1 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
Correspondence
Miguel Cámara
miguel.camara{at}nottingham.ac.uk
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ABSTRACT |
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INTRODUCTION |
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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
).
We have previously described a genetic locus (mexGHI-opmD), 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 multi-drug 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-quinolone-dependent 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.
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METHODS |
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TLC assays for C4-HSL and 3-oxo-C12-HSL detection.
Acidified supernatants from 18 h cultures (OD600 2) in LB at 37 °C 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-oxo-C12-HSL was detected using the AHL biosensor E. coli(pSB1075) (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 extracellular 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 µl 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 µl methanol. Ten-microlitre 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 °C 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 µl of a 10 mM stock).
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 µg per disk), tetracycline (30 µg), nalidixic acid (30 µg), chloramphenicol (30 µg), kanamycin (30 µg), rifampicin (15 µg) and carbenicillin (30 µg). Three millilitres of a cell suspension (5x106 c.f.u. ml1) 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 °C. 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 °C for 48 h. Pyocyanin was extracted from the agar medium and quantified according to Mavrodi et al. (2001).
Measurement of bioluminescence.
Bioluminescence was measured as a function of cell density using a combined automated luminometerspectrophotometer (LUCYI, Anthos Labtech). Overnight cultures of the P. aeruginosa lecA' : : luxCDABE reporter gene fusion were diluted to OD600 0·01 in LB and 200 µl of this dilution was added to each well of a LUCY plate. Where required, PQS (60 µM) or C4-HSL (100 µM) 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.
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
.
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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 °C with shaking for 24 h. Bacteria were harvested by centrifugation and embedded in alginate beads. Alginate beads (100 µl) 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 1x108 c.f.u. ml1, and for the mexI mutant 1·5x109 c.f.u. ml1. 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 °C, and washed in 10 mM MgSO4. Cells were then diluted to 107 and 108 c.f.u. ml1. 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 µM) was either co-injected with the bacteria or injected on its own. The plates were kept in a growth chamber at 28 °C and symptoms monitored daily for 5 days.
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RESULTS |
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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 mutations into a PAO1 strain harbouring a lecA' : : 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 lecA' : : lux expression to wild-type levels (Fig. 2
). Similar results were obtained with the ompD mutant in the lecA' : : 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 C4-HSL to these mutants further enhanced lecA' : : 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
).
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DISCUSSION |
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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 mexEF-oprN 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)
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 lecA' : : 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., 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 catecholvanadium 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 N-formylkynurenine, 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 (Resistance-Nodulation-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 MexGHI-OpmD efflux pump enhances the resistance of P. aeruginosa to a variety of antibiotics including
-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
-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 mexGHI-opmD 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.
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ACKNOWLEDGEMENTS |
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Received 20 September 2004;
revised 20 December 2004;
accepted 10 January 2005.
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