1 Clinical Research Group, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
2 Institute of Medical Microbiology and Hospital Epidemiology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
3 German Research Centre for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany
4 Department of Microbiology, Technical University Munich, Am Hochanger 4, D-85350 Freising, Germany
5 Department of Microbiology, Institute of Plant Biology, University Zürich, CH-8008 Zürich, Switzerland
Correspondence
Mario Juhas
juhas.mario{at}mh-hannover.de
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ABSTRACT |
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INTRODUCTION |
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Evidence has accumulated over the past few years that for the expression of pathogenic traits a quorum-sensing circuitry that is operating in P. aeruginosa is of central importance (for recent reviews, see de Kievit & Iglewski, 2000; Williams et al., 2000
; Camara et al., 2002
). This form of gene regulation ensures that virulence factors are expressed in a population-density-dependent manner. Two quorum-sensing systems that rely on diffusible N-acylhomoserine lactone (AHL) signal molecules have been identified in P. aeruginosa and were shown to orchestrate expression of virulence factors and participate in the development of biofilms: the las system consisting of the transcriptional activator LasR and the AHL synthase LasI which directs the synthesis of N-3-oxo-dodecanoyl-homoserine lactone (3-oxo-C12-HSL), and the rhl system consisting of RhlR and RhlI which directs the synthesis of N-butanoyl-homoserine lactone (C4-HSL). The two systems do not operate independently as the las system positively regulates expression of both rhlR and rhlI. Thus, the two quorum-sensing systems of P. aeruginosa are hierarchically arranged with the las system being on top of the signalling cascade (Latifi et al., 1996
; Pesci et al., 1997
). This quorum-sensing cascade controls the expression of a battery of extracellular virulence factors such as exoenzymes (elastase, alkaline protease), secondary metabolites (pyocyanine, hydrogen cyanide, pyoverdin) and toxins (exotoxin A) (Passador et al., 1993
; Winson et al., 1995
), as well as the development of biofilms (Davies et al., 1998
; Yoon et al., 2002
; Hentzer et al., 2003
a). The importance of quorum sensing for the pathogenicity of P. aeruginosa has been demonstrated in a number of animal models including the neonatal mouse model of pneumonia (Tang et al., 1996
), the burned mouse model (Rumbaugh et al., 1999
), the mouse agar bead model (Wu et al., 2000
) and a Caenorhabditis elegans model (Tan et al., 1999
). In all these infection models mutants defective in quorum sensing were substantially less pathogenic than their parental strains.
An additional signal molecule, the P. aeruginosa quinolone signal (PQS), adds a further level of complexity to the quorum-sensing circuitry, as it provides a link between the las and rhl systems (Pesci et al., 1999) by modulating expression of rhlRI and lasRI (McKnight et al., 2000
; Diggle et al., 2003
). Furthermore, the quorum-sensing circuitry is subject to regulation by a number of global regulators, including Vfr (Albus et al., 1997
), GacA (Reimmann et al., 1997
), RsaL (de Kievit et al., 1999
), MvaT (Diggle et al., 2002
), RsmA (Pessi et al., 2001
) and DksA (Jude et al., 2003
). In addition to the two AHL signal receptors LasR and RhlR, the P. aeruginosa genome encodes a third LuxR homologue, QscR, which has been shown to modulate the timing of quorum-sensing-controlled gene expression by repressing transcription of lasI (Chugani et al., 2001
).
Here we report on the characterization of a fourth LuxR homologue, which we designated VqsR (virulence and quorum-sensing regulator). It is shown that inactivation of the vqsR gene abrogates the production of AHL signal molecules, decreases the production of virulence factors and reduces the pathogenicity of P. aeruginosa in a nematode infection model system. A transcriptome analysis employing the Affymetrix GeneChip technology provided clear evidence that VqsR is involved in the regulation of the expression of certain sets of virulence and quorum-sensing-controlled genes in P. aeruginosa.
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METHODS |
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RNA isolation.
Cells were harvested from dialysis bags by centrifugation at 3800 g for 2 min at 4 °C. Total RNA was extracted using a modified hot phenol method (Tao et al., 1999). The bacteria were resuspended in water and lysed at 65 °C in a mixture of 5 ml phenol (pH 5·5)/2·5 ml 2 % SDS (w/v)/30 mM sodium acetate/3 mM EDTA by vigorous shaking for 10 min. The supernatant was purified by subsequent phenol/chloroform and chloroform extractions. Nucleic acids were precipitated overnight at 20 °C with ethanol and washed with 70 % ethanol. DNA was digested by the addition of 40 U DNase I (Roche) and 20 U SUPERaseIn (Ambion) in DNase I buffer (50 mM sodium acetate, 10 mM MgCl2, 2 mM CaCl2, pH 6·5) in a total volume of 200 µl for 30 min at 37 °C. RNA purification with RNeasy columns (Qiagen) was followed by quantification of its total yield by UV absorption spectrometry. RNA integrity was monitored by 1·2 % agarose gel electrophoresis with addition of 2 % (v/v) formaldehyde as denaturing agent.
Northern blot analysis.
The denatured RNA was separated by formaldehyde/agarose gel electrophoresis and transferred to a positively charged nylon membrane (Hybond-N+) using 20x SSC buffer for 2024 h. The dried and cross-linked membrane was pre-hybridized with 10 ml hybridization buffer (50 % formamide, 5x Denhardt's solution, 5x SSC, 1 % SDS, 100 µg herring sperm DNA ml1) for 2 h at 42 °C and afterwards hybridized with 10 ml of hybridization buffer containing denatured DIG-labelled probe (615 bp of amplified vqsR sequence) for 1624 h at 42 °C in a hybridization oven. Non-specifically bound probe was removed by several washing steps with 6x SSC+3 % SDS/2x SSC+3 % SDS/0·2x SSC+3 % SDS and hybridization signals were detected by washing of the membrane with anti-DIGalkaline phosphatase (Roche) and CDP Star as described previously (Allefs et al., 1990).
cDNA generation, labelling and GeneChip hybridization.
RNA from three independent isolations was pooled to minimize the influence of handling. cDNA synthesis, fragmentation and labelling with Biotin-ddUTP were performed as described in the protocol provided by Affymetrix. Briefly, 10 µg of RNA was mixed together with random primers (Invitrogen) and 10 non-pseudomonal control transcripts (kindly provided by S. Lory and co-workers, Harvard) and incubated for 10 min at 70 °C and 10 min at 25 °C. The cDNA mixture [5x first strand buffer, 10 mM DTT, 0·5 mM dNTPs, 25 U SuperScript II µl1 (Invitrogen), 0·5 U SUPERaseIn µl1 (Ambion)] was added to the reaction mixture and incubated for 10 min at 25 °C, 60 min at 37 °C, 60 min at 42 °C and 10 min at 70 °C. RNA was hydrolysed by the addition of 1 M NaOH and incubation for 30 min at 65 °C, followed by neutralization of the reaction by the addition of 1 M HCl. cDNA was purified with a Qiaquick column (Qiagen) and quantified by UV absorption at 260 nm. The 35 µg of cDNA was fragmented with 0·5 U DNase I (µg cDNA)1 in One Phor-All buffer (Amersham Pharmacia Biotech) for 10 min at 37 °C and the reaction was stopped by 10 min incubation at 98 °C. Gel electrophoresis and staining with 2 % SYBRGreen (Molecular Probes) were performed to ensure that the majority of cDNA fragments were within the required 50200 bp range. The fragmented cDNA was biotinylated with the Enzo BioArray Terminal Labelling Kit (Affymetrix) and hybridized to GeneChips in an Affymetrix hybridization oven. The Affymetrix fluidics station was used for washing and staining of GeneChips with streptavidin, antistreptavidin, streptavidin/phycoerythrin solutions. Hybridization signal intensities were scanned using an HP GeneArray scanner.
GeneChip analysis.
Data analysis was performed with the AFFYMETRIX MICROARRAY SUITE SOFTWARE 5.0 with Affymetrix default parameters. The average microarray hybridization signal intensity was scaled to 150. Experiments were carried out in duplicate; thus, a total of two GeneChips per strain and growth condition were scanned at 570 nm with 3 µm resolution by the Affymetrix scanner. Two GeneChips for each mutant and wild-type were compared by the four-comparison survival method (Chen et al., 2000). The data were imported into the Microsoft ACCESS database and selected by Wilcoxon rank test for genes with significant changes in their expression and at least twofold differential regulation. The arithmetic mean and standard deviation were calculated. Finally, a Bonferroni correction for multiple testing was applied as a rigorous criterion for significantly changed signal intensities. Significantly (P<0·05) differentially expressed ORFs were classified using latest internet annotation from the web site of the Pseudomonas Genome Project (http://www.pseudomonas.com).
Detection and characterization of AHLs.
As a simple assay for the production of AHLs, cross-streak experiments using GFP-based AHL biosensors were performed. Two biosensors, which, depending on the components used for construction, respond to different types of AHL molecules, were employed in this study: Pseudomonas putida F117(pKR-C12), which is most sensitive for 3-oxo-C12-HSL (Steidle et al., 2001), and E. coli JM105(pJBA89), which is most sensitive for 3-oxo-C6-HSL but detects a broad range of different AHLs (Andersen et al., 2001
). The monitor strain and the respective test strains were streaked onto the plates to form a T. Following incubation at 30 °C for 24 h, the plates were illuminated with blue light using an HQ 480/40 filter (AHF-Analysentechnik) in combination with a halogen lamp (Volpi) as a light source. Visualization of GFP fluorescence was accomplished in a dark box equipped with a light-sensitive camera (Hamamatsu Photonics) and an HQ 535/20 filter (AHF-Analysentechnik). For more detailed analysis, the AHL molecules were extracted from spent culture supernatants of the strains, separated by TLC and visualized by overlaying the TLC plates with soft agar seeded with the luxAB-based AHL biosensor E. coli MT102(pSB403) (Winson et al., 1998
) or biosensor Chromobacterium violaceum CV026 (McClean et al., 1997
) as described previously (Geisenberger et al., 2000
; Shaw et al., 1997
). Bioluminescent spots indicating AHL were detected by exposure to an X-ray film. On the basis of their mobilities (RF values) and by including appropriate reference compounds, a tentative identification of AHLs present in the culture extracts was possible.
Protease and pyocyanine secretion.
In order to investigate their pyocyanine secretion ability, tested bacterial strains were grown in King's A medium, pelleted by centrifugation and the amount of pyocyanine in the supernatant was quantified by measuring the A695 value (King et al., 1954). Protease production was examined by growing the analysed P. aeruginosa strains on M9 agar plates supplemented with 0·7 % casein (Cowell et al., 2003
). Elastase production was tested using the Elastin Congo red assay (Rust et al., 1994
).
Macrophage cytotoxicity assay.
THP-1 monocytes were grown and maintained in Readymix RPMI 1640 medium with 10 % FCS (PAA Laboratories) and 0·1 % antimicrobic/antimycotic solution (Gibco-BRL) and differentiated into macrophages using phorbol 12-myristate 13-acetate (PMA; Calbiochem). Wells of a 96-well plate were seeded with 3·5x105 cells per well and incubated with 16 nM PMA for 72 h. Prior to use, cells were washed twice with RPMI 1640 medium without FCS and antibiotic/antimycotic solution (PAA Laboratories), and P. aeruginosa strains were added to the cells at an m.o.i. of 30 and subsequently incubated in HEPES-buffered RPMI 1640 medium (PAA Laboratories) at 37 °C for 90 min. Two-and-a-half micrograms of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide in 20 µl RPMI 1640 medium was added to each well and the plate was incubated for another 30 min at 37 °C. The supernatant was removed by suction and the formazan solubilized in 20 µl DMSO and mixed with 80 µl H2O. The UV absorption of the mixture was measured at A572.
Nematode killing assays.
All Caenorhabditis elegans strains were maintained under standard culturing conditions on nematode growth medium agar with E. coli OP50 as a food source (Stiernagle, 1999). Synchronized cultures and staged animals were also obtained as described by Stiernagle (1999)
. Bristol N2 (wild-type) was used throughout this study and provided by the Caenorhabditis Genetics Center (University of Minnesota, St Pauls, MN, USA). C. elegans killing experiments were performed in a liquid-medium-based system using 24-well plates (D. Jordan, L. Wiehlmann, I. Ziegler, B. Tümmler, I. Attree & I. Steinmetz, unpublished results). Nematodes were exposed to a suspension (OD650 0·5) of bacterial cells for each strain tested.
Sequence analysis.
Codon usage and the codon adaptation index were calculated as described previously (Kiewitz et al., 2002). The bendability/curvature propensity plot was calculated with the BEND.IT server (http://hydra.icgeb.trieste.it/
kristian/dna/bend_it.html), using DNase I-based bendability parameters and consensus bendability scale as described previously (Brukner et al., 1995
).
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RESULTS AND DISCUSSION |
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vqsR possesses a las box (5'-AACTACCAGTTCTGGTAGGT-3') in its 157 to 138 upstream region (Wagner et al., 2003) which exhibits homology with the palindromic lux box DNA elements identified in Vibrio fischeri. las boxes are usually located upstream of LuxR-regulated genes and serve as binding sites for regulatory proteinautoinducer complexes. A bendability/curvature propensity plot revealed a low curvature DNA sequence in the upstream region of vqsR (data not shown). Hence, binding of a proteinautoinducer complex could promote more extensive bending of the upstream sequence leading to the transcriptional activation of this gene. Moreover, vqsR harbours a DNA-binding domain in its carboxy terminus that is typical for the LuxR group of transcriptional regulators (Fuqua et al., 1996
). These in silico findings suggest that the vqsR gene plays an important role in the quorum-sensing cascade of P. aeruginosa.
VqsR is essential for the production of autoinducer molecules
Quorum sensing in P. aeruginosa employs AHLs as autoinducer signalling molecules (Pearson et al., 1994). In order to verify the effect of vqsR on AHL synthesis, we streaked the P. aeruginosa TB wild-type strain and its Tn5 : : vqsR transposon mutant close to the GFP-based AHL sensor E. coli JM105(pJBA89). No AHL production was observed with the P. aeruginosa Tn5 : : vqsR transposon mutant (data not shown). P. aeruginosa possesses two different quorum-sensing acylhomoserine lactones: 3-oxo-C12-HSL and C4-HSL, which are the major components of the las and rhl systems, respectively (Wagner et al., 2003
). Cross-streak experiments were also performed with the biosensor P. putida F117(pKR-C12), which only detects a narrow range of long-chain AHLs, being most sensitive for 3-oxo-C12-HSL. As with E. coli JM105(pJBA89), the vqsR mutant did not provoke a positive signal (Fig. 1
a, b), supporting the view that VqsR plays a crucial role in the P. aeruginosa quorum-sensing cascade. Complementation of Tn5 : : vqsR in trans was performed in order to verify that the observed phenotype of the mutant was caused by the transposon mutation and not by any other secondary genetic event. For this purpose, the gene was cloned in the shuttle plasmid pME6010. This plasmid, based on the minimal pVS1 replicon, has been demonstrated to express proteins in both E. coli and P. aeruginosa (Heeb et al., 2000
). pME6010 was transformed into E. coli and subsequently introduced into the P. aeruginosa vqsR mutant via electroporation. Complementation in trans restored the AHL molecules secretion ability of the vqsR mutant to levels comparable to the wild-type (Fig. 1a, b
). For more detailed analysis, TLC was performed. Using this sensitive technique, the vqsR mutant was confirmed to be impaired in the production of AHLs, including C4-HSL (Fig. 1c
) and 3-oxo-C12-HSL (Fig. 1d
). Hence, our experiments revealed the important role of VqsR in the initial steps of the quorum-sensing cascade: in the production and secretion of quorum-sensing autoinducers.
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However, when we examined the cytotoxicity of P. aeruginosa towards human macrophages, no difference was seen among the wild-type, the vqsR mutant and the complemented strain, indicating that under the tested conditions VqsR does not play a role in the modulation of cytotoxicity.
VqsR has an impact on virulence in a C. elegans model
The nematode C. elegans has been used as a bacterial pathogenesis model for the identification of virulence-attenuated mutants in P. aeruginosa (Mahajan-Miklos et al., 1999; Tan et al., 1999
). In fact, it has been shown that the analysis of the interaction with this invertebrate host has the potential to predict disease outcome in the mammalian host. Testing of the wild-type and its Tn5 : : vqsR transposon mutant in our C. elegans killing model revealed a significant attenuation of the vqsR mutant compared to the wild-type (Fig. 4
). The killing activity of the complemented mutant was restored to wild-type levels (Fig. 4
), indicating an important role of the vqsR gene in virulence towards C. elegans.
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P. aeruginosa GeneChips were used to investigate the effect of VqsR on global changes in the gene expression profile. Total RNA, extracted from bacterial cultures cultivated in the presence of 10 % human blood serum or 10 mM H2O2, was hybridized on the DNA microarrays in duplicate. Numerous genes were differentially regulated in the vqsR mutant compared to the wild-type strain. By applying the stringent criteria outlined in Methods, the mutation of vqsR significantly influenced the expression of 151 genes in the presence of H2O2 and of 113 genes in the presence of human blood serum (Fig. 5). Only 26 genes were significantly regulated under both conditions. The products of these genes are involved in the transport of small molecules, pyochelin biosynthesis, pyoverdin synthesis, rhamnolipid biosynthesis, hydrogen cyanide biosynthesis and protease secretion (Fig. 6
).
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Expression of a wide variety of quorum-sensing-regulated genes and virulence factors is modulated by VqsR
VqsR modulates the expression of a broad spectrum of quorum-sensing and virulence genes. Fig. 5 lists the genes whose expression was found to be significantly up- and down-regulated in the Tn5 : : vqsR mutant according to the results of our GeneChip analysis. The mutant produced significantly less mRNA species of genes that are necessary for the synthesis of quorum-sensing quinolones and AHLs and of virulence factors under control thereof (Fig. 6
). Nearly half of the remaining genes belong to the class of conserved hypotheticals of unknown function. The genes with ascribed function predominantly encode enzymes required for siderophore biosynthesis (Lamont & Martin, 2003
; Ravel & Cornelis, 2003
) or membrane proteins, the latter of which are almost all known to be involved in antimicrobial resistance (Hancock & Speert, 2000
; Lambert, 2002
; Poole, 2001
).
In the presence of serum, the vqsR mutant overexpressed genes that belong either to the category of amino acid metabolism or to the category of conserved hypotheticals, presumably just reflecting the serum as a protein-rich carbon source. Exposure to H2O2 activated genes by more than 10-fold in the mutant that are either conserved hypotheticals or encode components of anaerobic energy metabolism. Nitrite and nitrate respiration have been reported to be repressed by quorum sensing (Wagner et al., 2003).
In summary, the disruption of vqsR repressed the expression of genes that are known to be promoted by quorum sensing and activated the expression of genes that are known to be repressed by quorum sensing. Moreover, the vqsR mutant harboured less mRNA transcript for the production of siderophores and membrane-bound elements of antibiotic resistance. Besides the cytotoxicity caused by elements of the type III secretion system (Frank, 1997), antibiotic resistance and extracellular virulence factors are the major determinants for morbidity and prognosis of infections with P. aeruginosa in humans. The protein encoded by PA2591 regulates several traits of pathogenicity; hence, we assigned the name vqsR (virulence and quorum-sensing regulator) to PA2591.
Inactivation of vqsR abrogated the production of AHLs and decreased LasI mRNA by about 10-fold with LasR mRNA levels being unaffected. In addition, vqsR contains a las box in its upstream region. Considering these data, we should like to conclude that vqsR is an essential element of the quorum-sensing hierarchy whose inactivation disrupts the production of AHLs, including 3-oxo-C12-HSL and C4-HSL. On the other hand, the presence of the las box located upstream of vqsR places this gene under the direct control of the regulatory LasR3-oxo-C12-HSL complex, thus creating an autoinducer feedback loop. This hypothesis is also supported by the GeneChip experiment performed by Wagner et al. (2003) where transcription of the vqsR gene (PA2591) was found to be reduced 5·7-fold in the P. aeruginosa mutant strain deficient in 3-oxo-C12-HSL and C4-HSL synthesis. As 3-oxo-C12-HSL and C4-HSL are involved in the initiation of quorum sensing, the disruption of 3-oxo-C12-HSL and C4-HSL production and secretion in the vqsR mutant inevitably has a negative effect on the whole quorum-sensing cascade. Indeed, our GeneChip experiments revealed significant down-regulation of the whole battery of quorum-sensing-regulated genes in the vqsR transposon mutant. Correspondingly, the mutant was compromised in phenotypic traits that are under quorum-sensing control such as elastase secretion and virulence in the C. elegans infection model. In summary, VqsR is a new member of the LuxR family that is involved in cell-to-cell communication and virulence of P. aeruginosa.
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ACKNOWLEDGEMENTS |
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Received 12 November 2003;
revised 13 January 2004;
accepted 20 January 2004.