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1 Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, VA, USA
2 Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Charlottesville, VA, USA
Correspondence
Joanna B. Goldberg
jbg2b{at}virginia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Two supplementary tables showing genes differentially expressed between the P. aeruginosa strains studied and QS-regulated genes whose expression was upregulated in strain 383, two supplementary figures showing functional classes of the differentially expressed genes and a sequence alignment between PA0122 and aegerolysin family proteins, and additional details of the methods used to analyse PA0122 are available with the online version of this paper.
The array data discussed in this publication have been deposited in the NCBI Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series accession number GSE9621.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). P. aeruginosa strains that initially infect CF patients have phenotypes similar to those found in the environment, which are characterized by having a non-mucoid phenotype. During colonization of the CF lung, P. aeruginosa undergoes a number of morphological changes (Smith et al., 2006), the most obvious of which is the conversion to a mucoid phenotype that is apparent in up to 90 % of the P. aeruginosa isolated from chronically infected patients (Doggett, 1969; Govan & Deretic, 1996). Mucoidy is due to the overproduction of the exopolysaccharide alginate.
Alginate production appears to protect P. aeruginosa from environmental stresses such as dehydration, as well as from the host immune system (Parsek & Singh, 2003). Mucoid strains can adopt a biofilm mode of growth and mucoid biofilms are significantly more resistant to the antibiotic tobramycin than isogenic non-mucoid biofilms (Hentzer et al., 2001). The overproduction of alginate also enhances adhesion to tracheal epithelial cells (Marty et al., 1998). In addition, this exopolysaccharide inhibits opsonic phagocytosis (Pier et al., 2001) and promotes resistance to reactive oxygen molecules (Borriello et al., 2004; Simpson et al., 1989). There is evidence that increased oxidative stress and enhanced lipid peroxidation activity in the CF lung result in the oxidation of membrane phospholipids and low-density lipoprotein (LDL) (Winklhofer-Roob et al., 1995; van der Vliet et al., 1997). These conditions favour the chronic state of infection in CF patients (Ramsey & Wozniak, 2005). Apart from an increase in alginate production that accompanies the conversion from the non-mucoid to the mucoid phenotype, there is also a decrease in expression of various virulence factors, including flagella, pili, the lipopolysaccharide O antigen and pyocyanin (Luzar et al., 1985; Hancock et al., 1983; Mahenthiralingam et al., 1994; De Vos et al., 2001).
The genome sequence of the prototype P. aeruginosa strain PAO1 has been determined (Stover et al., 2000). It is estimated that over one-third of the genes in the P. aeruginosa genome encode ‘hypothetical’ or ‘conserved hypothetical’ proteins. These proteins are recognized by the presence of an ORF that is predicted to encode a protein of unknown function that is either unique to an organism or present among various species.
The availability of the genome sequence along with microarray technology has allowed the detection of global gene expression changes in P. aeruginosa strains under various conditions (Chugani & Greenberg, 2007; Lizewski et al., 2004; Wolfgang et al., 2004; Firoved et al., 2002; Ochsner et al., 2002). Several groups have identified differences in gene expression between mucoid and non-mucoid strains of P. aeruginosa (Bragonzi et al., 2005; Lizewski et al., 2004; Firoved & Deretic, 2003). Others have utilized microarray technology to monitor the expression of genes associated with quorum sensing (QS) (Wagner et al., 2003; Schuster et al., 2003), interactions with host cells (Frisk et al., 2004; Ichikawa et al., 2000; Firoved et al., 2004) and biofilm formation (Bagge et al., 2004; Whiteley et al., 2001).
Our laboratory has previously reported a proteomic characterization of two genetically similar but phenotypically distinct strains of P. aeruginosa (Hanna et al., 2000). These strains were isolated from the sputum of a single CF patient and had phenotypes associated with early (383, non-mucoid) and late (2192, mucoid) stages of disease. We have extended our previous observations to the genomic level using P. aeruginosa GeneChip microarray technology and focused on hypothetical and conserved hypothetical proteins that show differential expression between these two strains. In the present study, we analysed a gene, PA0122, which has been recognized by others as being QS-regulated (Wagner et al., 2003; Schuster et al., 2003). We show that expression of the PA0122 gene and protein is growth phase-dependent and that it is more abundantly expressed in the non-mucoid strain 383 than in the mucoid strain 2192. We also report that PA0122 is a novel oxidized LDL (Ox-LDL) and lysophosphatidylcholine (lysoPC) binding protein in P. aeruginosa.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. P. aeruginosa strains 383 and 2192 are non-mucoid and mucoid, respectively. They were isolated 2 days apart from the sputum of the same CF patient (Hanna et al., 2000). Non-mucoid derivatives (nmd) of mucoid strains were isolated from their respective mucoid strains by repeated passage in static Luria–Bertani (LB) broth at room temperature followed by plating on tryptic soy agar (TSA) plates. Escherichia coli DH5
(Invitrogen) cells were used as recipients for plasmid constructs. For selection in E. coli, 50 µg ml–1 kanamycin (Km), 50 µg X-Gal ml–1 and 1 mM IPTG were added as required.
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The expression of the P. aeruginosa PA0122 gene was examined by qRT-PCR. Total RNA was isolated from strain 383 and strain 2192 cultures at different phases of growth, as well as from other mucoid strains and their nmd at OD600 0.5. qRT-PCR was performed using SYBR Green 1 chemistry in an Applied Biosystems 7900 HT Sequence Detection System. Target sequences were obtained from Affymetrix (https://www.affymetrix.com/analysis/netaffx/). Primers were designed using Primer Express (Applied Biosystems). For PA0122, the following primers were used: forward, 5'-ACCCAGGGCAGCTTCGA-3', and reverse, 5'-GGGTCATCCCAGCTGAAGGT-3'. Relative abundance was normalized against PA1777 (OprF) using forward primer 5'-CAGCTGGACGTGAAGTTCGA-3' and reverse primer 5'-GAAGTCAGCCAGGTTCTTGATGT-3'.
PA0122 cloning and recombinant protein expression in E. coli.
The PA0122 gene was amplified by PCR using genomic DNA from strain 383 as a template. Genomic DNA was isolated as previously described (Goldberg & Ohman, 1984). The primers (PA0122-HisF, 5'-GTTAACGGAATTCGACATGGCATACGCAG-3'; PA0122-HisR, 5'-GCTCCAGGTACTCGAGGGAGAAGCGGCCG-3') were designed using the sequence of PA0122 from the P. aeruginosa PAO1 genome (http://www.pseudomonas.com). To facilitate cloning into the pET-28b (+) expression vector (Novagen), the PA0122-HisF (forward primer) and PA0122-HisR (reverse primer) were engineered with EcoRI and XhoI restriction sites (indicated by bold type). After PCR amplification, samples were separated by 0.7 % agarose gel electrophoresis and stained with ethidium bromide. The resultant 0.45 kb DNA fragment was excised and purified using the QIAquick Gel Extraction kit (Qiagen) and cloned into the pCR2.1-TOPO vector (Invitrogen), as described by the manufacturer's protocol. TOPO-ligated product was transferred into DH5 cells, and colonies were selected on LB agar containing Km, X-Gal and IPTG. Plasmids purified using the QIAprep Spin Miniprep kit (Qiagen) were digested with EcoRI and XhoI, and the 0.45 kb fragment was isolated and inserted into pET-28b(+). The resultant construct was transformed into DH5, then selection for Km-resistant colonies was performed. Plasmids isolated from transformations were designated pET-PA0122-his. The presence of the nucleotides encoding six histidine residues at the 3' end of the gene (his-tag) in pET-PA0122-his was confirmed by DNA sequencing at the University of Virginia Biomolecular Research Facility. Plasmids were transferred into NovaBlue (DE3) strains of E. coli (EMD Biosciences Novagen), which was used as an expression host.
For expression in E. coli, a single colony of NovaBlue (DE3) containing pET-PA0122-his was inoculated into LB broth and grown to OD600 0.5. Cells were induced with 1 mM IPTG and harvested after reaching OD600 1.0. After IPTG induction, the total lysate of E. coli containing pET-PA0122-his was separated by SDS-PAGE and blotted onto nitrocellulose membranes. An expressed protein of 
16 kDa was detected with Ni-NTA-peroxidase, indicating the presence of the his-tag (data not shown).
The purification of the his-tagged recombinant PA0122 (r-PA0122) protein was carried out under native conditions by gravity-flow using BD TALON immobilized metal affinity chromatography (IMAC) resin, following the manufacturer's specification (BD Biosciences Clontech). The purified r-PA0122 protein was quantified by the method of Bradford (1976) (Bio-Rad), while the purity of the protein was assessed by using SDS-PAGE gels stained with Coomassie blue R-250 or silver (Chen et al., 1993; Shevchenko et al., 1996). Purified r-PA0122 protein was used for immunization and binding assays.
Polyclonal r-PA0122 antibody production and characterization.
Polyclonal antibody was raised against r-PA0122 in 6–8 week old BALB/c mice (Jackson Laboratories). Prior to immunization, blood samples were collected from each animal through the tail vein and separated serum (pre-immune serum) was stored at –20 °C. Primary immunization was carried out with 100 µg r-PA0122 protein emulsified with Freund's complete adjuvant (1 : 1, v/v) and administered subcutaneously. Two subsequent boosters consisting of 50 µg r-PA0122 emulsified in incomplete Freund's adjuvant (1 : 1) were administered at intervals of 21 days. Ten days after the final booster, blood was collected from the tail vein and separated serum was stored at –20 °C. Animal immunizations were conducted with the approval of the Animal Research Committee at the University of Virginia School of Medicine in accordance with Institute of Laboratory Animal Resources Commission on Life Sciences (1996) and other relevant publications.
For some Western blot analyses, pooled anti-r-PA0122 mouse polyclonal antiserum was adsorbed with NovaBlue (DE3) cells containing the pET-28b(+) cloning vector. For this, 4 ml E. coli grown in LB was pelleted and resuspended in 4 ml PBS and sonicated. Cell lysate (1 mg ml–1) was mixed with antiserum, diluted 1 : 50 and adsorbed overnight at 4 °C, followed by pelleting at 20 800 g. This step was repeated twice. The resulting supernatant was filtered using a 0.22 µm pore-size syringe filter (Millipore). A final dilution (1 : 2500) of adsorbed antiserum was made in killer filler (KF) reagent (1.8 l 1x PBS with 200 ml 0.1 M NaOH, 10 g casein and 10 g BSA, adjusted to pH 7.4; 0.2 g phenol red and 3.6 g sodium azide were then added).
Total proteins from whole-cell lysate.
A 1 ml volume of an overnight culture, or of cultures at different cell densities, was pelleted, resuspended and sonicated with Tris-EDTA buffer (50 mM Tris, pH 8.3, 5 mM EDTA) containing 50 µl of a cocktail of bacterial protease inhibitors (Sigma) and 1 µl benzonase (Sigma; 5000 U ml–1). Cell debris was removed by centrifugation at 20 800 g for 10 min. Protein concentrations were determined by Bradford assay.
Analysis of supernatant proteins.
Supernatants were collected from cultures grown in LB medium at different cell densities by centrifugation at 3220 g for 15 min, and filtered through 0.22 µm pore-size filters. The supernatant was added to cold acetone (1 : 10) and allowed to stand overnight at –20 °C. Acetone-precipitated proteins were centrifuged at 3220 g for 15 min. Precipitated protein pellets were resuspended in 1.0 ml 50 mM Tris-EDTA (pH 8.3) buffer containing a cocktail of bacterial protease inhibitors and dialysed exhaustively against Tris-EDTA at 4 °C. Protein samples were concentrated to 100 µl using Amicon centrifugation devices (Millipore), followed by protein quantification, and stored at –20 °C.
Subcellular compartment separation.
P. aeruginosa 383 and 2192 were grown overnight at 37 °C in LB (200 r.p.m.), subcultured (1 : 100) in 200 ml LB and grown to OD600 1.5. Bacterial cells were pelleted and various subcellular compartments were isolated as described elsewhere (Hancock & Nikaido, 1978). In brief, bacterial pellets were resuspended in 15.0 ml ice-cold 10 mM Tris/HCl (pH 8.0) containing 50 µl of a cocktail of protease inhibitors and 5 µl benzonase (346 U µl–1). This suspension was disrupted by a French pressure cell press (American Instrument Company) at 18 000 p.s.i. (124 MPa) three times, and further incubated for 30 min at room temperature. The disrupted cell suspension was centrifuged at 3220 g for 15 min at 4 °C and the cell debris removed. The clear supernatant suspension was designated whole-cell extract (WEx) and subjected to ultracentrifugation at 100 000 g for 6 h in an SW41Ti rotor (Beckman) at 4 °C. The pellets were classified as total membranes (TMEx) and the supernatant as the cytosolic/periplasmic fraction; both were stored at –20 °C.
The TMEx were resuspended in 5 ml 20 % (w/w) sucrose in 10 mM Tris/HCl (pH 8.0) containing 1 mM EDTA and 200 µM DTT. Discontinuous eight-step sucrose density gradients were created in 12 ml Ultra-Clear centrifuge tubes (Beckman). Each gradient was layered with 1 ml of 75, 60, 55, 50, 45, 40 and 30 % sucrose. The 5 ml volume of 20 % sucrose that contained the membrane suspension was applied to the top of the sucrose step gradient (Skaar et al., 2002). The gradient was centrifuged using an SW41Ti rotor at 190 000 g for 46 h at 4 °C. After centrifugation, 1 ml fractions were subsequently collected from the top, and stored at –20 °C. Protein concentrations of each fraction were determined by the Bradford assay. NADH oxidase activity was used as a marker for inner-membrane fractions, while a mouse mAb to the outer-membrane protein OprF (a kind gift of Dr Robert E. Hancock, Department of Microbiology, University of British Columbia, Vancouver, Canada) was used as a marker for the outer membrane (Mutharia & Hancock, 1983).
Enzyme assay.
NADH oxidase activity was determined by spectrophotometer analysis of absorbance decreases at 340 nm (Tielker et al., 2005). Briefly, 900 µl reaction buffer (50 mM Tris/HCl, pH 7.5, containing 0.2 mM DTT and 0.12 mM NADH) was added to 100 µl of each fraction. Samples were incubated at 25 °C for 5 min and the absorbance was monitored.
SDS-PAGE and Western blot analysis.
For SDS-PAGE analysis, protein samples were mixed with Laemmli buffer (Laemmli, 1970), boiled for 10 min and separated on 10 % Tris-Tricine SDS-PAGE (Schagger & von Jagow, 1987) gels using the Criterion gel system (Bio-Rad). For Western blot analysis, proteins were transferred onto 0.2 µm nitrocellulose membranes (Bio-Rad) at 100 V for 45 min. Membranes were blocked with either 5 % (w/v) fat-free milk in PBS-T [10 mM PBS (pH 7.4) with 0.05 % Tween-20] or KF reagent for 1 h at room temperature.
Blots were probed with either unabsorbed or absorbed anti-r-PA0122 pooled mouse serum diluted in PBS-T or KF, and incubated overnight at 4 °C. Immunodetection was performed with alkaline phosphatase (AP)-conjugated goat anti-mouse IgG (Southern Biotechnology Associates) or peroxidase-conjugated rabbit anti-mouse IgG secondary antibody (Sigma) at a dilution of 1 : 5000 in PBS-T. The blots were washed three times with PBS-T followed by PBS for 5 min each. Finally, reactivity was visualized with chromogenic substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT; Sigma) for alkaline phosphatase or with TMB (3,3',5,5'-tetramethylbenzidine and 0.01 % H2O2; Kirkegaard & Perry Laboratories) for peroxidase. In some blots, the peroxidase reaction product was visualized using enhanced chemiluminescence (ECL) according to the manufacturer's protocol (Amersham). To locate outer membrane-containing fractions following sucrose gradients, blots were probed with OprF mAb (1 : 2000), followed by peroxidase-conjugated rabbit anti-mouse IgG antibody (Sigma) at a dilution of 1 : 5000 in PBS-T, and developed with ECL.
Binding of r-PA0122 protein to Ox-LDL.
Human-plasma-derived LDL was purchased from EMD Biosciences, and the oxidation of LDL was carried out with hypochlorous acid, as described previously (Coleman et al., 2004). For the binding assay, r-PA0122 and Ox-LDL were used and complex formation was detected using Western blotting, as described elsewhere (Kudo et al., 2001). In brief, various concentrations (2, 4 and 8 µg) of r-PA0122 protein were incubated with 10 µg Ox-LDL in 50 µl PBS at 37 °C for 5 h. After incubation, the reaction complex was separated by electrophoresis on a 5 % native polyacrylamide gel (Bio-Rad) using the Criterion gel system (Bio-Rad). The proteins were transferred onto 0.2 µm nitrocellulose membranes (as described above), blocked with 5 % (w/v) fat-free milk in PBS-T for 1 h at room temperature, and probed with anti-r-PA0122 antisera (1 : 5000) overnight at 4 °C on a shaker. Immunodetection was carried out using anti-mouse IgG-peroxidase (Sigma) at 1 : 5000 for 1 h at room temperature. Bound complexes and free protein were visualized by using the ECL detection kit (Amersham).
Immobilization of r-PA0122 protein on NTA and CM5 sensor chips.
Surface plasmon resonance (SPR) was performed using the BIAcore 3000 system (BIACore) to analyse interactions and make kinetic measurements between r-PA0122 protein with Ox-LDL and lysoPC (lysophosphatidylcholine; 1-hexanoyl-2-hydroxy-sn-glycero-3-phosphocholine), which is a synthetic short carbon chain (C:6), water-soluble lipid (Avanti Polar Lipids). For direct binding, an NTA sensor chip (BIAcore) was used to immobilize r-PA0122 through the C-terminal his-tag of the protein. Attachment of the ligand was performed following the manufacturer's protocol. Initially, the chip was regenerated by injecting buffer containing 0.01 % HEPES, 0.15 M NaCl, 0.35 M EDTA and 0.005 % Surfactant P20, pH 8.3, for 1 min. After washing, the system was pulsed for 1 min in 500 µM NiCl2 running/eluent buffer (0.01 % HEPES, 0.15 M NaCl, 0.35 M EDTA, 0.005 % Surfactant P20, pH 7.4) to saturate NTA with Ni2+ ions at the flow rate of 50 µl min–1. His-tagged r-PA0122 protein (5 µg ml–1) was dissolved in running buffer, injected by 1 min pulse (flow rate 50 µl min–1) and immobilized on the nickel surface.
For the interaction analysis of LDL and Ox-LDL, ligands were dissolved (5 µg ml–1) in running/eluent buffer, and injected at the flow rate of 50 µl min–1 at different time points and observed for Ox-LDL binding onto the r-PA0122 surface.
For the kinetic study, r-PA0122 protein was covalently immobilized on a research grade CM5 chip (BIAcore) through amino groups. Briefly, the chip was activated for 5 min with a mixture of N-hydroxysuccinimide (NHS) and N-ethyl-N'-dimethyl-aminopropyl-carbodiimide (EDC). The r-PA0122 his-tag protein (100 µg ml–1), dissolved in buffer containing 10 mM sodium acetate (pH 4.5), was injected over the activated chip for 5 min at 25 °C. Approximately 0.75 ng r-PA0122-his-tag protein was immobilized per chip and the unbound active sites were deactivated with 1 M ethanolamine-HCl (pH 8.5) for 5 min.
Kinetic analysis of BIAcore sensorgram data.
All kinetic data were collected from the BIAcore 3000 system at 25 °C at a flow rate of 50 µl min–1 with running buffer [10 mM HEPES (pH 7.4), 150 mM NaCl, 3 mM ZnCl2, 0.005 % Surfactant P20]. To determine the kinetic constants, Ox-LDL and lysoPC were dissolved in running buffer. Ox-LDL or lysoPC was injected to measure association (contact time with the chip was 1 min) and dissociation, which were followed in the same buffer for 5 min. After each kinetic injection, the chip was regenerated with 50 mM NaOH with 1 M NaCl. BIAcore sensorgrams were used according to a 1 : 1 Langmuir binding model, and the BIAevaluation 4.1 software package was applied to calculate kinetic constants.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Schuster et al., 2003) (see Supplementary Table S2). Three of these genes have annotated ORFs that encode ‘hypothetical’ or ‘conserved hypothetical’ proteins. Among these transcripts, we selected PA0122, a ‘conserved hypothetical’ gene, for further analysis, since it exhibited an 8.02-fold increase in expression in 383 compared to 2192, and had limited homology to previously described proteins.
qRT-PCR for the PA0122 gene
The relative gene expression of PA0122 in 383 and 2192 was confirmed by qRT-PCR, which revealed that expression was 8.8-fold higher in 383 than in 2192 (Fig. 1a). PA0122 gene expression was followed during growth in strains 383 and 2192. In 383, the highest level of PA0122 gene expression was observed at early stationary phase (OD600 1.5), and was sixfold higher than that at mid-exponential phase (OD600 0.5); the expression of PA0122 decreased at OD600 2.3. The pattern was similar for 2192, but there was comparatively less PA0122 gene expression than in 383 (Fig. 1b). These results suggest that PA0122 is regulated by growth phase in both 383 and 2192.
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). These results indicate that PA0122 is expressed at greater levels in these isogenic non-mucoid strains than in the parental mucoid strains. However, different pairs showed different levels of expression.
Analysis of the PA0122 gene product
The PA0122 gene is 411 bp long and is predicted to encode a 136 aa protein with a theoretical molecular mass of 14.58 kDa and a pI of 4.65. PA0122 had limited homology with known proteins from the protein database, but contained a motif analogous to fungal proteins classified in the aegerolysin family of proteins (see Supplementary Fig. S2). PA0122 had 42.52 % identity and 53.54 % similarity (E-value 2e–23) with the Asp-haemolysin protein from Aspergillus fumigatus (S46523) and 37.40 % identity and 53.43 % similarity (E-values 4e–13 and 1e–12) to two haemolysin-like proteins from Clostridium bifermentans (CAA71483 and CAA71484). Other similar fungal proteins were Aa-Pri1 from Agrocybe aegerita (AAC02265) with 37.8 % identity and 50.7 % similarity (E-value 5e–15), PriA from Pleurotus ostreatus (AAL57035) with 34.0 % identity and 51.5 % similarity (E-value 1e–13), and a hypothetical protein from Neurospora crassa (XP_32273) with 31.8 % identity and 46.6 % similarity (E-value 6e–12).
Recognition of native PA0122 protein in P. aeruginosa
Antibodies to r-PA0122 were raised in mice and used to detect native PA0122 from P. aeruginosa. Western analysis of total protein extracts from overnight cultures of 383 and 2192 revealed an immunoreactive band at 16 kDa, a size consistent with the inferred molecular mass of PA0122 (Fig. 2a). This protein was more abundant in strain 383 than in strain 2192. These results suggest that the PA0122 ORF encodes a protein in P. aeruginosa, and support the microarray and qRT-PCR analyses that indicate that its expression is greater in 383 than in 2192.
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16 kDa was observed at OD600 1.6 and showed a decreased intensity at OD600 2.0 (Fig. 2b). A similar pattern of growth phase-dependent expression was seen in strain 2192, but the protein expression level was much lower (data not shown). An immunoreactive protein of the same molecular mass was detected in acetone-precipitated extracts from the supernatant of 383 (Fig. 2c). In the supernatant fraction, the expression of this protein was apparent at OD600 1.0, but showed greatest expression at OD600 1.6 and no expression at OD600 2.0. In addition, a second immunoreactive band was evident at OD600 1.6 in both the cellular and supernatant fractions as detected by Western blotting, which may suggest proteolytic degradation of the PA0122 protein.
Subcellular localization of PA0122
To determine the subcellular location of PA0122, P. aeruginosa strains 383 and 2192 were disrupted by French press. A 16 kDa immunoreactive band was detected by Western blotting of whole-cell extracts, cytoplasmic extracts and total membrane extracts of P. aeruginosa strain 383 (Fig. 3). After further separation of the PA0122 protein by discontinuous (20–75 %) sucrose density gradient, each fraction was analysed for NADH oxidase activity as a marker for the inner membrane (Tielker et al., 2005), while the presence of OprF was used as a marker for the outer membrane. We observed a prominent immunoreactive band in fractions 6 and 7, while a faint band was seen in other fractions of strain 383 (Fig. 4a). The outer-membrane protein OprF was detected in fractions 9–12, while NADH oxidase activity was confined to fractions 4–6 (Fig. 4b, c). The band corresponding to PA0122 was absent in the total membrane fractions from 2192, while a band of reduced intensity was detected in 2192 whole-cell and cytoplasmic extracts (Fig. 3). PA0122 was not detected in sucrose gradients of membrane fractions from strain 2192 (data not shown). Thus, PA0122 is present in the cytoplasm of 383, and also associated with the inner and, to a lesser extent, the outer membrane. In strain 2192, PA0122 is only detected in the cytoplasm.
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, 2002). To determine whether r-PA0122 exhibits similar binding properties, we monitored the binding of different concentrations of r-PA0122 to Ox-LDL at 37 °C under non-denaturing conditions. The formation of r-PA0122–Ox-LDL complexes was detected by Western blot analysis using anti-r-PA0122 antisera as a probe. In this assay, free r-PA0122 protein was detected as a faster-migrating band, while the reactivity of complex formation was concentration-dependent (Fig. 5); no binding was seen to LDL. These results indicate that r-PA0122 is able to bind to Ox-LDL, but not to LDL.
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). Binding of r-PA0122 to LDL was not detected (Fig. 6b), while binding of r-PA0122 to Ox-LDL was observed (Fig. 6c).
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, 2002). To determine the kinetics of binding of Ox-LDL or lysoPC to r-PA0122, the association (Ka) and dissociation (Kd) rates were measured, and the dissociation constant (KD) was calculated by SPR. Fig. 6(d) shows sensorgrams for Ox-LDL. Ox-LDL had a KD of 1.36x10–9 M to r-PA0122. The possible binding subcomponent of Ox-LDL was determined by using water-soluble synthetic short-chain (C:6) lysoPC. Fig. 6(e) depicts the sensorgrams for lysoPC with a KD of 2.94x10–5 M. These calculated values indicate that lysoPC is a bioactive subcomponent of Ox-LDL that associates at a rate similar to that of Ox-LDL, but dissociates more slowly from r-PA0122. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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In our microarray analysis, we found 115 genes that were differentially regulated between 383 and 2192 that were classified as encoding ‘hypothetical’ or ‘conserved hypothetical’ proteins. Twelve of the 73 genes that were upregulated in strain 383 have been reported elsewhere as being QS-regulated (Wolfgang et al., 2004; Schuster et al., 2003) (Supplementary Table S2); three of these were annotated as encoding ‘hypothetical’ or ‘conserved hypothetical’ proteins. In P. aeruginosa, QS regulation plays an important role in the production of virulence factors, the development of biofilms and immune modulation during chronic lung infection (Venturi, 2006; Schuster & Greenberg, 2006; Wagner et al., 2006). In addition, QS also controls the expression of exoproducts such as proteases, exotoxin A, rhamnolipids and pyocyanin (Kong et al., 2005). Other investigators have identified a number of genes that are regulated by the las and/or rhl QS systems (Venturi, 2006). The acyl-homoserine lactone (AHL)-dependent transcriptome has been revealed using microarray analysis (Wagner et al., 2003; Schuster et al., 2003). These studies have shown that approximately 20 % of genes can be identified as being QS-regulated in P. aeruginosa strain PAO1. PA0122 has been recognized as being QS-regulated (Schuster & Greenberg, 2006), and shown to contain a las box upstream of the ORF (Schuster et al., 2004; Wagner et al., 2003). PA0122 is upregulated in the stationary phase of planktonic growth and in developed (or confluent) biofilms (Waite et al., 2006).
In this study, we selected PA0122 for further investigation based on its recognition as being QS-regulated, its differential expression pattern in 383 vs 2192 and its limited homology with previously described proteins. Using qRT-PCR, we recapitulated the growth phase-dependent pattern of PA0122 transcription levels in the non-mucoid strain 383 and the mucoid strain 2192. Immunoblot analysis showed that PA0122 protein expression is also regulated in a growth phase-dependent manner with the peak of protein expression found at OD600 1.6. Cell fractionation of strain 383 showed that PA0122 protein was detectable in the culture supernatant, where it reached a maximum at OD600 1.6. However, at OD600 2.0, PA0122 protein levels were reduced in the cell extracts and absent in the supernatant (Fig. 2b). The reduction in the protein level at OD600 2.0 could be due to proteolysis of this protein.
We showed that non-mucoid revertants of three mucoid strains had increased PA0122 gene expression when compared to the mucoid parental strains. In addition, cell fractionation studies in 383 and 2192 revealed differences in the localization of PA0122. In 383, PA0122 was present in the cytoplasmic and membrane fractions, while this protein was only observed in the cytoplasmic fraction of 2192. PA0122 was not detected in crude membrane fractions of strain 2192, even when five times the amount of protein (100 µg) was loaded per lane (data not shown). The mechanism for the differential expression and localization of PA0122 in these strains is currently under investigation.
It should be noted that prior to this work, PA0122 was only recognized as an ORF putatively encoding a conserved hypothetical protein. Our Western blot analysis confirmed that the gene encodes a protein product that is associated with the cytoplasm, the inner membrane and supernatant fraction of strain 383. To our knowledge, this is the first report of a P. aeruginosa protein interacting with Ox-LDL or lysoPC. Previously, P. aeruginosa has been reported to bind to two types of phospholipids, phosphatidyl inositol (PI) and phosphatidylserine (PS), located on the plasma membrane of corneal epithelium (Panjwani et al., 1996). The authors suggest that association of P. aeruginosa with PI or PS on the surface of host cells could trigger signalling events that might increase the expression of bacterial adhesion and virulence factors. Another study showed that the lysophospholipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (also called monopalmitoylphosphatidic acid) inhibits expression of several secreted virulence factors, including alginate, pyoverdin, elastase and LasA from P. aeruginosa PAO1 (Laux et al., 2002). None of these studies has reported a receptor for oxidized phospholipid (Ox-PL) binding to P. aeruginosa proteins.
A possible role for PA0122 during infection is suggested from studies of the homologous haemolytic proteins called aegerolysins (Marchler-Bauer & Bryant, 2004), and in particular Asp-haemolysin, which is a haemolytic toxin produced by A. fumigatus (Ebina et al., 1985). The toxic effect of Asp-haemolysin has been demonstrated on various cell types, including macrophages, endothelial cells and red blood cells from humans, rabbits and sheep (Kumagai et al., 1999, 2005; Fukuchi et al., 1998). It has also been noted that recombinant Asp-haemolysin specifically binds to Ox-LDL and recognizes lysoPC (Kumagai et al., 2006; Kudo et al., 1999, 2002), but does not exhibit haemolytic activity (Kumagai et al., 2002). Similarly, r-PA0122 did not exhibit haemolytic activity (data not shown). Using SPR, a high binding affinity (1.36x10–9 M) was obtained for r-PA0122 and Ox-LDL. A similar binding affinity (1.2x10–9 M) has been reported for r-Asp-haemolysin and Ox-LDL (Kudo et al., 2001). In addition, a lower binding affinity (2.94x10–5 M) was obtained for r-PA0122 and synthetic C:6-lysoPC. The molecular masses of aegerolysin proteins are reported to be between 14 and 17 kDa, with pIs between 4.0 and 5.0 (Berne et al., 2002). These characteristics are similar to those we observed for PA0122. Thus, our findings are in agreement with earlier studies reporting the function of r-Asp-haemolysin.
In conclusion, PA0122 is a novel Ox-LDL/lysoPC-binding protein identified in P. aeruginosa. It is more abundant in the non-mucoid strain 383 and localized to different compartments, including the cell membrane and culture supernatant, suggesting that it might interact with Ox-PL in vivo. The role of PA0122 in P. aeruginosa pathogenesis is currently under investigation.
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ACKNOWLEDGEMENTS |
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Edited by: M. A. Curtis
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Berne, S., Krizaj, I., Pohleven, F., Turk, T., Macek, P. & Sepcic, K. (2002). Pleurotus and Agrocybe hemolysins, new proteins hypothetically involved in fungal fruiting. Biochim Biophys Acta 1570, 153–159.[Medline]
Borriello, G., Werner, E., Roe, F., Kim, A. M., Ehrlich, G. D. & Stewart, P. S. (2004). Oxygen limitation contributes to antibiotic tolerance of Pseudomonas aeruginosa in biofilms. Antimicrob Agents Chemother 48, 2659–2664.
Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72, 248–254.[CrossRef][Medline]
Bragonzi, A., Worlitzsch, D., Pier, G. B., Timpert, P., Ulrich, M., Hentzer, M., Andersen, J. B., Givskov, M., Conese, M. & Doring, G. (2005). Nonmucoid Pseudomonas aeruginosa expresses alginate in the lungs of patients with cystic fibrosis and in a mouse model. J Infect Dis 192, 410–419.[CrossRef][Medline]
Chen, H., Cheng, H. & Bjerknes, M. (1993). One-step Coomassie brilliant blue R-250 staining of proteins in polyacrylamide gel. Anal Biochem 212, 295–296.[CrossRef][Medline]
Chugani, S. & Greenberg, E. P. (2007). The influence of human respiratory epithelia on Pseudomonas aeruginosa gene expression. Microb Pathog 42, 29–35.[CrossRef][Medline]
Coleman, L. G., Jr, Polanowska-Grabowska, R. K., Marcinkiewicz, M. & Gear, A. R. L. (2004). LDL oxidized by hypochlorous acid causes irreversible platelet aggregation when combined with low levels of ADP, thrombin, epinephrine, or macrophage-derived chemokine (CCL22). Blood 104, 380–389.
Darzins, A. & Chakrabarty, A. M. (1984). Cloning of genes controlling alginate biosynthesis from a mucoid cystic fibrosis isolate of Pseudomonas aeruginosa. J Bacteriol 159, 9–18.
Davies, J. C. (2002). Pseudomonas aeruginosa in cystic fibrosis: pathogenesis and persistence. Paediatr Respir Rev 3, 128–134.[CrossRef][Medline]
De Vos, D., De Chial, M., Cochez, C., Jansen, S., Tummler, B., Meyer, J. M. & Cornelis, P. (2001). Study of pyoverdine type and production by Pseudomonas aeruginosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and accumulation of pyoverdine-negative mutations. Arch Microbiol 175, 384–388.[CrossRef][Medline]
Doggett, R. G. (1969). Incidence of mucoid Pseudomonas aeruginosa from clinical sources. Appl Microbiol 18, 936–937.[Medline]
Ebina, K., Ichinowatari, S. & Yokota, K. (1985). Studies on toxin of Aspergillus fumigatus. XXII. Fashion of binding of Asp-hemolysin to human erythrocytes and Asp-hemolysin-binding proteins of erythrocyte membranes. Microbiol Immunol 29, 91–101.[Medline]
Firoved, A. M. & Deretic, V. (2003). Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa. J Bacteriol 185, 1071–1081.
Firoved, A. M., Boucher, J. C. & Deretic, V. (2002). Global genomic analysis of AlgU (
E)-dependent promoters (sigmulon) in Pseudomonas aeruginosa and implications for inflammatory processes in cystic fibrosis. J Bacteriol 184, 1057–1064.
Firoved, A. M., Ornatowski, W. & Deretic, V. (2004). Microarray analysis reveals induction of lipoprotein genes in mucoid Pseudomonas aeruginosa: implications for inflammation in cystic fibrosis. Infect Immun 72, 5012–5018.
Frisk, A., Schurr, J. R., Wang, G., Bertucci, D. C., Marrero, L., Hwang, S. H., Hassett, D. J. & Schurr, M. J. (2004). Transcriptome analysis of Pseudomonas aeruginosa after interaction with human airway epithelial cells. Infect Immun 72, 5433–5438.
Fukuchi, Y., Kudo, Y., Kumagai, T., Ebina, K. & Yokota, K. (1998). Oxidized low density lipoprotein inhibits the hemolytic activity of Asp-hemolysin from Aspergillus fumigatus. FEMS Microbiol Lett 167, 275–280.[CrossRef][Medline]
Goldberg, J. B. & Ohman, D. E. (1984). Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol 158, 1115–1121.
Govan, J. R. & Deretic, V. (1996). Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia. Microbiol Rev 60, 539–574.
Hancock, R. E. & Nikaido, H. (1978). Outer membranes of Gram-negative bacteria. XIX. Isolation from Pseudomonas aeruginosa PAO1 and use in reconstitution and definition of the permeability barrier. J Bacteriol 136, 381–390.
Hancock, R. E., Mutharia, L. M., Chan, L., Darveau, R. P., Speert, D. P. & Pier, G. B. (1983). Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains. Infect Immun 42, 170–177.
Hanna, S. L., Sherman, N. E., Kinter, M. T. & Goldberg, J. B. (2000). Comparison of proteins expressed by Pseudomonas aeruginosa strains representing initial and chronic isolates from a cystic fibrosis patient: an analysis by 2-D gel electrophoresis and capillary column liquid chromatography-tandem mass spectrometry. Microbiology 146, 2495–2508.
Hentzer, M., Teitzel, G. M., Balzer, G. J., Heydorn, A., Molin, S., Givskov, M. & Parsek, M. R. (2001). Alginate overproduction affects Pseudomonas aeruginosa biofilm structure and function. J Bacteriol 183, 5395–5401.
Ichikawa, J. K., Norris, A., Bangera, M. G., Geiss, G. K., van't Wout, A. B., Bumgarner, R. E. & Lory, S. (2000). Interaction of Pseudomonas aeruginosa with epithelial cells: identification of differentially regulated genes by expression microarray analysis of human cDNAs. Proc Natl Acad Sci U S A 97, 9659–9664.
Institute of Laboratory Animal Resources Commission on Life Sciences (1996). Guide for the Care and Use of Laboratory Animals. Washington DC: National Academy Press.
Kong, K. F., Jayawardena, S. R., Indulkar, S. D., del Puerto, A., Koh, C. L., Hoiby, N. & Mathee, K. (2005). Pseudomonas aeruginosa AmpR is a global transcriptional factor that regulates expression of AmpC and PoxB β-lactamases, proteases, quorum sensing, and other virulence factors. Antimicrob Agents Chemother 49, 4567–4575.
Kudo, Y., Kumagai, T., Fukuchi, Y., Ebina, K. & Yokota, K. (1999). Binding of Asp-hemolysin from Aspergillus fumigatus to oxidized low density lipoprotein. Biol Pharm Bull 22, 549–550.[Medline]
Kudo, Y., Fukuchi, Y., Kumagai, T., Ebina, K. & Yokota, K. (2001). Oxidized low-density lipoprotein-binding specificity of Asp-hemolysin from Aspergillus fumigatus. Biochim Biophys Acta 1568, 183–188.[Medline]
Kudo, Y., Ootani, T., Kumagai, T., Fukuchi, Y., Ebin, K. & Yokota, K. (2002). A novel oxidized low-density lipoprotein-binding protein, Asp-hemolysin, recognizes lysophosphatidylcholine. Biol Pharm Bull 25, 787–790.[CrossRef][Medline]
Kumagai, T., Nagata, T., Kudo, Y., Fukuchi, Y., Ebina, K. & Yokota, K. (1999). Cytotoxic activity and cytokine gene induction of Asp-hemolysin to murine macrophages. Nippon Ishinkin Gakkai Zasshi 40, 217–222.[Medline]
Kumagai, T., Kudo, Y., Fukuchi, Y., Ebina, K. & Yokota, K. (2002). Expression of a synthetic gene encoding the Asp-hemolysin from Aspergillus fumigatus in Escherichia coli. Biol Pharm Bull 25, 115–157.[CrossRef][Medline]
Kumagai, T., Ogawa, N., Tsutsumi, H., Ebina, K. & Yokota, K. (2005). A synthetic peptide (P-21) derived from Asp-hemolysin inhibits the induction of macrophage proliferation by oxidized low-density lipoprotein. Biol Pharm Bull 28, 1381–1384.[CrossRef][Medline]
Kumagai, T., Tsutsumi, H., Ogawa, N., Naito, S., Ebina, K., Yokota, K. & Nagata, K. (2006). Oxidized low-density lipoprotein-binding specificity of the Asp-hemolysin-related synthetic peptides from Aspergillus fumigatus. Biol Pharm Bull 29, 2181–2186.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Laux, D. C., Corson, J. M., Givskov, M., Hentzer, M., Moller, A., Wosencroft, K. A., Olson, J. C., Krogfelt, K. A., Goldberg, J. B. & Cohen, P. S. (2002). Lysophosphatidic acid inhibition of the accumulation of Pseudomonas aeruginosa PAO1 alginate, pyoverdin, elastase and LasA. Microbiology 148, 1709–1723.
Lizewski, S. E., Schurr, J. R., Jackson, D. W., Frisk, A., Carterson, A. J. & Schurr, M. J. (2004). Identification of AlgR-regulated genes in Pseudomonas aeruginosa by use of microarray analysis. J Bacteriol 186, 5672–5684.
Luzar, M. A., Thomassen, M. J. & Montie, T. C. (1985). Flagella and motility alterations in Pseudomonas aeruginosa strains from patients with cystic fibrosis: relationship to patient clinical condition. Infect Immun 50, 577–582.
Mahenthiralingam, E., Campbell, M. E. & Speert, D. P. (1994). Nonmotility and phagocytic resistance of Pseudomonas aeruginosa isolates from chronically colonized patients with cystic fibrosis. Infect Immun 62, 596–605.
Marchler-Bauer, A. & Bryant, S. H. (2004). CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32, W327–W331.
Marty, N., Pasquier, C., Dournes, J. L., Chemin, K., Chavagnat, F., Guinand, M., Chabanon, G., Pipy, B. & Montrozier, H. (1998). Effects of characterised Pseudomonas aeruginosa exopolysaccharides on adherence to human tracheal cells. J Med Microbiol 47, 129–134.
Mutharia, L. M. & Hancock, R. E. (1983). Surface localization of Pseudomonas aeruginosa outer membrane porin protein F by using monoclonal antibodies. Infect Immun 42, 1027–1033.
Ochsner, U. A., Wilderman, P. J., Vasil, A. I. & Vasil, M. L. (2002). GeneChipR expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol Microbiol 45, 1277–1287.[CrossRef][Medline]
Ohman, D. E. & Chakrabarty, A. M. (1981). Genetic mapping of chromosomal determinants for the production of the exopolysaccharide alginate in a Pseudomonas aeruginosa cystic fibrosis isolate. Infect Immun 33, 142–148.
Panjwani, N., Zhao, Z., Raizman, M. B. & Jungalwala, F. (1996). Pathogenesis of corneal infection: binding of Pseudomonas aeruginosa to specific phospholipids. Infect Immun 64, 1819–1825.[Abstract]
Parsek, M. R. & Singh, P. K. (2003). Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 57, 677–701.[CrossRef][Medline]
Pier, G. B., Coleman, F., Grout, M., Franklin, M. & Ohman, D. E. (2001). Role of alginate O acetylation in resistance of mucoid Pseudomonas aeruginosa to opsonic phagocytosis. Infect Immun 69, 1895–1901.
Ramsey, D. M. & Wozniak, D. J. (2005). Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis. Mol Microbiol 56, 309–322.[CrossRef][Medline]
Schagger, H. & von Jagow, G. (1987). Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem 166, 368–379.[CrossRef][Medline]
Schuster, M. & Greenberg, E. P. (2006). A network of networks: quorum-sensing gene regulation in Pseudomonas aeruginosa. Int J Med Microbiol 296, 73–81.[CrossRef][Medline]
Schuster, M., Lostroh, C. P., Ogi, T. & Greenberg, E. P. (2003). Identification, timing, and signal specificity of Pseudomonas aeruginosa quorum-controlled genes: a transcriptome analysis. J Bacteriol 185, 2066–2079.
Schuster, M., Hawkins, A. C., Harwood, C. S. & Greenberg, E. P. (2004). The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol Microbiol 51, 973–985.[CrossRef][Medline]
Shevchenko, A., Wilm, M., Vorm, O. & Mann, M. (1996). Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68, 850–858.[Medline]
Simpson, J. A., Smith, S. E. & Dean, R. T. (1989). Scavenging by alginate of free radicals released by macrophages. Free Radic Biol Med 6, 347–353.[CrossRef][Medline]
Skaar, E. P., Tobiason, D. M., Quick, J., Judd, R. C., Weissbach, H., Etienne, F., Brot, N. & Seifert, H. S. (2002). The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. Proc Natl Acad Sci U S A 99, 10108–10113.
Smith, E. E., Buckley, D. G., Wu, Z., Saenphimmachak, C., Hoffman, L. R., D'Argenio, D. A., Miller, S. I., Ramsey, B. W., Speert, D. P. & other authors (2006). Genetic adaptation by Pseudomonas aeruginosa to the airways of cystic fibrosis patients. Proc Natl Acad Sci U S A 103, 8487–8492.
Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J., Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J. & other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature 406, 959–964.[CrossRef][Medline]
Tielker, D., Hacker, S., Loris, R., Strathmann, M., Wingender, J., Wilhelm, S., Rosenau, F. & Jaeger, K. E. (2005). Pseudomonas aeruginosa lectin LecB is located in the outer membrane and is involved in biofilm formation. Microbiology 151, 1313–1323.
van der Vliet, A., Eiserich, J. P., Marelich, G. P., Halliwell, B. & Cross, C. E. (1997). Oxidative stress in cystic fibrosis: does it occur and does it matter? Adv Pharmacol 38, 491–513.[Medline]
Venturi, V. (2006). Regulation of quorum sensing in Pseudomonas. FEMS Microbiol Rev 30, 274–291.[CrossRef][Medline]
Wagner, V. E., Bushnell, D., Passador, L., Brooks, A. I. & Iglewski, B. H. (2003). Microarray analysis of Pseudomonas aeruginosa quorum-sensing regulons: effects of growth phase and environment. J Bacteriol 185, 2080–2095.
Wagner, V. E., Frelinger, J. G., Barth, R. K. & Iglewski, B. H. (2006). Quorum sensing: dynamic response of Pseudomonas aeruginosa to external signals. Trends Microbiol 14, 55–58.[CrossRef][Medline]
Waite, R. D., Paccanaro, A., Papakonstantinopoulou, A., Hurst, J. M., Saqi, M., Littler, E. & Curtis, M. A. (2006). Clustering of Pseudomonas aeruginosa transcriptomes from planktonic cultures, developing and mature biofilms reveals distinct expression profiles. BMC Genomics 7, 162[CrossRef][Medline]
Whiteley, M., Bangera, M. G., Bumgarner, R. E., Parsek, M. R., Teitzel, G. M., Lory, S. & Greenberg, E. P. (2001). Gene expression in Pseudomonas aeruginosa biofilms. Nature 413, 860–864.[CrossRef][Medline]
Winklhofer-Roob, B. M., Ziouzenkova, O., Puhl, H., Ellemunter, H., Greiner, P., Muller, G., Van't Hof, M. A., Esterbauer, H. & Shmerling, D. H. (1995). Impaired resistance to oxidation of low density lipoprotein in cystic fibrosis: improvement during vitamin E supplementation. Free Radic Biol Med 19, 725–733.[CrossRef][Medline]
Wolfgang, M. C., Jyot, J., Goodman, A. L., Ramphal, R. & Lory, S. (2004). Pseudomonas aeruginosa regulates flagellin expression as part of a global response to airway fluid from cystic fibrosis patients. Proc Natl Acad Sci U S A 101, 6664–6668.
Received 10 July 2007;
revised 3 October 2007;
accepted 1 November 2007.
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