Quorum sensing inbacteria Annu Rev Microbiol 55 165ndash199

Miller MB Skorupski K Lenz DH Taylor RK andBassler BL (2002) Parallel quorum sensing systems con-verge to regulate virulence in Vibrio cholerae Cell 110303ndash314

Murray PR (1995) Manual of Clinical Microbiology Wash-ington DC American Society for Microbiology Press

Ochsner UA and Reiser J (1995) Autoinducer-mediatedregulation of rhamnolipid biosurfactant synthesis inPseudomonas aeruginosa Proc Natl Acad Sci USA 926424ndash6428

Ochsner UA Fiechter A and Reiser J (1994) Isolationcharacterization and expression in Escherichia coli of thePseudomonas aeruginosa rhlAB genes encoding a rham-nosyltransferase involved in rhamnolipid biosurfactant syn-thesis J Biol Chem 269 19787ndash19795



Ohtani K Hayashi H and Shimizu T (2002) The luxSgene is involved in cell-cell signalling for toxin productionin Clostridium perfringens Mol Microbiol 44 171ndash179

Passador L Cook JM Gambello MJ Rust L andIglewski BH (1993) Expression of Pseudomonas aerug-inosa virulence genes requires cell-to-cell communicationScience 260 1127ndash1130

Pechersquore J-C (2001) New perspectives on macrolide anti-biotics Int J Antimicrob Agents 18 S93ndashS97

Pesci EC Milbank JB Pearson JP McKnight SKende AS Greenberg EP and Iglewski BH (1999)Quinolone signaling in the cell-to-cell communication sys-tem of Pseudomonas aeruginosa Proc Natl Acad Sci USA96 11229ndash11234

Rajan S and Saiman L (2002) Pulmonary infections inpatients with cystic fibrosis Semin Respir Infect 17 47ndash56

Saiman L (2002) Do macrolides have an anti-inflammatoryeffect In 16th Annual North American Cystic Fibrosis Con-ference S124 New Orleans Louisiana October 3ndash6 2002

Schauder S Shokat K Surette MG and Bassler BL(2001) The LuxS family of bacterial autoinducers biosyn-thesis of a novel quorum-sensing signal molecule MolMicrobiol 41 463ndash476

Schoumlni MH (2003) Macrolide antibiotic therapy in patientswith cystic fibrosis Swiss Med Wkly 133 297ndash301

Schuster M Lostroh CP Ogi T and Greenberg EP(2003) Identification timing and signal specificity ofPseudomonas aeruginosa quorum-controlled genes atranscriptome analysis J Bacteriol 185 2066ndash2079

Shreve MR Butler S Kaplowitz HJ Rabin HR StokesD Light M and Regelmann WE (1999) Impact ofmicrobiology practice on cumulative prevalence of respira-tory tract bacteria in patients with cystic fibrosis J ClinMicrobiol 37 753ndash757

Singh PK Schaefer AL Parsek MR Moninger TOWelsh MJ and Greenberg EP (2000) Quorum-sensingsignals indicate that cystic fibrosis lungs are infected withbacterial biofilms Nature 407 762ndash764

Smith RS and Iglewski BH (2003) P aeruginosa quorum-sensing systems and virulence Curr Opin Microbiol 6 56ndash60

Soni R Marks G Henry DA Robinson M Moriarty CParsons S et al (2002) Effect of Burkholderia cepaciainfection in the clinical course of patients with cystic fibro-sis a pilot study in a Sydney clinic Respirology 7 241ndash245

Stevenson B and Babb K (2002) LuxS-mediated quorumsensing in Borrelia burgdorferi the Lyme disease spiro-chete Infect Immun 70 4099ndash4105

Stintzi A Johnson Z Stonehouse M Ochsner U MeyerJM Vasil ML and Poole K (1999) The pvc gene clusterof Pseudomonas aeruginosa role in synthesis of thepyoverdine chromophore and regulation by PtxR andPvdS J Bacteriol 181 4118ndash4124

Stover CK Pham XQ Erwin AL Mizoguchi SD War-rener P Hickey MJ et al (2000) Complete genomesequence of Pseudomonas aeruginosa PA01 an opportu-nistic pathogen Nature 406 959ndash964

Sundin GW Shankar S Chugani SA Chopade BAKavanaugh-Black A and Chakrabarty AM (1996)Nucleoside diphosphate kinase from Pseudomonas aerug-inosa characterization of the gene and its role in cellulargrowth and exopolysaccharide alginate synthesis MolMicrobiol 20 965ndash979

Surette MG and Bassler BL (1998) Quorum sensing inEscherichia coli and Salmonella typhimurium Proc NatlAcad Sci USA 95 7046ndash7050

Surette MG Miller MB and Bassler BL (1999) Quorumsensing in Escherichia coli Salmonella typhimurium andVibrio harveyi a new family of genes responsible for auto-inducer production Proc Natl Acad Sci USA 96 1639ndash1644

Tanaka K and Takahashi H (1994) Cloning analysis andexpression of an rpoS homologue gene from Pseudomo-nas aeruginosa PAO1 Gene 150 81ndash85

Tancrede C (1992) Role of human microflora in health anddisease Eur J Clin Microbiol Infect Dis 11 1012ndash1015

Wagner VE Bushnell D Passador L Brooks AI andIglewski BH (2003) Microarray analysis of Pseudomonasaeruginosa quorum-sensing regulons effects of growthphase and environment J Bacteriol 185 2080ndash2095

West SE Schweizer HP Dall C Sample AK andRunyen-Janecky LJ (1994) Construction of improvedEscherichia-Pseudomonas shuttle vectors derived frompUC1819 and sequence of the region required for theirreplication in Pseudomonas aeruginosa Gene 148 81ndash86

Whitehead NA Barnard AM Slater H Simpson NJand Salmond GP (2001) Quorum-sensing in Gram-negative bacteria FEMS Microbiol Rev 25 365ndash404

Winzer K Sun YH Green A Delory M Blackley DHardie KR et al (2002) Role of Neisseria meningitidisluxS in cell-to-cell signaling and bacteremic infection InfectImmun 70 2245ndash2248

Wolter JM Bowler SD and McCormack JG (1999) Areantipseudomonal antibiotics really beneficial in acute res-piratory exacerbations of cystic fibrosis Aust NZ J Med29 15ndash21

Xavier KB and Bassler BL (2003) LuxS quorum sensingmore than just a numbers game Curr Opin Microbiol 6191ndash197

Yahr TL Barbieri JT and Frank DW (1996) Geneticrelationship between the 53- and 49-kilodalton forms ofexoenzyme S from Pseudomonas aeruginosa J Bacteriol178 1412ndash1419

Yahr TL Vallis AJ Hancock MK Barbieri JT andFrank DW (1998) ExoY an adenylate cyclase secretedby the Pseudomonas aeruginosa type III system Proc NatlAcad Sci USA 95 13899ndash13904

Yoon SS Hennigan RF Hilliard GM Ochsner UAParvatiyar K Kamani MC et al (2002) Pseudomonasaeruginosa anaerobic respiration in biofilms relationshipsto cystic fibrosis pathogenesis Dev Cell 3 593ndash603

Zhu J Miller MB Vance RE Dziejman M BasslerBL and Mekalanos JJ (2002) Quorum-sensing regula-tors control virulence gene expression in Vibrio choleraeProc Natl Acad Sci USA 99 3129ndash3134

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et al



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ing oropharyngeal flora (OF) bacteria such as viridansgroup streptococci and coagulase-negative staphylococciwhich only colonize the upper respiratory tracts in healthyadults To understand the pathogenesis of CF infection itis necessary to learn the molecular interactions and cellndashcell communications in this complex microbial communityin the lungs of CF patients

Quorum sensing is a cell density-dependent cellndashcellsignalling system that uses small extracellular moleculestermed autoinducers to co-ordinate gene expression in abacterial community In Gram-negative bacteria autoin-ducers are typically acyl-homoserine lactone (HSL)molecules and in Gram-positive bacteria typically oli-gopeptides (Miller and Bassler 2001)

P aeruginosa

pos-sesses two acyl-homoserine lactone quorum-sensingsystems

lasRlasI

(Passador

et al

1993) and

rhlRrhlI

(Brint and Ohman 1995 Latifi

et al

1995 Ochsner andReiser 1995) and one 2-heptyl-3-hydroxy-4-quinolone-based signalling system (denoted PQS for

Pseudomonas

quinolone signal) (Pesci

et al

1999) Quorum sensing in

P aeruginosa

affects a broad range of genes and is centralto its pathogenesis Up to 11 of genes in the genome arecontrolled by quorum sensing (Schuster

et al

2003 Wag-ner

et al

2003) Quorum sensing regulates a range ofimportant cellular and secreted virulence factors and playsimportant roles in the development of biofilms (Fuqua

et al

2001 Miller and Bassler 2001 Whitehead

et al

2001Smith and Iglewski 2003) Recent data suggest that

Paeruginosa

lives in biofilms in the lungs of CF patients(Singh

et al

2000) and biofilms may be primarily anaer-obic (Yoon

et al

2002)

P aeruginosa

autoinducers aredetectable in the lungs suggesting that quorum sensingcontrols

in vivo

pathogenicity (Singh

et al

2000 Erickson

et al

2002 Favre-Bonte

et al

2002 Middleton

et al

2002)

The LuxS-dependent quorum-sensing system is aunique system the signalling molecule of which termedautoinducer-2 (AI-2) results from spontaneous cyclizationof the LuxS product (45-dihydroxy-23-pentanedione)(Surette

et al

1999 Schauder

et al

2001) The

luxS

family of genes is spread widely among Gram-positiveand Gram-negative bacteria and AI-2 has been proposedto be an interspecies signalling molecule harbouring theinformation of both density and fitness of a bacterial com-munity (Bassler 1999 Schauder

et al

2001 Xavier andBassler 2003) Recent studies indicate that AI-2 is ableto regulate a range of genes and cellular processes

(DeLisa

et al

2001 Stevenson and Babb 2002) It hasbeen linked to virulence in a number of bacteria including

Neisseria meningitidis

(Winzer

et al

2002)

Streptococ-cus pyogenes

(Lyon

et al

2001)

Vibrio cholerae

(Miller

et al

2002 Zhu

et al

2002)

Clostridium perfringens

(Ohtani

et al

2002) and

Actinobacillus actinomycetem-comitans

(Fong

et al

2001) Evidence has shown that AI-2 is involved in mixed-species biofilm formation (McNab

et al

2003) and interspecies gene regulation (Fong

et al

2001) The

P aeruginosa

PAO1 genome however lacksan identifiable

luxS

gene homologue (Stover

et al

2000)and therefore does not produce AI-2

To understand the contribution of microbial interactionsto

P aeruginosa

pathogenesis and host diseases animalstudies have been carried out to analyse the effect of OFon

P aeruginosa

virulence The molecular interactionsbetween pathogenic

P aeruginosa

and avirulent strainsisolated from sputum samples of CF patients have beeninvestigated with an

in vitro

system

P aeruginosa

genesresponding to OF bacteria have been identified bygenome-wide gene expression profiling in the presenceof OF strains The role of AI-2-mediated quorum sensingin the bacterial interactions has also been assessed Evi-dence is presented that suggests important contributionsof host microflora to

P aeruginosa

infection via modulationof its gene expression

Results

Mixed microbial species in CF lungs shown by clinical microbiology data

Present in the lungs of CF patients is a complex microbialcommunity dominated by chronic infecting pathogensmost frequently

P aeruginosa

The constitution of thismicrobial community is influenced by the physiologicalstatus of the patients and antibiotic treatments Becausenon-pathogenic species are often neglected in clinicallaboratories (Shreve

et al

1999) many other speciesmay be present in the CF lungs This view is supportedby recent findings that uncommon bacterial species livein the lungs of CF patients (Coenye

et al

2002)The microbial dynamics in the lungs of CF patients over

an extended period is reflected in the microbiological datagathered from sputum samples of CF patients This isdemonstrated by the data from the Adult CF Clinic inFoothills Hospital Calgary Alberta Canada Figure 1A

Fig 1

Microbiological analysis of sputum samples from CF patientsA Longitudinal microbiology data from a single CF patient colonized primarily by

P aeruginosa

showing the transient infections by secondary pathogens and the persistent colonization of

Pseudomonas

and normal oropharyngeal flora Bacterial levels are in colony-forming units (cfu)ml

-

1

sputumB Concentration of

Pseudomonas

(solid bars) and normal oropharyngeal flora (open bars) from CF patients The average cfu ml

-

1

sputum was calculated for eight patients (with a minimum of 40 sputum samples per patient) collected over a 10-year span The error bars represent standard deviation in cfu

Modulation of

P aeruginosa


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et al



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P aeruginosa


Strepto-coccus

and

Staphylococcus


H influenzae

S aureus

and

Candida albicans


P aeruginosa


Pseudomonas


Pseudomonas


-

1


Pseudomonas

In vivo
















1482 K Duan et al













































































(6) Secretedprotein







Table 1 cont


1484 K Duan et al





















1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al


































































Modulation of

P aeruginosa


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et al



50

1477ndash1491


P aeruginosa


Strepto-coccus

and

Staphylococcus


H influenzae

S aureus

and

Candida albicans


P aeruginosa


Pseudomonas


Pseudomonas


-

1


Pseudomonas

In vivo
















1482 K Duan et al













































































(6) Secretedprotein







Table 1 cont


1484 K Duan et al





















1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al


































































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K Duan

et al



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1477ndash1491


P aeruginosa


Strepto-coccus

and

Staphylococcus


H influenzae

S aureus

and

Candida albicans


P aeruginosa


Pseudomonas


Pseudomonas


-

1


Pseudomonas

In vivo
















1482 K Duan et al













































































(6) Secretedprotein







Table 1 cont


1484 K Duan et al





















1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al










































































1482 K Duan et al













































































(6) Secretedprotein







Table 1 cont


1484 K Duan et al





















1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al































































































(6) Secretedprotein







Table 1 cont


1484 K Duan et al





















1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al


































































1484 K Duan et al





















1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al











































































1486 K Duan et al



Discussion







Fold inductiona

CF004 AI-2















1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al













































































1488 K Duan et al




























Acknowledgements


References



















1490 K Duan et al










































































Acknowledgements


References



















1490 K Duan et al


































































1490 K Duan et al


































































Modulation of Pseudomonas aeruginosa gene expression by host

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