Microbiology (2005), 151, 1113–1125
DOI 10.1099/mic.0.27631-0
The MexGHI-OpmD multidrug efflux pump
controls growth, antibiotic susceptibility and
virulence in Pseudomonas aeruginosa via
4-quinolone-dependent cell-to-cell communication
Séverine Aendekerk,1,2 Stephen P. Diggle,2 Zhijun Song,3 Niels Høiby,3
Pierre Cornelis,1 Paul Williams2 and Miguel Cámara2
1
Correspondence
Miguel Cámara
[email protected]
Laboratory of Microbial Interactions, Department of Molecular and Cellular Interactions,
Flanders Interuniversity Institute for Biotechnology, Vrije Universiteit Brussel, Building E,
Room 6.6, Pleinlaan 2, B-1050 Brussels, Belgium
2
Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences,
University of Nottingham, Nottingham NG7 2RD, UK
3
Dept Clinical Microbiology 9301, University Hospital of Copenhagen, Rigshospitalet,
DK-2100 Copenhagen, Denmark
Received 20 September 2004
Revised
20 December 2004
Accepted 10 January 2005
In Pseudomonas aeruginosa the production of multiple virulence factors depends on
cell-to-cell communication through the integration of N-acylhomoserine lactone (AHL)- and
2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)- dependent signalling. Mutation of genes encoding
the efflux protein MexI and the porin OpmD from the MexGHI-OpmD pump resulted in the inability
to produce N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and PQS and a
marked reduction in N-butanoyl-L-homoserine lactone levels. Both pump mutants were impaired
in growth and exhibited enhanced rather than reduced antibiotic resistance. Provision of
exogenous PQS improved growth and restored AHL and virulence factor production as well as
antibiotic susceptibility, indicating that the pump mutants retained their capacity to respond to PQS.
RT-PCR analysis indicated that expression of the PQS biosynthetic genes, phnA and pqsA,
was inhibited when the mutants reached stationary phase, suggesting that the pleiotropic
phenotype observed may be due to intracellular accumulation of a toxic PQS precursor.
To explore this hypothesis, double mexI phnA (unable to produce anthranilate, the precursor of
PQS) and mexI pqsA mutants were constructed; the improved growth of the former suggested
that the toxic compound is likely to be anthranilate or a metabolite of it. Mutations in mexI and
opmD also resulted in the attenuation of virulence in rat and plant infection models. In plants,
addition of PQS restored the virulence of mexI and opmD mutants. Collectively, these results
demonstrate an essential function for the MexGHI-OpmD pump in facilitating cell-to-cell
communication, antibiotic susceptibility and promoting virulence and growth in P. aeruginosa.
INTRODUCTION
Pseudomonas aeruginosa is an ubiquitous Gram-negative
c-proteobacterium capable of causing disease in humans,
animals and plants (Cao et al., 2001a; Lyczak et al., 2002).
P. aeruginosa produces a vast array of extracellular virulence
factors, the majority of which are regulated both in a cell
population density-dependent manner, via cell-to-cell
communication or ‘quorum sensing’ (Swift et al., 2001;
Withers et al., 2001; Bassler, 2002; Smith & Iglewski, 2003),
and in a growth phase-dependent manner (Diggle et al.,
Abbreviations: AHL, N-acylhomoserine lactone; C4-HSL, N-butanoyl-Lhomoserine lactone; 3-oxo-C12-HSL, N-(3-oxododecanoyl)-L-homoserine
lactone; PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone.
0002-7631 G 2005 SGM
2002, 2003). P. aeruginosa possesses two N-acylhomoserine
lactone (AHL)- dependent quorum sensing systems
(reviewed by Winzer & Williams, 2001; Cámara et al.,
2002). The las system comprises the N-(3-oxododecanoyl)L-homoserine lactone (3-oxo-C12-HSL) synthase, LasI and
its cognate response regulator LasR, whilst the rhl system
involves the N-butanoyl-L-homoserine lactone (C4-HSL)
synthase and the response regulator RhlR (Winzer &
Williams, 2001; Cámara et al., 2002). The las system
regulates the production of extracellular virulence determinants such as elastase, the LasA protease, alkaline protease and exotoxin A (Winzer & Williams, 2001; Cámara
et al., 2002). It also plays a role in controlling the expression
of the xcpP and xcpR genes involved in the regulation of the
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1113
S. Aendekerk and others
Type II general secretion pathway (Chapon-Hervé et al.,
1997) and has been implicated in the maturation of P.
aeruginosa biofilms (Davies et al., 1998). The rhl system
controls the production of rhamnolipids, elastase, LasA
protease, hydrogen cyanide, pyocyanin, siderophores and
the cytotoxic lectins PA-IL and PA-IIL (Diggle et al., 2002;
Latifi et al., 1995, 1996; Winson et al., 1995). The las and
the rhl systems are hierarchically organized such that the
las system exerts transcriptional control over the rhl
system (Latifi et al., 1996). However, the rhl system can
also function independently of the las system (Diggle et al.,
2003). Three independent transcriptome analyses have
revealed that the las and the rhl systems influence the
expression of more than 5 % of the P. aeruginosa PAO1
genome (Schuster et al., 2003; Wagner et al., 2003; Hentzer
et al., 2003). Recently, an additional LuxR homologue,
VqsR (virulence and quorum sensing regulation) has been
shown to control the expression of a subset of quorum
sensing regulated genes and to be needed for the full
expression of siderophore biosynthesis and uptake genes
(Juhas et al., 2004; Cornelis & Aendekerk, 2004).
P. aeruginosa also releases into the extracellular milieu
2-heptyl-3-hydroxy-4(1H)-quinolone, the pseudomonas
quinolone signal (PQS), a molecule which closely resembles
the 4-quinolone family of synthetic antimicrobials. The
synthesis and action of PQS are modulated by the las and
rhl systems respectively (Pesci et al., 1999). LasR regulates
the production of PQS, and the addition of exogenous
PQS to P. aeruginosa cultures enhances the expression of
the elastase gene lasB, rhlI and rhlR as well as the alternative sigma factor, RpoS (Diggle et al., 2003; Pesci et al.,
1999; McKnight et al., 2000). These findings suggest that
PQS functions as a regulatory link between the las and
rhl AHL-dependent quorum sensing systems and the stationary phase (via RpoS). The overlap between the quorum
sensing and the RpoS regulons has been confirmed by a
recent transcriptome analysis of the genes controlled by
RpoS in P. aeruginosa (Schuster et al., 2004). Although the
rhl system is active in the absence of PQS, production of
certain rhl-dependent phenotypes such as PA-IL lectin
and pyocyanin strictly depends on the presence of PQS at
the onset of stationary phase (Diggle et al., 2003).
PQS is derived from anthranilate (Calfee et al., 2001), and
the structural genes required for PQS biosynthesis have
recently been identified (pqsABCD, pqsH plus the anthranilate synthase genes phnAB) together with the transcriptional regulator MvfR (PqsR) and a proposed effector,
PqsE (Gallagher et al., 2002; Déziel et al., 2004). The transcription of pqsH is regulated by the las quorum sensing
system, linking AHL-dependent quorum sensing with PQS
regulation (Gallagher et al., 2002). Evidence that PQS is
produced during human infections has been obtained by
the direct detection of PQS in P. aeruginosa strains from
cystic fibrosis (CF) patients (Collier et al., 2002) and by data
indicating that PQS synthesis is increased in P. aeruginosa
isolates from CF airways (Guina et al., 2003).
1114
We have previously described a genetic locus (mexGHIopmD), conferring resistance to vanadium in P. aeruginosa
(Aendekerk et al., 2002), of which the gene products MexH,
MexI and OpmD are highly conserved in relation to other
components of P. aeruginosa antibiotic efflux pumps (Poole,
2002). Recently, it was shown that a plasmid expressing
mexH, mexI and opmD conferred elevated resistance to
norfloxacin, ethidium bromide, acriflavine and rhodamine
6G, confirming that MexHI-OpmD functions as a multidrug efflux pump (Sekiya et al., 2003). We knew from our
previous work that mutants in the non-coding region,
upstream of mexGHI-opmD, in mexI and opmD were
severely affected in the production of AHL-regulated
exoproducts including elastase, rhamnolipids and pyocyanin (Aendekerk et al., 2002). In addition, the expression of
lecA, which encodes the cytotoxic, galactophilic lectin PA-IL
of P. aeruginosa, was markedly reduced by a Tn5 insertion
into the mexGHI-opmD operon (Diggle et al., 2002). Here
we demonstrate that the MexI efflux protein and the OpmD
porin play a key role in controlling P. aeruginosa growth,
antibiotic susceptibility and virulence via 4-quinolonedependent cell-to-cell communication. We also show that
loss of the pump by mutation may result in the accumulation of a toxic PQS precursor which could be responsible
for the severely attenuated phenotype observed.
METHODS
Growth conditions and strains. All the bacterial strains and
plasmids used in this study are listed in Table 1. The P. aeruginosa
strain PA14 mexI and opmD mutants and the PAO1 strain mexI
phnA and mexI pqsA double mutants were constructed by allelic
exchange as described for the corresponding single mexI PAO1
mutants by Aendekerk et al. (2002). P. aeruginosa strains were
grown in Luria broth (LB), Casamino acids medium (CAA) or ox
broth (Statens Serum Institut, Copenhagen, Denmark) at 37 uC.
When required, synthetic PQS was added to a final concentration
of 60 mM, the synthetic AHLs 3-oxo-C12-HSL and C4-HSL at concentrations of 10 or 100 mM, and anthranilate at concentrations of
100 nM to 100 mM. Growth was continuously monitored in the
Bioscreen apparatus (Life Technologies), using the following parameters: culture volume 300 ml, temperature 37 uC, shaking for 10 s
every 3 min, and reading every 20 min. As inoculum, an overnight
culture of PAO1 in CAA was diluted in order to achieve a final
OD600 of 0?001. Each culture was inoculated in triplicate and each
experiment was repeated three times.
Synthesis of AHLs and PQS analogues. C4-HSL and 3-oxo-
C12-HSL were synthesized as described previously (Chhabra et al.,
1993, 2003), and PQS was synthesized as described by Diggle et al.
(2003) using the procedures of Pesci et al. (1999).
TLC assays for C4-HSL and 3-oxo-C12-HSL detection. Acidified
supernatants from 18 h cultures (OD600 2) in LB at 37 uC were
extracted with dichloromethane as described previously (Diggle et al.,
2002; Yates et al., 2002) and analysed by TLC. Ten-microlitre samples
were spotted onto reverse-phase silica RP-18 F254S (Merck) and separated using a methanol/H2O (60 : 40) system. C4-HSL was assayed
using an Escherichia coli strain harbouring the AHL reporter plasmid
pSB536 (Swift et al., 1997). For the detection of 3-oxo-C12-HSL,
samples were spotted onto RP-2 plates and the TLC was resolved
using a mixture of methanol/H2O (45 : 55, v/v). In this case 3-oxoC12-HSL was detected using the AHL biosensor E. coli(pSB1075)
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Microbiology 151
P. aeruginosa antibiotics, virulence and growth
Table 1. Bacterial strains and plasmids
Strain or plasmid
P. aeruginosa strains
PAO1
PAO1 mexI
PAO1 opmD
PAO1 phnA
PAO1 pqsA
PAO1 mexI phnA
PAO1 mexI pqsA
PAO1 lecA9 : : lux
PAO1 lecA9 : : lux mexI
PAO1 lecA9 : : lux opmD
PA14
PA14 mexI
PA14 opmD
Plasmids
pSB536
pSB1075
Genotype or phenotype
Wild-type
Mutant with a Gm cassette inserted in the gene mexI
Mutant with a Gm cassette inserted in the gene opmD
Mutant in the phnA gene
Mutant in the pqsA gene
Derivative of the phnA mutant with the mexI mutation
Derivative of the pqsA mutant with the mexI mutation
lecA9 : : luxCDABE chromosomal reporter fusion in PAO1
Derivative of the PAO1 lecA9 : : lux mutant with the mexI mutation
Derivative of the PAO1 lecA9 : : lux mutant with the opmD mutation
Wild-type able to infect different host
Mutant with a Gm cassette inserted in the mexI gene
Mutant with a Gm cassette inserted in the opmD gene
AHL biosensor ahyR ahyI9 : : luxCDABE in pAHP13 (AmpR) used for the
detection of C4-HSL
AHL biosensor lasR lasI9 : : luxCDABE in pAHP13 (AmpR) used for the
detection of 3-oxo-C12-HSL
(Winson et al., 1998). For both biosensors, AHLs were visualized as
bright spots on a dark background when viewed with a Luminograph
LB980 (Berthold) photon video camera.
TLC analysis of PQS production. For the detection of extra-
cellular PQS, cell-free spent supernatants were prepared from 18 h
cultures (10 ml at OD600 2) and extracted with 10 ml acidified ethyl
acetate. The organic phase was dried and resuspended in 50 ml
methanol. Intracellular PQS was detected after disruption by sonication of a cell pellet prepared from a 40 ml overnight culture resuspended in 20 ml LB. PQS was then extracted from the supernatant
of the cell lysate using equal volume of acidified ethyl acetate. The
solvent was evaporated and resuspended in 100 ml methanol. Tenmicrolitre samples of each extracellular and intracellular extract were
spotted onto normal-phase silica 60F254 (Merck) TLC plates, pretreated by soaking in 5 % K2HPO4 for 30 min and activated at
100 uC for 1 h. Extracts were separated using a dichloromethane/
methanol (95 : 5, v/v) system until the solvent front reached the top
of the plate. PQS was visualized under UV light and identified by
comparison with a synthetic standard (5 ml of a 10 mM stock).
Source or reference
ATCC 15692
Aendekerk et al. (2002)
Aendekerk et al. (2002)
University of Washington
Genome Center
This work
This work
This work
Winzer et al. (2000)
This work
This work
Tan et al. (1999)
This work
This work
Swift et al. (1997)
Winson et al. (1998)
of bioluminescence. Bioluminescence was
measured as a function of cell density using a combined automated
luminometer–spectrophotometer (LUCYI, Anthos Labtech). Overnight cultures of the P. aeruginosa lecA9 : : luxCDABE reporter gene
fusion were diluted to OD600 0?01 in LB and 200 ml of this dilution
was added to each well of a LUCY plate. Where required, PQS
(60 mM) or C4-HSL (100 mM) or both were added. Bioluminescence
and optical density were determined. Luminescence is given, in relative light units (RLU) divided by OD495, indicating the approximate
light output per cell.
Measurement
RT-PCR. RNA was extracted from P. aeruginosa PAO1, mexI and
opmD grown in LB using the High Pure RNA Isolation Kit (Roche
Diagnostics). cDNA was synthesized using the First-Strand cDNA
Synthesis Kit (Amersham Pharmacia). For PQS biosynthesis gene
expression, RT-PCR was done using two sets of primers: pqsA1 and
pqsA2 or phnA1 and phnA2 (Table 2). The primers used for lasI
and rhlI transcript detection are also shown in Table 2. As a control,
RT-PCR was done using primers oprL-1 and oprL-2 for the amplification of the housekeeping gene oprL (Lim et al., 1997). The complete list of primers used in this study is shown in Table 2.
Antibiotic resistance test. Antibiotic resistance was measured by
the filter-disk assay method using commercially available disks
(Fluka). The following antibiotics were tested: spectinomycin (15 mg
per disk), tetracycline (30 mg), nalidixic acid (30 mg), chloramphenicol
(30 mg), kanamycin (30 mg), rifampicin (15 mg) and carbenicillin
(30 mg). Three millilitres of a cell suspension (56106 c.f.u. ml21)
was added to a LB plate and left for 30 min, after which the cell
suspension was removed and the plate dried under laminar flow.
The disks were applied to the plates (four per plate) and the plates
incubated for 18 h at 37 uC. The diameter of the inhibition ring was
then measured. Each experiment was done in triplicate.
Quantification of pyocyanin. For pyocyanin analysis bacteria
were spread onto PAB agar (Difco) in numbers that enabled confluent growth and incubated at 37 uC for 48 h. Pyocyanin was extracted
from the agar medium and quantified according to Mavrodi et al.
(2001).
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Construction of the PAO1 pqsA, mexI pqsA and mexI phnA
mutants. A pqsA chromosomal deletion mutant in PAO1 lacking
1172 internal nucleotides was constructed as follows. Using PAO1
DNA as a template, a 2600 bp fragment containing the intact pqsA
gene (1554 bp) was amplified using the primer pair pqsAUF
containing a SalI restriction site (59-TAGGTGTCGACTTCGGCAGGCTCGCC-39) and pqsADR containing a HindIII restriction site
(59-GCGTAAAGCTTCGGGCAATCCAGGTT-39). The resulting
PCR product was digested with SalI and HindIII and cloned into
similarly digested pUC18, resulting in the plasmid pUCpqsA. To
introduce a deletion of the recombinant pqsA gene, the primer
pair DpqsAUP (59-TCGGGACGTAATCGAGGCGGAACAG-39) and
DpqsADR (59-TTCCGTACGTACGGGCATGTTGATTC-39) was
used in conjunction with inverse PCR using pUCpqsA DNA as a
template. The resulting blunt-ended PCR product containing a
1172 bp deletion in pqsA was self-ligated, resulting in the plasmid
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1115
S. Aendekerk and others
Table 2. Primers used for RT-PCR
Target gene
oprL
lasI
rhlI
pqsA
phnA
Primer designation
Primer sequence
oprL-1
oprL-2
lasI-1
lasI-2
rhlI-1
rhlI-2
pqsA-1
pqsA-2
phnA-1
phnA-2
59-ATGGAAATGCTGAAATTCGGC-39
59-CTTCTTCAGCTCGACGCGACG-39
59-TACAAATTGGTCGGCGCGAA-39
59-TAAAGCGCGATCTGGGTCTT-39
59-ATGATCGAATTGCTCTCTGAA-39
59-TCACACCGCCATCGACAGCGGTA-39
59-AATGCCGGACCTACATTCT-39
59-TCCTCGATAGTGTGTCCT-39
59-ACTGGAAGAAAGTCTGGA-39
59-TGGGGTTGCGCAGGCACAA-39
pUCDpqsA. The PCR product was excised from the vector using SalI
and XbaI, and cloned into the suicide vector pDM4 (Milton et al.,
1996) digested with SalI and XbaI, resulting in the plasmid
pDM4DpqsA. Allelic exchange using pDM4DpqsA contained in E.
coli S17-1 lpir with PAO1 resulted in a P. aeruginosa strain (PAO1
pqsA) containing an in-frame deletion of the pqsA gene. This deletion was confirmed by PCR analysis (data not shown). The phnA
deletion mutant is a transposon mutant from PAO1 obtained from
the University of Washington Genome Center. The mexI pqsA and
mexI phnA double mutants were made by inserting a GmR cassette
in the mexI gene of the pqsA and phnA mutants as previously
described (Aendekerk et al., 2002).
Virulence assays in plants and animals
Animals. Lung infection in rats was performed as previously
reported (Song et al., 1998). PAO1 and mexI strains were first
cultured on agar plates, and then one colony of each strain was
inoculated into ox broth and cultured at 37 uC with shaking for
24 h. Bacteria were harvested by centrifugation and embedded in
alginate beads. Alginate beads (100 ml) containing either PAO1 or
the mexI mutant were instilled intratracheally to the deep left
bronchus for each rat (female, 7-week-old Lewis rats with a body
weight of between 150 and 200 g). The challenge concentration for
PAO1 strain was 16108 c.f.u. ml21, and for the mexI mutant
1?56109 c.f.u. ml21. Challenge concentrations were determined
from a series of pilot studies. The mortality rate was followed up to
7 days post-challenge, after which all animals were sacrificed. Lung
pathology was expressed as lung index of macroscopic pathology
(LIMP) and pathological scoring. LIMP represents the lung area
with pathological changes versus the area of the whole lung. Lung
scores can be divided into four grades: score 1, normal; score 2, only
lung atelectasis <10 mm2; score 3, lung atelectasis <40 mm2; score
4, lung abscess or lung atelectasis >40 mm2.
Plants. P. aeruginosa strain PA14 and the isogenic mexI and opmD
mutants were grown overnight in LB medium or until they reached
the stationary phase at 37 uC, and washed in 10 mM MgSO4. Cells
were then diluted to 107 and 108 c.f.u. ml21. Ten microlitres of
diluted cells was inoculated with a micropipette into the stems of
Romaine lettuce plants. The stems were washed with 0?1 % bleach
and placed on 15 cm diameter Petri dishes containing a Whatman
filter impregnated with 10 mM MgSO4. The midrib of each lettuce
leaf was inoculated with each of the three P. aeruginosa strains to be
tested. For some experiments, PQS (60 mM) was either co-injected
with the bacteria or injected on its own. The plates were kept in a
growth chamber at 28 uC and symptoms monitored daily for 5 days.
1116
RESULTS
MexI and OpmD are required for AHL and PQS
production
We have previously shown that mutations in the P.
aeruginosa genes mexI and opmD from the MexGHIOpmD efflux system result in the inability to produce
detectable levels of C6-HSL and C4-HSL using the AHL
biosensor strain Chromobacterium violaceum CV026
(Aendekerk et al., 2002). However, CV026 has a relatively
low sensitivity for C4-HSL (McClean et al., 1997) and is
not activated by 3-oxo-C12-HSL. To gain better insights
into the production of AHLs by the mexI and opmD
mutants, two alternative and more sensitive AHL biosensor strains were used in conjunction with TLC (Diggle
et al., 2002; Yates et al., 2002). These were the lux-based
E. coli(pSB536) for the detection of C4-HSL and E.
coli(pSB1075) for 3-oxo-C12-HSL (Swift et al., 1997;
Winson et al., 1998). We also investigated the effect of the
mexI and opmD mutations on PQS production using the
TLC-based method described by Calfee et al. (2001). Fig. 1
shows that the mexI and opmD mutants produced no
detectable 3-oxo-C12-HSL or PQS and markedly reduced
levels of C4-HSL in spent-culture supernatants. However,
the addition of 60 mM synthetic PQS to cultures of mexI
and opmD mutants resulted in the restoration of both 3oxo-C12-HSL (Fig. 1a) and C4-HSL production (Fig. 1b),
although in the mexI mutant, 3-oxo-C12-HSL levels did
not return to wild-type (Fig. 1a, lane 5). In contrast, exogenous provision of 3-oxo-C12-HSL did not restore PQS
synthesis in these mutants (data not shown), demonstrating that the PQS-negative phenotype was not simply due
to a non-functioning las system. This is believed to be the
first time that 3-oxo-C12-HSL synthesis has been shown
to be PQS-dependent. Furthermore, no intracellular PQS
(Fig. 1c) or AHLs (data not shown) could be detected in
either the mexI or opmD mutants, indicating that the loss
of the AHLs was not a consequence of the inability to
export 3-oxo-C12-HSL or PQS.
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P. aeruginosa antibiotics, virulence and growth
(a)
1
2
3
4
5
6
7
(b)
1
2
3
4
5
6
7
(c)
1
2
3
4
5
6
7
Fig. 1. (a) Detection of 3-oxo-C12-HSL by TLC using the
AHL biosensor strain E. coli(pSB1075). Lanes 1 to 7 contain
synthetic 3-oxo-C12-HSL (1), extracts from spent medium of P.
aeruginosa PAO1 (2), PAO1 grown in the presence of 60 mM
PQS (3), mexI mutant (4), mexI mutant grown in the presence
of 60 mM PQS (5), opmD mutant (6) and opmD mutant grown
in the presence of 60 mM PQS (7). (b) Detection of C4-HSL
by TLC using the AHL biosensor strain E. coli(pSB536). Lane
1 contains synthetic C4-HSL, and lanes 2 to 7 are as for (a).
(c) Detection of PQS by TLC and UV light. Lanes 1 to 7 contain synthetic PQS (1), extracts from spent medium of PAO1
(2), mexI mutant (4) and opmD mutant (6), and intracellular
extracts from PAO1 (3), mexI mutant (5) and opmD mutant (7).
All extractions were from cells grown for 18 h (OD600 2).
Expression of quorum sensing regulated
virulence determinants in the mexI and ompD
mutants can be restored by exogenously
supplied PQS
The fact that mutations in mexI and opmD abolish or
drastically reduce the production of quorum sensing signal
molecules suggests that this may also result in a decreased
production of quorum sensing regulated virulence determinants in these mutants. If this is the case, since PQS has
a central role in quorum sensing-mediated gene expression
(Diggle et al., 2003) and can restore AHL production in the
mexI and opmD mutants, addition of PQS to these mutants
should restore the production of virulence determinants.
In our previous work, we showed that the production of
elastase and pyocyanin was drastically reduced in the mexI
and opmD mutants.
We have also shown before that expression of lecA (encoding the cytotoxic PA-IL lectin) is strictly under the control
of the rhl quorum sensing system (Winzer et al., 2000). In
addition, lecA expression has an absolute requirement for
PQS (Diggle et al., 2003). In both mexI and opmD mutants,
the loss of PQS and reduced level of C4-HSL suggested
that expression of lecA should be severely down-regulated.
To test this hypothesis we first introduced mexI and opmD
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Fig. 2. Expression of the lecA9 : : luxCDABE transcriptional
fusion in P. aeruginosa PAO1 and mexI mutant. Strains were
grown at 37 6C for 22 h in LUCYI (Anthos Labtech). X, No
addition (PAO1); &, no addition (mexI); n, 100 mM C4-HSL
(mexI); 6, 60 mM PQS (mexI). In all experiments, RLU and
OD495 were determined as described in Methods. The graphs
represent single experiments although each one was repeated
three times with similar results.
mutations into a PAO1 strain harbouring a lecA9 : : lux
chromosomal fusion (Winzer et al., 2000). Interestingly,
mutations in mexI resulted in the abolition of lecA
expression, which could only partially be restored upon
addition of high concentration of C4-HSL (Fig. 2). In
contrast, the provision of exogenous PQS to the mexI
mutant restored lecA9 : : lux expression to wild-type levels
(Fig. 2). Similar results were obtained with the ompD
mutant in the lecA9 : : lux strain (data not shown). This is
consistent with our recent work (Diggle et al., 2003), in
which we showed that inhibition of PQS biosynthesis, in
the presence of a functional rhl system, abolished PA-IL
production. Addition of a combination of PQS and C4HSL to these mutants further enhanced lecA9 : : lux expression (data not shown). In contrast, 3-oxo-C12-HSL had
no effect on lecA transcriptional levels (data not shown),
as shown previously (Winzer et al., 2000).
mexI and opmD mutants show enhanced
antibiotic resistance which can be reversed
upon addition of exogenous PQS
We have previously described the role of the mexGHI-opmD
locus in resistance to vanadium in P. aeruginosa (Aendekerk
et al., 2002). Also, a mutant (ncr, with an insertion in the
non-coding upstream region) unable to express mexGHIopmD was shown to be more resistant to tetracycline and
to the combination of ticarcillin and clavulanic acid
(Aendekerk et al., 2002). To investigate whether mutations
in mexI and opmD confer altered susceptibility to other
antibiotics, we tested a range of these against the two
mutants. Confirming and extending our previous results,
we observed that the mexI and opmD mutants became
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S. Aendekerk and others
Table 3. Antibiotic sensitivity of wild-type, mexI and opmD mutants in the absence and presence of PQS
Numbers represent the diameter of inhibition (cm) around the point of addition of the antibiotic. PQS concentration in the medium was
60 mM. Results are means±SD of 3 replicates.
Antibiotic
Kanamycin
Spectinomycin
Carbenicillin
Nalidixic acid
Rifampicin
Tetracycline
Chloramphenicol
PAO1
PAO1+PQS
mexI
mexI+PQS
opmD
opmD+PQS
1?35±0?05
2?9±0?1
3?6±0?1
2?55±0?05
2?9±0?1
2?85±0?05
3?55±0?05
2?0±0?01
4?1±0?1
4?9±0?1
2?65±0?05
3?35±0?05
5?0±0?01
4?8±0?1
0
0
1?15±0?05
1?45±0?05
1?75±0?05
2?15±0?05
3?0±0?01
1?55±0?05
1?25±0?05
4?25±0?05
2?1±0?1
3?0±0?01
4?75±0?05
4?35±0?05
0
0
1?15±0?05
1?35±0?05
1?6±0?1
2?2±0?1
3?05±0?05
1?65±0?05
1?3±0?1
4?4±0?1
2?15±0?05
3?2±0?1
4?6±0?1
4?25±0?05
resistant to kanamycin and spectinomycin, while they
displayed a decreased sensitivity to carbenicillin, nalidixic
acid, tetracycline, chloramphenicol and rifampicin (Table 3).
Furthermore, the sensitivity towards all antibiotics was
partially or totally restored upon provision of exogenous
PQS to the mutants, in a way similar to complementation by
the mexGHI-opmD genes in trans (Aendekerk et al., 2002).
Interestingly, antibiotic sensitivity also increased in the
wild-type strain upon supplementation of the medium with
PQS (Table 3). Hence the increased resistance to several
antibiotics in the P. aeruginosa mexI and opmD mutants is
governed by the absence of PQS in these mutants.
PQS biosynthetic gene expression is switched
off during the growth of the mexI and opmD
mutants
To understand the mechanism leading to the reduction in
the production of virulence determinants in the MexGHIOpmD mutants, and as the mexI and opmD mutations
exert a profound effect on cell-to-cell communication, we
investigated whether this was due to a reduction in the
expression of lasI, rhlI (encoding the 3-oxo-C12-HSL and
the C4-HSL synthases, respectively) or pqsA and phnA,
which are two key genes in the biosynthesis of PQS
(Gallagher et al., 2002; Déziel et al., 2004). Detection of
transcripts for these genes was carried out using RT-PCR.
After 18 h growth (OD600 2), the cDNAs corresponding
to all four genes were present in the parent PAO1 strain
while in both the mexI and opmD mutants, only the lasI
and rhlI transcripts were present (Fig. 3a). A more detailed
analysis of pqsA and phnA expression as a function of
growth revealed that the corresponding transcripts could
be detected at OD600 0?6 and 1?2, but not at OD600 1?6. We
conclude that, the absence of PQS production in the mexI
and opmD mutants is a result of the inhibition of the
expression of PQS biosynthetic genes during growth. The
fact that the absence of 3-oxo-C12-HSL did not correlate
with the inhibition of lasI transcription suggested the
presence of an alternative, post-transcriptional mechanism
preventing the biosynthesis of this quorum sensing signal
molecule.
1118
Mutation of mexI and opmD results in growth
deficiency which is restored upon addition of
exogenous PQS
To investigate whether mutations in mexI and opmD
affected the growth of P. aeruginosa, which could also
account for the increase in antibiotic resistance observed,
the growth of both mutants was monitored. As shown in
Fig. 4, in LB liquid medium at 37 uC, an extended lag
phase was observed for both mutants (20–40 h) compared
with the wild-type (8 h), although the growth rate was
(a) WT
mexI
(b)
opmD
oprL
WT mexI
opmD OD600
0.6
lasI
1.2
rhlI
1.6
pqsA
phnA
Fig. 3. RT-PCR analysis of transcripts. (a) Transcripts for oprL
(house-keeping control gene), lasI, rhlI, pqsA and phnA, in
wild-type, mexI mutant and opmD mutant strains (left to right).
The cultures were grown in LB medium until they reached an
OD600 of 2. (b)Transcripts for pqsA in wild-type, mexI and
opmD mutants as a function of the OD600 in LB medium (indicated on the right). Identical results were obtained for the
phnA transcripts (results not shown).
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P. aeruginosa antibiotics, virulence and growth
Fig. 4. Growth of wild-type ($), mexI (#) and opmD (n) mutants in LB medium in the absence (no additions) and in the
presence of exogenously added 100 mM 3-oxo-C12-HSL (+3-oxo-C12-HSL) or 60 mM PQS (+PQS). The graphs
represent the mean of three independent measurements using the Bioscreen growth monitoring apparatus as described in
Methods. Each OD600 value is the mean of three wells using an identical inoculum. For clarity, the variation between wells is
not indicated, but it never exceeded 10 %. The growth experiments were repeated three times in different experiments and the
trend was similar.
similar between the mutants and the wild-type. Interestingly, whilst addition of 10 mM 3-oxo-C12 to the medium
had no effect, the presence of 60 mM PQS considerably
shortened the lag phase (Fig. 4). This suggests that only
exogenously supplied PQS can overcome the growth
defect in the P. aeruginosa pump mutants. Although these
experiments were done in the Bioscreen apparatus, where
the cultures are under lower oxygen tension, similar results
(extended lag phases for mexI and opmD mutants and
growth stimulation by addition of PQS) were obtained
when the cultures were incubated in flasks and shaken at
150 r.p.m. (results data not shown).
pqsA mutant (which accumulates anthranilate and does
not produce PQS) was characterized by a very long lag phase
and a diminished growth rate (Fig. 5), in contrast to the
single phnA or pqsA mutants, which did not present any
The extended lag phase of mexI and opmD
mutants is probably due to the accumulation of
a toxic PQS precursor
It has recently been shown that anthranilate, the product
of the anthranilate synthase PhnAB, is a biosynthetic precursor of PQS, thought to be modified by PqsA during the
initial steps in the biosynthesis of this 4-quinolone signal
molecule (Essar et al., 1990; Gallagher et al., 2002; Déziel
et al., 2004). In addition, it has been demonstrated that a
pqsA mutant accumulates and excretes anthranilate into
the growth medium (Déziel et al., 2004), suggesting the
existence of an efflux mechanism for this PQS precursor.
Hence the inhibition of pqsA transcription in the pump
mutants may result in the accumulation of anthranilate or
a metabolic derivative which may exert a toxic effect on
the cells and hence affect growth. This would suggest that
mexGHI-opmD is involved in the excretion of these toxic
metabolites. To investigate this hypothesis we first constructed a P. aeruginosa mexI phnA double mutant (which
does not produce anthranilate, from the PhnAB pathway,
and therefore does not produce PQS via the pqsA-E operon)
and observed improved growth compared with the single
mexI mutant (Fig. 5). Conversely, the growth of a mexI
http://mic.sgmjournals.org
Fig. 5. Growth of wild-type (PAO1), phnA, pqsA, mexI, mexI
phnA and mexI pqsA mutants in LB medium. The graphs represent the mean of three independent measurements. Each
OD600 value is the mean of three wells using an identical
inoculum. For clarity, the variation between wells is not indicated, but it never exceeded 10 %. The growth experiments
were repeated three times in different experiments and the
trend was similar.
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S. Aendekerk and others
growth alterations (Fig. 5). Interestingly, although exogenous PQS reduced the lag phase of the mexI mutant
(Fig. 4) it had no effect on the mexI phnA or mexI pqsA
double mutants (data not shown). Taken together, these
data indicate that the delayed growth observed in the
pump mutants is caused by the accumulation of anthranilate or a metabolic derivative and that the mexGHI-opmD
pump might be involved in the detoxification of these
compounds.
presence of multiple abscesses in the animals infected with
the wild-type PAO1 while only a single abscess was detected
in the lungs from the rats infected with the mexI mutant.
The mexI and opmD mutations were then transferred by
allelic exchange to the highly plant- and animal-virulent
P. aeruginosa PA14 strain, and the virulence of the mutants
was tested in a lettuce infection assay where PA14, in
contrast to PAO1, is known to induce necrotic lesions
(Rahme et al., 1995). Fig. 6(b) shows that the mexI and
opmD mutants did not cause any necrosis of the leaves
whilst typical disease signs (necrosis and maceration) were
clearly observed for the wild-type. The fact that PQS can
restore the quorum sensing regulated phenotypes tested
suggests that one of the reasons for the reduced virulence
of the P. aeruginosa mexI and opmD mutants is the absence
of this signal molecule. To investigate whether this is the
case we tested whether co-inoculation of the PA14 mexI
and opmD mutants with PQS could restore their ability
to cause necrotic lesions in the lettuce infection assay.
Fig. 6(b) shows that the addition of PQS indeed restored
virulence in both of the mutants. However for the mexI
mutant this restoration was only partial. Inoculation of
PQS alone did not cause any necrosis (results not shown).
Mutation of mexI and opmD severely
compromises virulence in P. aeruginosa
The fact that a mutation in the MexGHI-OpmD pump
affected the production of some quorum sensing regulated
virulence determinants suggested that it could also have
significant implications in the ability of this organism
to cause disease. Consequently, we tested the effect that
mutations in mexI and ompD had on the virulence of P.
aeruginosa using both an animal and a plant model of
infection. As the animal model we used a rat lung infection model (Song et al., 1998) in which rats were infected
intratracheally in the left lung with either the wild-type
PAO1 (40 animals) or the mexI mutant (20 animals). Whilst
32 (80 %) animals infected with the wild-type died, only 1
rat infected with the mexI did (Fig. 6a). Analysis of bacterial numbers present in the lung at the end of the experiments revealed large differences between the mexI mutant
(46106 c.f.u. per lung) and the wild-type (26108 c.f.u.
per lung) even though 15 times more cells from the mexI
mutant were used for the infection. Pathological examination of the lungs from the infected rats (Fig. 6a) showed the
DISCUSSION
In this work, we have demonstrated a key role for the
MexI and OpmD proteins, two components of a newly
discovered efflux system in P. aeruginosa (Aendekerk
et al., 2002; Sekiya et al., 2003), in the production of
4-quinolone and AHL quorum sensing signal molecules
(a)
(b)
PA14
mexl
1
opmD
PA14
mexl
mexl+PQS
PA14
2
opmD opmD+PQS
3
Fig. 6. (a) Lungs from rats infected with the wild-type P. aeruginosa PAO1 (right) and the mexI mutant (left). Multiple
abscesses are present in the wild-type-infected lung while only one is visible for the mexI-infected lung (shown by arrows).
The table shows a summary of the results from these virulence studies. (b) Plant (lettuce) infection assay with P. aeruginosa
PA14 wild-type and its corresponding mexI and opmD mutants (1), wild-type PA14, mexI and mexI co-inoculated with 60 mM
PQS (2) and wild-type PA14, opmD and opmD co-inoculated with PQS (3).
1120
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Microbiology 151
P. aeruginosa antibiotics, virulence and growth
in this organism, and in promoting cell growth and in
virulence. Furthermore, we have shown that loss by mutation of the pump enhances rather than reduces resistance
to several different classes of antibiotics, a process which
can be reversed by the provision of the PQS signal molecule. We believe that this is the first example of mutants
in an efflux system displaying such strong phenotypes,
including obvious growth defects.
Previous studies have suggested a role for P. aeruginosa
efflux pumps in the transport of quorum sensing signal
molecules to the extracellular milieu. The MexAB-OprM
efflux system of P. aeruginosa has been proposed to be
involved in the efflux of 3-oxo-C12-HSL while C4-HSL
appears to diffuse freely out of the cells (Pearson et al., 1999).
In addition this efflux pump has been shown to play an
important role in the invasiveness of P. aeruginosa and it
has been suggested to be involved in the export of virulence determinants (Hirakata et al., 2002). However the
mechanism involved was not elucidated. Köhler et al. (2001)
have also proposed that the inducible MexEF-OprN system
might be responsible for the efflux of PQS. These authors
found that overexpression of this pump, achieved via the
nfxC mutation, restored the activity of the MexT activator
and resulted in the decreased transcription of rhlI and a
reduction of PQS and C4-HSL production (Köhler et al.,
1999, 2001). They suggested that either PQS or a precursor
might be the substrate of the MexEF-OprN pump. It is
remarkable that the phenotypes described by Köhler and
co-workers for the nfxC mutant overexpressing mexEFoprN are similar to those we describe here and in our
previous work (Aendekerk et al., 2002), including decreased
production of virulence factors and signal molecules, and
lack of virulence in a model of acute pneumonia in mice
(Cosson et al., 2002). Recently, Ramsey & Whiteley (2004)
described a genetic locus, dad6 (dynamic attachment
deficient), corresponding to the PA2491 gene and encoding
an oxidoreductase; mutants in this gene showed a defect
in the establishment of biofilms. This gene is located
upstream of the mexT regulator gene and the mexEF-oprN
genes and its inactivation results in increased expression
of the mexEF-oprN genes and decreased expression of the
PQS biosynthesis genes (Ramsey & Whiteley, 2004). In
contrast, our data presented here are believed to be the
first to conclusively show that the absence of an efflux
pump dramatically affects the production of quorum
sensing signal molecules via the repression of PQS biosynthesis genes in P. aeruginosa while the data of Köhler
et al. (2001) and Ramsey & Whiteley (2004) suggest that
overproduction of MexEF-OprN results in the same
phenotype. Microarray analysis of the expression of
different efflux porin genes showed that, from the genes
encoding members of the Opm porin family (Hancock &
Brinkman, 2002; Jo et al., 2003) only oprM, oprJ, opmA,
opmD and opmL are highly transcribed (Jo et al., 2003).
However, that study was done in the PAK strain of P.
aeruginosa, which does not produce PQS (Lépine et al.,
2003). Using P. aeruginosa PAO1, Murata et al. (2002)
http://mic.sgmjournals.org
detected the production of OpmB and OpmD by Western
blot analysis, with OpmD increasing at the onset of the
stationary phase, corresponding to the time of PQS
appearance.
Our studies also demonstrate that the absence of the main
components of the MexGHI-OpmD pump does not affect
the response to exogenous PQS as demonstrated by the
restoration of pyocyanin production (data not shown) and
the expression of a lecA9 : : lux fusion, both traits known to
be controlled by PQS and the Rhl system (Diggle et al.,
2003; Gallagher et al., 2002; Déziel et al., 2004; D’Argenio
et al., 2002).
An unexpected finding was the growth-phase-dependent
shut-down of transcription of the PQS biosynthetic genes
pqsA and phnA in the mexI and opmD mutants. This result
may suggest the involvement of the pump in the efflux of
a toxic compound (explaining the reduced growth rate)
linked to the production of PQS. As already noted, no PQS
could be detected inside the cells of the mexI and opmD
mutants. The precursor of PQS is anthranilate (Calfee et al.,
2001). The phnA and phnB genes encode the two components of an anthranilate synthase and were first described as being important for the production of phenazines,
including pyocyanin (Essar et al., 1990, Anjaiah et al., 1998).
Subsequently it was demonstrated that phnAB are necessary for PQS biosynthesis, providing an explanation as to
why mutants in these genes failed to produce phenazines
(Gallagher et al., 2002).
In a previous report we described that a trpC mutant
failed to produce phenazines and excreted anthranilate
into the medium (Anjaiah et al., 1998). Addition of
increasing amounts of tryptophan to the trpC mutant
restored the production of phenazines while decreasing the
concentration of anthranilate in the spent medium, due to
feedback inhibition of the TrpEG anthranilate synthase
(Essar et al., 1990). These results suggest that (i) anthranilate synthesized by the TrpEG anthranilate synthase cannot be channelled into the synthesis of PQS, (ii) high
anthranilate concentrations inhibit the production of
phenazines, eventually by repressing phnA transcription
and (iii) anthranilate is excreted from the cells. One
possibility is that, in the absence of efflux mediated by
MexI-OpmD, accumulation of intracellular anthranilate
leads to the progressive transcriptional shutdown of the
PQS biosynthesis genes. Our physiological studies of the
double mexI phnA and mexI pqsA mutants indicate that
the accumulation of anthranilate in the cell may be responsible for the growth defect in the absence of the pump,
since the mexI pqsA mutant is almost non-viable, in contrast to a pqsA mutant which grows in a manner similar to
the parent strain. Anthranilate is also a direct precursor of
catechol biosynthesis. Catechols have been shown to exert
toxic effects in cells such as generation of reactive oxygen
species by redox reactions, oxidative DNA damage and
protein damage by thiol arylation or oxidation, and they
can also interfere with electron transport (Schweigert et al.,
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1121
S. Aendekerk and others
2001). Catechols can also complex metals, such as iron
(Schweigert et al., 2001). Accumulation of catechols inside
the cells in the absence of the MexGHI-OpmD pump
could also explain the observed increased sensitivity to
vanadium in a mutant affected in this pump (Aendekerk
et al., 2002), which could be due to the accumulation of
catechol–vanadium complexes. The incomplete restoration
of growth in the case of the mexI phnA double mutant could
be explained by the fact that a second route exists in
pseudomonads for the production of anthranilate via the
degradation of tryptophan by the enzyme tryptophan
2,3-dioxygenase (TDO, encoded by PA 2579) into Nformylkynurenine, followed by the conversion of this
molecule into N-kynurenine by a formamidase (PA2081),
and finally the degradation of N-kynurenine into anthranilate by a kynureninase (PA2080) (Kurnasov et al., 2003;
Matthijs et al., 2004). Supporting this proposition, it has
been shown that a transposon insertion in the TDO gene
suppresses the autolysis phenotype caused by an overproduction of PQS in P. aeruginosa (D’Argenio et al., 2002).
It is also worth noting a recently published report showing
the presence of an efflux system in E. coli that acts as a
‘metabolic relief valve’ by pumping out p-hydroxybenzoic
acid, which is a precursor of ubiquinones (Van Dyk et al.,
2004).
Despite the loss of AHL production in the mexI and opmD
mutants, both the lasI and rhlI transcripts were present in
both the exponential and the stationary phase. Previously,
it has been shown that the absence of MvfR (PqsR), which
is a positive regulator of PQS biosynthesis, does not affect
the transcription of lasR or rhlR (Cao et al., 2001b). These
data suggest that the restoration of AHL production in
the pump mutants by exogenous PQS must therefore be
occurring at the post-transcriptional level. It is possible
that this involves the post-transcriptional regulatory protein RsmA, which influences the expression of both lasI
and rhlI (Pessi et al., 2001), since PQS is capable of overcoming the RsmA-dependent repression of lecA, a gene
which is known to be positively regulated by both C4-HSL
and PQS (Diggle et al., 2003). Clearly, addition of PQS
to the opmD mutant results in the complete restoration
of production of 3-oxo-C12, while the effect is less pronounced in the case of the mexI mutant. One possibility is
that MexI is the most important component of the pump
and that PQS stimulates the transcription of the pump.
Indeed, as opmD is the last gene of the operon, mexI should
be transcribed in the opmD mutant (Aendekerk et al., 2002).
Analysis of the P. aeruginosa PAO1 genome sequence
suggests that this opportunistic human pathogen, which
exhibits innate resistance to multiple antibiotic classes,
possesses at least ten different RND (ResistanceNodulation-Cell Division) type multi-drug efflux pumps
(Stover et al., 2000). For those pumps that have been
extensively characterized, genetic disruption generally
leads to increased susceptibility to antimicrobial agents.
Paradoxically, here we show that disruption of the
1122
MexGHI-OpmD efflux pump enhances the resistance of
P. aeruginosa to a variety of antibiotics including b-lactams,
aminoglyclosides and quinolones (Table 3).
Interestingly, Maseda et al. (2004) showed that overproduction of the MexEF-OprN pump results in increased
resistance to quinolones, but hypersusceptibility to most
b-lactams. The same authors demonstrated that C4-HSL
is needed for maximal expression of mexAB-oprM in
stationary phase and that this is antagonized by the MexT
regulator. For both the mexI and opmD mutants, the
provision of exogenous PQS restored susceptibility to for
example kanamycin and spectinomycin. These data indicate that PQS-dependent cell-to-cell communication in
P. aeruginosa is also involved in controlling susceptibility
to antimicrobial agents. This is further highlighted by the
increase in susceptibility of the parent PAO1 strain cultured
in the presence of PQS. The mechanism involved is not
known but may be a consequence of the PQS-dependent
repression of other multi-drug efflux pumps. Although
there is no published information on the regulation of
any of the RND efflux pumps by PQS, both the mexGHIopmD gene cluster (qsc133; Whiteley et al., 1999) and the
mexAB-oprM cluster (Maseda et al., 2004) are positively
regulated by AHL-dependent quorum sensing. Alternatively,
PQS may directly regulate genes involved in controlling cell
envelope permeability. Further work will clearly be required
to determine the mechanism by which PQS modulates
antibiotic susceptibility.
Mutation of both mexI and opmD rendered P. aeruginosa
unable to cause necrosis and macerate plant tissues.
Likewise, the mexI mutant was found to be avirulent in an
acute rat lung infection model. This is in agreement with a
previous study which showed that biosynthetic mutants
unable to produce PQS are avirulent in a pathogenicity
assay employing the nematode Caenorhabditis elegans
(Gallagher & Manoil, 2001). Interestingly, in the plant
infection model, the virulence of both mexI and opmD
mutants could be restored to different levels by the provision of exogenous PQS. The fact that PQS only partially
restored virulence in the mexI mutant could be explained
by the inability of PQS to fully restore 3-oxo-C12-HSL
production in this mutant. The restoration of virulence
confirms that PQS uptake occurs via an alternative pathway to export and that PQS uptake is not dependent on
the presence of a functional MexGHI-OpmD pump or the
quorum sensing circuitry. These results clearly reveal the
central role played by PQS as the primary quorum sensing
signal molecule required for the healthy growth and full
virulence of P. aeruginosa. As such 4-quinolone-dependent
cell-to-cell communication, as well as the MexGHI-OpmD
pump, make attractive targets for the development of novel
anti-pseudomonal drugs.
ACKNOWLEDGEMENTS
This work was supported in part by an IWT fellowship and a Marie
Curie training fellowship (to S. A.), and by a grant (QLK3-2000-01759)
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Microbiology 151
P. aeruginosa antibiotics, virulence and growth
from the European Union (Vth Framework Programme) and a grant
from the Biotechnology and Biological Sciences Research Council
(UK). We would like to thank Ram Chhabra, Chris Harty and Alex
Truman for synthesizing the AHLs and PQS, and Mavis Daykin for
technical support.
Davies, D. G., Parsek, M. R., Pearson, J. P., Iglewski, B. H.,
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295–298.
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R. G. & Rahme, L. G. (2004). Analysis of Pseudomonas aeruginosa
4-hydroxy-2-alkylquinolines (HAQs) reveals a role for 4-hydroxy-2heptylquinoline in cell-to-cell communication. Proc Natl Acad Sci
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