1 Australian Proteome Analysis Facility, Level 4, Building F7B, Macquarie University, Australia 2109
2 ARC Special Research Centre for Functional and Applied Genomics, Institute for Molecular Bioscience, The University of Queensland, Australia 4072
3 Department of Microbiology, Colorado State University, Fort Collins, Colorado 80523-1677, USA
Correspondence
Stuart J. Cordwell
s.cordwell{at}proteome.org.au
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ABSTRACT |
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Present address: Children's Medical Research Institute, Westmead, Australia 2145.
Present address: Department of Human Anatomy and Genetics, University of Oxford, Oxford, UK.
Present address: Department of Medicine, Division of Infectious Diseases, University of California, San Francisco, CA 94143, USA.
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INTRODUCTION |
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The las and rhl QS systems are also connected, since the LasRAHL complex regulates expression of the lasI gene (Seed et al., 1995), and also the entire rhl QS system via positive transcription of rhlR (Latifi et al., 1996
; Pesci et al., 1997
). Therefore, mutants lacking a functional las system cannot fully activate the downstream genes regulated by the rhl system. Genes regulated by QS in P. aeruginosa contain specific promoter regions to which the regulatory proteinAHL complex binds (Whiteley et al., 1999
; Whiteley & Greenberg, 2001
). These promoter regions are referred to as lux-boxes, based on the discovery of the promoter in the luxI gene from a comparable QS system in Vibrio fischeri (Devine et al., 1989
). In P. aeruginosa, QS has been proposed as a global mechanism for controlling virulence factor expression and the development of biofilms (Davies et al., 1998
). Several genes are regulated by QS, including toxA (exotoxin A; Gambello et al., 1993
), lasB (elastase; Brint & Ohman, 1995
; Latifi et al., 1995
; Passador et al., 1993
; Pearson et al., 1997
) and lasA (LasA protease; Toder et al., 1991
), aprA (alkaline metalloproteinase; Gambello et al., 1993
; Latifi et al., 1995
), rhlAB, encoding proteins involved in the synthesis of rhamnolipid (Ochsner et al., 1994
; Pearson et al., 1997
), katA and sodA (catalase and superoxide dismutase; Hassett et al., 1999
), lecA (lectin; Winzer et al., 2000
) and genes involved in pyocyanin formation (Brint & Ohman, 1995
; Latifi et al., 1995
) and twitching motility (Glessner et al., 1999
). However, studies of QS-regulated gene expression, utilizing las and/or rhl mutants, may be inaccurate due to the accumulation of secondary mutations in global regulators such as vfr and algR that are directly responsible for some of these phenotypes (e.g. twitching motility; Beatson et al., 2002b
).
Mutants lacking genes of the las and/or rhl systems are significantly less virulent towards host cells (Tang et al., 1996; Wu et al., 2001
), and this appears to be independent of surface colonization (Reimmann et al., 2002
). As QS is cell-density-dependent, regulation of virulence factors by this mechanism may allow the organism to mount an attack only when the number of cells present is sufficient to overwhelm the host. Therefore, QS systems and the genes they regulate represent potential targets for new antimicrobial agents (Dong et al., 2001
). Furthermore, the las QS system is positively regulated by the vfr (virulence factor regulator) gene-product, a homologue of the cyclic AMP receptor protein (CRP) from Escherichia coli (Albus et al., 1997
; Beatson et al., 2002a
), which positively regulates the transcription of lasR. Similarly, the rhl QS system is subject to regulation by other systems, such as GacA, a two-component response regulator (Reimmann et al., 1997
), MvaT (Diggle et al., 2002
) and by the Pseudomonas quinolone signal (PQS) molecule (McKnight et al., 2000
; Calfee et al., 2001
).
The P. aeruginosa PAO1 genome has been fully sequenced and annotated (Croft et al., 2000; Stover et al., 2000
). Nearly 50 % of the open reading frames (ORFs) have no known function. Transcriptomics and proteomics (reviewed by Harrington et al., 2000
; Cordwell et al., 2001
) can be applied to gauge the expression of mRNA and protein gene-products in response to altered genetic or biological conditions. Both approaches have recently been used to monitor the molecular basis of biofilm development in P. aeruginosa (Whiteley et al., 2001
; Sauer et al., 2002
). The effects of QS on gene expression have not yet been fully determined, although it has been hypothesized that approximately 4 % of the P. aeruginosa genome (over 200 genes) may be QS-regulated (Whiteley et al., 1999
). Therefore, global approaches such as transcriptomics and proteomics are the most appropriate ways to examine the influence of QS and to identify novel QS-regulated genes.
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METHODS |
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Two-dimensional gel electrophoresis (2-DE).
Proteins were solubilized in a 2-DE compatible buffer, consisting of 5 M urea, 2 M thiourea, 2 % (w/v) CHAPS, 2 % (w/v) sulfobetaine 310, 2 mM tributyl phosphine, 1 % (v/v) pH 310 carrier ampholytes (Bio-Rad), 40 mM Tris and a trace amount of bromophenol blue. The mixture was vortexed briefly, and centrifuged (20 000 g, 10 min at 20 °C) to remove any insoluble material prior to isoelectric focusing. Proteins were loaded by in-gel rehydration on 17 cm pH 47 immobilized pH gradient (IPG) strips (Bio-Rad) using a maximum volume of 500 µl (500 µg protein ml-1). pH 611 IPGs (Amersham Biosciences) were rehydrated in sample solution for a minimum of 6 h. An aliquot of the protein sample (100 µl; 1 mg ml-1) was cup-loaded at the acidic end of the IPG prior to focusing. Isoelectric focusing was conducted as described previously (Nouwens et al., 2000). For pH 47 gradients, proteins were focused for a total of 25 kV h. For pH 611 gradients, proteins were focused for a total of 60 kV h. Following isoelectric focusing, IPG strips were reduced, alkylated and detergent-exchanged, and second dimension SDS-PAGE was carried out as described previously (Nouwens et al., 2000
). Gels were fixed in 40 % (v/v) methanol/10 % (v/v) acetic acid for 1 h, and then stained with Sypro Ruby (Molecular Probes) overnight. Gels were de-stained for 2 h in 10 % (v/v) methanol/7 % (v/v) acetic acid and imaged using a Bio-Rad Molecular Imager Fx. After scanning, gels were double-stained using Coomassie G-250 (Cordwell, 2002
). For comparative analyses, statistical data were acquired using PD-QUEST Version 7.0 (Bio-Rad) and Z3 (Compugen) as described previously (Cordwell et al., 2002
). Briefly, proteins were acquired from separate duplicate cultures and each gel was run in duplicate. Statistical analyses were performed on triplicate gels.
Peptide mass mapping.
Protein spots were excised and placed in a 96-well microtitre plate. Gel pieces were washed three times with 120 µl of 25 mM ammonium bicarbonate (pH 7·8)/50 % (v/v) acetonitrile and dried using a SpeedVac (Savant Instruments) centrifuge for 20 min. Gel pieces were rehydrated with 8 µl of porcine modified trypsin (Promega, 10 ng µl-1) in 25 mM ammonium bicarbonate (pH 7·8) and left for 10 min at room temperature. Once gel pieces were rehydrated, proteins were digested at 37 °C for 16 h. Peptides were extracted from the gel in 8 µl of 50 % acetonitrile/1 % trifluoroacetic acid (TFA) by sonication (10 min). A small aliquot (1·0 µl) of each extract was spotted onto a target plate, and 1 µl of -cyano-4-hydroxycinnamic acid (Sigma, 8 mg ml-1 in 50 % acetonitrile/1 % TFA) was placed on top and allowed to dry. Trypsin, wash, extraction and matrix solutions were delivered to the microtitre and target plates using a Multiprobe 104 robotic system (Packard Instruments). Peptide mass maps of tryptic peptides were generated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) using a Micromass TOF-Spec 2E. All spectra were obtained in reflectron mode using an accelerating voltage of 20 kV. Mass calibration was performed using [Glu1] Fibrinopeptide B, 1570·68 [M+H+], as an external standard.
Database searching and protein identification.
Data generated from peptide mass maps were compared to the complete translated ORFs for P. aeruginosa PAO1 (http://www.pseudomonas.com) using PROTEINLYNX software (Micromass). The parameters for a successful identification included a mass tolerance of 150 p.p.m. per peptide and a minimum of three matching peptides. Generally, a set of matching peptides leading to a total sequence coverage of greater than 30 % was required to provide further confidence in the obtained match, although low and high mass proteins, and those that are the result of protein fragmentation, may not always conform to this rule. For proteins generating poor spectra, a concentration and desalting step as described in Gobom et al. (1999) was used and the concentrated peptides were again characterized by MALDI-TOF MS. Sequences from identified proteins were examined for a predicted signal sequence using the program SIGNALP (http://www.cbs.dtu.dk/services/SignalP/).
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RESULTS |
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Comparative 2-DE of extracellular proteins
Extracellular proteins from all strains of P. aeruginosa were solubilized and separated using 2-DE across the pH ranges 47 and 611. This provided an effective separation width of 35 cm in the first dimension with a single pH unit overlap between pH 6 and 7 (Fig. 1). The major extracellular proteins of strain PAO1, based on summed spot intensities, were elastase (lasB), LasA protease (lasA) and an aminopeptidase (PA2939). LasB appeared to be extensively processed, with 11 spot variants' visible on 2-D gels. These included a major region corresponding to the mature 33 kDa enzyme (spots 1.11.6 in Fig. 1
). Proteins from stationary phase culture supernatants were used to perform comparative 2-DE. Image and statistical analyses, using the PD-QUEST and Z3 software packages, were performed to detect differences in protein expression between wild-type and mutant strains (Fig. 2
). An increase or decrease in visible spot intensity of greater than twofold, averaged over three gels and normalized using the intensities of 10 spots from each pH range with no apparent change in abundance, defined the selection of differentially expressed proteins.
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Flagellar hook-associated protein (flgK) was absent from PAO1 culture supernatants but was present in all other strains. However, rhlI,
rhlR and
rhlRI showed a different cluster of spot variants corresponding to FlgK than were detected in strains containing a non-functional las system (Fig. 4
). Furthermore, the las deletion FlgK variant was also present in
las/
rhl combined mutants. A cluster of spots, identified as fragments of type B flagellin (fliC), was only present on 2-D gels from strains deficient in the las system. Lipase (lipA) was not present in wild-type or
rhl deletion strains; however, two major LipA variants were present in all strains that contained a las deletion, including those that also contained rhl deletions (Fig. 5
). A 95 kDa fragment of PA0041 (two-partner secretion exoprotein) was only present in strains that did not contain a functional regulator of either QS system (
lasR,
lasRI,
rhlR,
rhlRI,
lasR
rhlR and
lasRI
rhlRI).
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DISCUSSION |
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The products of 13 genes were present at reduced extracellular abundance in our QS mutants, including known QS-regulated genes such as lasB, lasA (Toder et al., 1991) and aprA (Gambello et al., 1993
), as well as genes not previously associated with QS (including PA2939 and prpL). Other well-characterized QS-regulated extracellular virulence determinants most probably require specific interactions with host factors for their expression and were not identified here. PA2939 encodes a recently characterized, secreted, zinc-dependent metalloprotease (Cahan et al., 2001
). Culture supernatants derived from mutants of the las QS system (
lasR,
lasI,
lasRI) and
vfr did not contain any of the major isoforms of this protein. PA2939 contains a putative signal peptide indicating that it is secreted via the Xcp-mediated apparatus and
xcp mutants appear to lack PA2939 in their culture supernatants (Braun et al., 1998
). Several xcp genes are regulated by QS (Chapon-Herve et al., 1997
), suggesting the loss of PA2939 in
las mutants may be due to defective or inhibited secretion. However, PA2939 levels were unaffected in
rhl mutants, despite the role of the rhl QS system in regulating Xcp-mediated secretion. Furthermore, several other proteins secreted via the Xcp machinery could be detected in the culture supernatants from our
las mutants. We suggest, therefore, that a reduction in Xcp-mediated secretion is not solely responsible for the absence of PA2939 in
las mutants. Furthermore, preliminary analysis of membrane and cytosolic protein fractions from these mutants did not reveal an intracellular increase in PA2939 levels consistent with inhibited secretion (data not shown).
Aminopeptidases, such as PA2939, act by generating free amino acids from short peptides under nutrient limitation. Any linkage of this protein to QS-regulated virulence may be via the complementary role of endoproteinases, which cleave host-surface proteins into smaller peptides. PA4175 contains sequence similarity to bacterial endoproteinases and was recently renamed PrpL (PvdS-regulated endoprotease; Wilderman et al., 2001). PrpL is able to cleave lactoferrin, transferrin, elastin and casein, suggesting that it may have a role in virulence. prpL is under the transcriptional control of PvdS, an alternate sigma factor that regulates genes involved in the production of pyoverdine (Cunliffe et al., 1995
; Leoni et al., 2000
; Wilson et al., 2001
). PrpL was present in reduced abundance or absent from the culture supernatants of strains containing a
las mutation, but was unaffected in
rhlI,
rhlR and
rhlRI. Several pyoverdine synthetases appear to be QS-regulated (Whiteley et al., 1999
); however, it is unknown whether QS directly regulates the expression of these genes, or whether it does so via an intermediary factor. The reduction of PrpL levels seen here could reflect direct QS regulation of prpL or a secondary effect of QS on regulators, such as pvdS.
Two apparently QS-regulated proteins (PA0572 and PA3611), which were previously designated as hypothetical, were detected. PA0572 is absent from strains containing a las mutation, yet is present in strains containing only a
rhl mutation, while PA3611 is only present in the wild-type. PA0572 contains some similarity to zinc metalloproteinases (S. A. Beatson, personal communication). Three other proteins (azurin, CbpD and PA4944) were detected only in wild-type supernatants. Chitin-binding protein (CbpD) has adhesin-like properties and is found primarily in clinical isolates of P. aeruginosa (Folders et al., 2000
). The 43 kDa mature form of CbpD is protected from the proteolytic activity of elastase when bound to chitin. Elastase cleaves CbpD into smaller products (including 30 and 23 kDa forms) that are incapable of binding chitin (Folders et al., 2000
). The 30 and 23 kDa products were both identified here, which is not surprising since no specific efforts to inhibit elastase activity were undertaken. Regulation of cbpD by QS has been characterized by Whiteley et al. (1999)
, who showed that the loss of either the las or rhl system reduced cbpD transcription to 15 % (
las) and 1 % (
rhl) of that measured in the wild-type. We were unable to observe CbpD in any mutants suggesting that (i) the reduction in cbpD transcription results in levels of CbpD that are too low to be detected by fluorescence staining of 2-DE separated proteins and/or (ii) the secretion system required to export CbpD also requires both QS systems. All mutants were deficient in the expression of PA4944, a protein with significant sequence similarity to host factor I (HF-I), an RNA-binding protein that regulates enterotoxin production in Yersinia enterocolitica (Nakao et al., 1995
) and several virulence factors in Brucella abortus (Robertson & Loop, 1999
). Previous reports have suggested that genes under the control of QS may also be regulated by other factors, including the alternative sigma factors RpoS (Van Delden & Iglewski, 1998
) and PvdS (Ochsner et al., 1996
). RpoS may regulate QS itself via repression of rhlI transcription post-stress (Whiteley et al., 2000
). PA4944 may be another protein in this family of regulators.
PA0807 (N-acetylmuramoyl-L-alanine amidase), PA0040 and PA4233 (a hypothetical protein with similarity to multi-drug efflux transporters) low-mass fragments were present in culture supernatants derived from PAO1, but were not present in culture supernatants derived from the mutant strains. Since they are membrane-associated, rather than truly secreted proteins, and since we could not identify the whole product of any of these fragments, we cannot distinguish between the possibility that the genes encoding these products are truly regulated by QS, or that the proteins themselves are the substrates of QS-regulated extracellular proteases. PA0040 and PA0041 are linked together both genetically and functionally (Bordetella pertussis filamentous haemagglutinin-like two-partner secretion transporter and exoprotein, respectively; Croft et al., 2000; Jacob-Dubuisson et al., 2001
), yet they display different regulation here. A possible hypothesis for these apparently conflicting data is that the transporter (PA0040) is enzymically processed in strain PAO1 (hence the presence of a PA0040 fragment) by QS-regulated proteases (e.g. LasB). In QS mutants, this processing might not occur, allowing the complete transporter to export substantially greater amounts of the exoprotein (PA0041). Increased levels of a second two-partner secretion exoprotein, PA4625, were also detected in several QS mutants.
QS has not previously been reported to negatively regulate gene expression in P. aeruginosa; however, such regulation has been shown in Yersinia paratuberculosis (Atkinson et al., 1999). In our study, several proteins appeared only in QS mutant strains. The majority of these proteins belonged to the Gram-negative outer membrane or periplasm (FlgK, FliC, PhoA, AotJ, DsbA and GlpQ). The possibility that some of these proteins are negatively regulated by QS must take into account the following arguments: (i) the reduction in abundance of truly secreted proteins (e.g. LasB) in QS mutants enhances their detection by 2-DE; (ii) that QS mutations lead to structural damage to the outer membrane causing hyper-release of these proteins; and (iii) that QS-regulated proteases are absent in QS mutants allowing these proteins a longer half-life in the culture supernatant following cell autolysis. However, QS-regulated genes that encode for products in the V. fischeri periplasm have been determined previously (Callahan & Dunlap, 2000
).
The differences in electrophoretic mobility for FlgK and the absence of FliC fragments in the wild-type and rhl mutants suggest an involvement of QS in P. aeruginosa swimming and/or swarming (Köhler et al., 2000
). Both
las and
rhl mutants display defective swarming (Köhler et al., 2000
) and this may partly be due to effects on flagella and pili; however,
rhl mutants remain capable of swimming and thus the negative swarming phenotype may be independent of effects on flagella. Furthermore, exogenous AHL destruction in the presence of P. aeruginosa does not affect flagella or twitching motility (Reimmann et al., 2002
). Lipase (lipA) is regulated by the rhl QS system (Reimmann et al., 1997
) and was not detected in wild-type extracellular fractions, or in those from any
rhl mutant, but was clearly identified as two major spots in supernatants derived from all
las mutants, including those that also contained a
rhl mutation. This result suggests that lipA is negatively regulated by the las system or that the las system represses a downstream inducer of lipA. The net effect of this lipAlas interaction is stronger than the positive control of the rhl system on lipA. Our results appear to conflict with those seen by Reimmann et al. (1997)
, who have shown that increased levels of RhlR and LasR result in enhanced levels of LipA; however, this may be a result of increased RhlR and independent of the presence of LasR.
This study has been the first to examine the role of QS in P. aeruginosa extracellular protein production using a proteomics approach. Similar studies conducted on whole cells of V. fischeri (Callahan & Dunlap, 2000) discovered five novel QS-regulated proteins. We examined only the extracellular fraction with the aim of discovering potential QS-regulated secreted virulence factors. The products of 13 genes, including seven not previously associated with QS, were identified. We conclude that the role of QS in P. aeruginosa gene expression is yet to be fully understood and that further global analyses, such as proteome analysis of intracellular and membrane-associated proteins, and transcriptomics will be needed to shed further light on this complex regulatory mechanism.
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ACKNOWLEDGEMENTS |
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Received 29 August 2002;
revised 10 January 2003;
accepted 14 January 2003.