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1 Department of Biochemistry and Microbiology, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV 25755-9320, USA
2 Department of Biology and Environmental Science, West Virginia Wesleyan College, Buckhannon, WV 26201, USA
3 Department of Pediatrics, Joan C. Edwards School of Medicine at Marshall University, Huntington, WV 25701-3655, USA
4 Progenesis Technologies, LLC, Bldg 740, Rm 4136, Dow Technology Park, 3200 Kanawha Turnpike, South Charleston, WV 25303, USA
Correspondence
Hongwei D. Yu
yuh{at}marshall.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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A supplementary table, listing oligonucleotides used in this study, is available with the online version of this paper.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In CF patients, the respiratory tract system is impaired not only by defective CFTR but also by microbial infections with a variety of pathogens, such as Pseudomonas aeruginosa, due to decreased mucociliary clearance (Govan & Deretic, 1996). Chronic lung infection with P. aeruginosa leads to increased morbidity and mortality in CF (Lyczak et al., 2002). Biofilm formation in CF lungs by P. aeruginosa facilitates survival through resistance to host immune responses and increased antibiotic resistance (Govan & Deretic, 1996). Biofilm formation in CF lungs is also dependent upon bacterial communication or quorum sensing (QS) (Singh et al., 2000).
Conversion of P. aeruginosa to mucoid phenotype or overproduction of exopolysaccharide alginate has clearly been shown to be protective for survival (Govan & Deretic, 1996). MucA is a negative regulator of alginate production that sequesters the alginate master regulator, ECF sigma factor AlgU (Schurr et al., 1996), to the inner membrane (Rowen & Deretic, 2000). Mutations in mucA cause constitutive production of alginate (Martin et al., 1993) due to loss of MucA repression of AlgU. AlgU activates transcription of the algD biosynthetic operon (Deretic et al., 1987), which then leads to alginate production (Wozniak & Ohman, 1994). Alginate production can also occur independently of mucA mutations through proteolytic derepression of MucA by the protease AlgW (Qiu et al., 2007).
The two-component response regulator AlgB (PA5483) controls alginate production at the algD promoter (Wozniak & Ohman, 1994). AlgB and KinB (PA5484) are encoded on the chromosome in an operon, and KinB has been shown to phosphorylate AlgB (Ma et al., 1997). However, phosphorylation of AlgB is not required for alginate production (Ma et al., 1998; Damron et al., 2009). AlgB is required for mucoidy (Goldberg & Ohman, 1984) and transcriptional activation of the algD biosynthetic operon (Leech et al., 2008). Recently, we have observed that inactivation of kinB causes strain PAO1 to produce copious amounts of alginate (Fig. 1) (Damron et al., 2009). Inactivation of kinB causes loss of AlgU repression by MucA and alginate production that is dependent upon AlgW, AlgB and the alternative sigma factor RpoN (
54) (Damron et al., 2009). Alginate production provides protection for P. aeruginosa; however, alginate-independent, AlgU-dependent gene products are responsible for the detrimental inflammation (Firoved et al., 2004). Of the 5567 proteins encoded in the PAO1 genome there are 113–186 predicted lipoproteins (Babu et al., 2006). In mucoid strains, 70 % of genes with a >30-fold increase in expression encode lipoproteins (Firoved et al., 2004). AlgU-dependent lipoproteins or lipotoxins cause activation of NF-
B in human lung epithelial cells through Toll-like receptor (TLR)2 (Firoved et al., 2004). Lipotoxins have been shown to stimulate inflammatory responses (Firoved et al., 2002, 2004). However, the physiological roles of these lipotoxins have not been characterized.
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lptF exhibits decreased adherence to A549 human lung epithelial cells. The studies presented here suggest that LptF in P. aeruginosa is an important survival factor. |
METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Escherichia coli strains were grown at 37 °C in Lennox broth (LB) or LB agar supplemented, when necessary, with carbenicillin or tetracycline at concentrations of 100 and 20 µg ml–1, respectively. P. aeruginosa strains were grown at 37 °C in LB or on Pseudomonas isolation agar (PIA) plates (Difco). When necessary, the PIA plates were supplemented with carbenicillin or tetracycline at concentrations of 300 and 200 µg ml–1, respectively. Amplicon sequencing of plasmids and gene deletions were performed by the Marshall University Genomics Core Facility. The sequences of the oligonucleotides utilized this study are listed in Supplementary Table S1.
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SDS-PAGE, total protein preparation and peptide mass spectrometric sequencing.
Total protein preparations were obtained by processing cell lysates with Epicentre ReadyPreps. Protein concentrations were determined using the Bio-Rad DC Protein Assay. SDS-PAGE (14 % polyacrylamide) was performed to separate total cell lysates for staining with R250 Coomassie stain. Selected upregulated protein bands were excised from the gel for direct mass spectrometric sequencing. Gel pieces were destained with Protea Silver destaining solution (Protea Biosciences). The pieces were dehydrated and then rehydrated with acetonitrile and 50 mM ammonium bicarbonate, respectively. Proteins in the gel pieces were reduced and alkylated with 250 mM DTT (60 min, 55 °C) and 650 mM iodoacetamide (60 min at room temperature in the dark), respectively. Digestion was performed with 625 ng trypsin in 50 mM ammonium bicarbonate buffer overnight. Extraction of peptides was performed using 5 % formic acid in 50 % acetonitrile and with 50 mM ammonium bicarbonate. Three cycles of dehydration, rehydration and supernatant collection were performed, and the recovered peptides were dried down in a lyophilizer to be purified with an acetic acid rinse in addition to a final lyophilization.
The LC-MALDI MS system used was an ABI Tempo LC MALDI spotter with Tempo LC MALDI v.2.00.09 data acquisition and processing software. Lyophilized-digested samples were reconstituted, and 5 µl was injected onto a Chromolith CapRod monolith column 150x0.1 mm (Merck). The peptides were eluted from the column using an acetonitrile/trifluoroacetic acid gradient (2–72 % acetonitrile in 25 min) and spotted directly onto a MALDI plate. The MALDI spots were analysed using an ABI 4800 MALDI-TOF/TOF analyser operated with 4000 Series Explorer software. The MS acquisition was in reflector mode positive ion mode with 400 laser shots per spectrum performed. The 15 strongest precursors were chosen for MS-MS, and the MALDI spot was interrogated until at least four peaks in the MS-MS spectra achieved a signal : noise ratio 
70. The resulting MS/MS spectra were analysed using ABI Protein ProteinPilot software 2.0. The spectral data were compared with the Pseudomonas Genome Project version 2 database for identification of the peptides and corresponding proteins.
Analysis of outer membrane proteins.
P. aeruginosa strains were streaked on PIA and cultured for 24 h at 37 °C. The cells were scraped from the plates and suspended in PBS (pH 7.4, Sigma-Aldrich). The cells were harvested by centrifugation at 7000 g. The cell pellet was suspended in 2 % sarkosyl with 2 mM PMSF protease inhibitor in PBS. The cells were lysed by sonication for 1 min on ice. The lysate was clarified by low-speed centrifugation. The supernatant was taken and centrifuged at 40 000 g for 1 h. The resulting pellet, containing outer membrane proteins, was resuspended in Tris-buffered saline (TBS; Protea Biosciences). The protein concentration was determined. The preparations were separated by SDS-PAGE and visualized by silver staining with Bio-Rad Silver Stain Plus.
β-Galactosidase activity assay of PlpteEF–lacZ promoter fusion.
The MiniCTX-lacZ (Hoang et al., 2000) integration gene delivery vector was used for inserting promoter fusions into the CTX phage attB site on the P. aeruginosa chromosome. A 949 bp length upstream of the lptF start site was cloned into the HindIII/EcoRI sites of MiniCTX-lacZ. The construct was sequenced to show that no mutations had occurred during the cloning. MiniCTX-PlptEF–lacZ was transferred to recipient strains by pRK2013 conjugation. Strains with integration into the attB site were selected on PIA supplemented with tetracycline (200 µg ml–1) and were passed through three isolations. The β-galactosidase activity assay was based on the method originally described by Miller (1972), with the following modification. The cells were grown on PIA with antibiotics for selection in triplicate for 24 h at 37 °C and harvested in PBS. Cell density was measured by OD600. The β-galactosidase activity was assayed after toluene permeabilization of the cells. The reported values represent the means of samples in triplicate from three independent experiments with standard error indicated.
Mutant strain construction.
For in-frame deletion of lptF, the upstream and downstream sequence fragments (1 kb) flanking lptF were PCR-amplified and fused using the crossover PCR method. The PCR products with the in-frame deletion of the target gene were then cloned into pCR4-TOPO. The subcloned in-frame deletion fragment was then digested and ligated into the pEX100T-NotI vector. The resulting vectors were sequenced to show that no mutations had occurred apart from the intended specific gene deletion. A two-step allelic exchange procedure was employed with the pEX100T constructs for gene disruption or in-frame deletion. The single-crossover merodiploid exconjugants were selected based on carbenicillin resistance and sensitivity on 10 % sucrose (sacB). After incubation of the merodiploids in LB, the double-crossover recombinants were isolated from the PIA plates supplemented with 10 % (w/v) sucrose. The disruption or in-frame deletion of the target gene was confirmed by antibiotic-resistance assays, PCR amplification of the flanking region of the target gene with multiple sets of primers, and amplicon sequencing.
Analysis of alginate production.
P. aeruginosa strains were grown at 37 °C on PIA plates in triplicate for 24 h. The resulting bacterial growth was removed from plates and suspended in PBS. The OD600 of the suspension in PBS was measured. The suspensions were assayed for the amount of uronic acid in comparison with a standard curve made with D-mannuronic acid lactone (Sigma-Aldrich), as previously described (Damron et al., 2009).
Susceptibility to killing by hydrogen peroxide and hypochlorite.
Sensitivity to hydrogen peroxide and hypochlorite was determined by measuring the radius of the growth inhibition zone surrounding filter disks (6 mm diameter, BBL). A 25 ml volume of LB agar was poured into 100x15 mm plates. Overnight cultures were diluted with LB, and 100 µl OD600 0.1 culture was added to 3 ml molten 0.6 % soft agar and gently mixed. The culture–soft agar suspension was then overlaid on the 25 ml of LB agar. Disks were soaked with 10 µl fresh stock solutions of 10 % hydrogen peroxide or 6 % hypochlorite. The disks were then applied to the soft agar-containing plate. The zone of inhibition was scored after 24 h incubation at 37 °C by measuring the radius.
Cell culture methods.
A549 lung epithelial cells (ATCC catalogue no. CCL-185) were purchased from ATCC. The cells were cultivated in F-12K medium supplemented with 10 % fetal bovine serum (ATCC) and antibiotics (pen-strep, MP Biomedicals) in 100x20 mm tissue culture treated dishes (Greiner Bio-One) and subcultured every 2–3 days. One day prior to experimental use, they were grown to 80–90 % confluence and split at a ratio of 1 : 1.
A549 epithelial cell adherence assay.
Adherence was measured by incubation of A549 cells with GFP-tagged P. aeruginosa harbouring pMRPQ-1 (Davies et al., 1998). A549 cells were harvested by treatment with 1 ml trypsin (0.25 %, Hyclone) for 10 min followed by gentle pipetting to remove any adherent cells. Live harvested cells were quantified by using erythrosin B (10 % in PBS, Fisher) exclusion dye and counted on a haemocytometer. A549 cells (1.5x105) were resuspended in 300 µl F-12K medium plus 10 % fetal bovine serum. GFP-tagged P. aeruginosa was added to the cells at a ratio of 100 : 1, and the mixture was rotated end over end at room temperature in a 1.5 ml microcentrifuge tube for 15 min. The cells were washed twice with 500 µl FACS buffer (3 % BSA, 0.02 % sodium Azide, 1 mM EDTA in PBS) and analysed for GFP fluorescence using a Becton Dickinson FACSAria cell sorter. Ten thousand cells were counted in each sample. Data were analysed using Flowjo software 8.8.2. Threshold gates were drawn based on a no-bacteria control. Results were reported as a percentage of PAO1-treated cells. All experiments were conducted in triplicate with three independent trials. Within each trial, data were normalized to the average adherence percentage of PAO1. The average normalized percentage for each trial was then calculated and used in statistical analysis. Student's t tests were performed to determine reported P values.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). In our previous studies, we observed that inactivation of alginate regulator kinB in PAO1 caused alginate overproduction (Fig. 1
) (Damron et al., 2009). This suggested that KinB is a negative regulator of alginate production in the wild-type mucA strain PAO1. Since inactivation of kinB causes mucoidy (PAO1kinB : : aacC1), we hypothesized that AlgU-dependent gene products, as well as genes controlled by KinB–AlgB, would be upregulated. We sought to identify proteins upregulated in the kinB mutant to discover members of the KinB regulon. To do so, we subjected total protein extracts to MudPIT analysis. MudPIT analysis utilizes two liquid-column chromatographic separations and tandem MALDI-TOF MS peptide fingerprinting to identify peptides in a complex sample. The conditions of these experiments allowed the identification of the peptides present in the highest concentrations in the proteomes analysed.
Alginate production by the kinB mutant requires rpoN (Damron et al., 2009). Therefore, we compared the proteomes of the kinB mutant and the nonmucoid kinB/rpoN double mutant. In mucoid PAO1kinB : : aacC1, AlgD was present, but it was absent in the kinB/rpoN double mutant (Table 2). AlgD, or GDP-mannose 6-dehydrogenase, is responsible for the initial enzymic steps leading to alginate production in P. aeruginosa. Another differentially expressed peptide observed between the proteomes of the kinB mutant and the kinB/rpoN double mutant was azurin (PA4922) (Table 2). Azurin is a QS-regulated redox protein that is located in the periplasm (Nouwens et al., 2003; Sriramulu et al., 2005). Azurin is secreted by P. aeruginosa in response to eukaryotic proteins and induces apoptosis of macrophages (Zaborina et al., 2000). In the kinB mutant, azurin formed 1.5 % of the peptides identified; however, in the kinB/rpoN mutant, azurin represented 15.4 % of peptides identified.
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). QS-regulated proteins were observed in the kinB mutant, but only one (azurin) in the kinB/rpoN double mutant. PA4739 is a small periplasmic hypothetical protein that has been shown to be upregulated in response to QS signals (Schuster et al., 2003) and hydrogen peroxide (Salunkhe et al., 2005). PA0041 is similar to Bordetella pertussis haemagglutinin exoprotein (Jacob-Dubuisson et al., 2001). PA0041 was detected in the kinB mutant but not the kinB/rpoN double mutant (Table 2). Since PA0041 is a secreted protein, it may be a component of the exopolysaccharide matrix of the kinB mutant (Fig. 1).
Identification of mucoidy-coupled lipotoxin F
The periplasmic chaperone SurA was identified in the kinB mutant (Table 2). SurA has been shown to assist in folding of outer membrane proteins OmpA, OmpF and LamB in E. coli (Lazar & Kolter, 1996). In our analysis, only one potential outer membrane protein was observed, PA3692 or LptF (Firoved et al., 2004). Many lipoproteins or lipotoxins have been shown to be upregulated in mucoid mucA mutants (Firoved et al., 2004) and in the presence of the cell wall inhibitor D-cycloserine (Wood et al., 2006). According to the Pseudomonas Genome Database version 2 (http://www.pseudomonas.com), LptF (PA3692) is a conserved OmpA-like lipoprotein. The C-terminal 110 residues are 49 % identical to P. aeruginosa major porin OprF. We observed that LptF was upregulated in the kinB mutant; however, it was absent from the kinB/rpoN double mutant (Table 2). To validate the observations from the MudPIT analysis, total protein extracts of PAO1 and PAO1kinB : : aacC1 were separated by SDS-PAGE and visualized by Coomassie staining (data not shown). A significantly upregulated protein was observed in PAO1kinB : : aacC1 total protein extracts with an apparent mass of 27 kDa (Fig. 2, lane 2). The protein was identified as LptF (PA3692) by direct peptide fingerprint analysis.
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). To confirm that LptF is in fact an outer membrane protein, outer membrane proteins from PAO1 and PAO1kinB : : aacC1 were prepared by the sarkosyl method. Total protein extracts and sarkosyl-insoluble proteins were separated and visualized by silver staining (Fig. 2). LptF is upregulated in the outer membrane protein fraction of PAO1kinB : : aacC1; however, it is also present in PAO1 (Fig. 2, lanes 3 and 4). Lipotoxins have been shown to activate the host inflammatory response (Firoved et al., 2004); however, their physiological functions have not been investigated, and therefore we further characterized lipotoxin F.
Expression of PlptEF is AlgU-dependent and upregulated in CF isolates
We reasoned that since LptF was upregulated in mucA mutants (Firoved et al., 2004) and in the mucoid kinB mutant, it was likely to be AlgU-dependent. LptE and LptF are encoded in the genome as an operon (Firoved et al., 2004). Interestingly, the lptEF promoter does not contain an AlgU consensus sequence (Firoved et al., 2004). A lacZ fusion with the lptEF promoter was constructed and integrated into the P. aeruginosa chromosome to compare expression of PlptEF in various strains. PlptEF was active in nonmucoid strains PAO1 and PA14 (Fig. 3). PlptEF expression was observed to be AlgU-dependent and could be restored upon expression of AlgU in trans (Fig. 3). Also deletion of algU from PAO1kinB : : aacC1 caused complete loss of detectable PlptEF (Fig. 3).
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) and strain 2192 is a strain isogenic to 383 but with a mucA mutation (Hanna et al., 2000). Interestingly, PlptEF was upregulated in both nonmucoid CF isolate 383 and mucoid CF isolate 2192 (Fig. 3) compared with lab strain PAO1 (Fig. 3). However, no PlptEF expression was detected in nonmucoid CF149. This indicates that CF149 harbours an algU mutation. When we sequenced algU and mucA in CF149, we found that this strain carries both algU and mucA mutations. The algU gene of CF149 has a missense mutation (C182 to T182), resulting in an amino acid change from Ala61 to Val61. The mucA mutation is a deletion of a C at 374, which causes a frameshift with the formation of a premature stop at TGA386 (GenBank accession number FJ649224). This further suggests that PlptEF expression is AlgU-dependent. Several other CF isolates showed high lptEF expression (Fig. 3). Even within one CF sputum sample, several morphologies were observed, and each exhibited a different level of lptEF expression (Fig. 3, strains CFO23o, s and w). These results show that the lptEF promoter is AlgU-dependent and upregulated in CF isolates.
LptF is not required for alginate production
Envelope proteins such as MucE can activate alginate overproduction in P. aeruginosa through regulated proteolysis of MucA by the serine protease AlgW (Qiu et al., 2007). Since LptF was highly upregulated in the mucoid kinB mutant, we examined whether LptF expression plays a role in the signal transduction which leads to AlgW-dependent alginate production of this strain (Damron et al., 2009). To test this, lptF was deleted from PAO1kinB : : aacC1. However, both the kinB mutant and the kinB/lptF double mutant produced approximately 100 micrograms of alginate per millilitre per OD600 unit. Furthermore, overexpression of lptF in PAO1 from the PBAD promoter of pHERD20T did not stimulate alginate production above the normal nonmucoid level (30 micrograms alginate per millilitre per OD600 unit). These results suggested that LptF does not activate alginate production. Therefore, we concluded that LptF is likely to be co-expressed with alginate and is not involved in the signalling pathway that leads to alginate production.
Deletion of lptF causes increased resistance to hydrogen peroxide in PAO1, but increased susceptibility to hypochlorite
In the CF lung, P. aeruginosa produces alginate for protection (Govan & Deretic, 1996). Since LptF is upregulated along with alginate production, we hypothesized that LptF serves as a protective factor. We first generated a PAO1 lptF deletion mutant and observed no changes in growth rate compared with PAO1, showing that lptF is not an essential gene (data not shown). We next examined whether LptF has a protective role against hydrogen peroxide and hypochlorite. To test the role of lptF regarding cell membrane integrity, susceptibility assays were performed with hydrogen peroxide and hypochlorite (Table 3). Interestingly, PAO1lptF was more resistant to hydrogen peroxide than PAO1 (Table 3). However, deletion of algU did not result in the same level of resistance to hydrogen peroxide. Deletion of lptF caused significantly increased susceptibility to hypochlorite (Table 3). Neutrophils utilize the generation of oxidants to kill microbes, and mucoid mutants are more resistant to hypochlorite killing (Learn et al., 1987). Deletion of algU and lptF caused increased susceptibility to hypochlorite (Table 3). These data suggest that the AlgU-dependent proteins such as LptF protect P. aeruginosa from hypochlorite killing.
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). E. coli OmpA can participate in adhesion to surfaces and interactions with cells (Smith et al., 2007); therefore, we were interested to see if LptF also has a role in adhesion. To test this hypothesis, we performed adherence assays with A549 lung epithelial cells. A constitutively GFP-expressing plasmid pMRPQ-1 (Davies et al., 1998) was conjugated into PAO1, PAO1rpoN, PAO1lptF and mucoid strain PAO1kinB : : aacC1. Pili and flagella expression are controlled by rpoN (Ishimoto & Lory, 1989; Totten et al., 1990). TLR5, which is expressed on A549 cells, recognizes flagellin and promotes adherence of bacteria to cell surfaces (Hayashi et al., 2001). Thus, PAO1rpoN serves as a negative control for adhesion for our experiments. Epithelial cells were incubated with the indicated bacteria strains for 15 min at room temperature. The cells were then washed twice and analysed immediately by flow cytometry. Threshold gating was used to determine the percentage of GFP-positive cells (Fig. 4a), which is indicative of the adherence of the bacteria to the A549 cells. PAO1 readily adhered to A549 cells, and as expected, deletion of rpoN substantially decreased adhesion (Fig. 4b, c). In the absence of lptF, adhesion to A549 cells decreased to 71.5 %±7.9 compared with PAO1 (Fig. 4b). These data suggest that lptF is required in PAO1 for maximal adhesion to A549 cells. LptF is highly upregulated in PAO1kinB : : aacC1, which produces copious amounts of alginate (Fig. 1). However, PAO1kinB : : aacC1 adherence is reduced compared with PAO1 (Fig. 4b).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). LptF upregulation in mucoid cells (Firoved et al., 2002, 2004; Firoved & Deretic, 2003; Wood et al., 2006) suggests that LptF has roles in establishment of mucoid biofilms. Collectively, these data warranted further investigation.
We first examined lptF expression and confirmed that it is controlled by AlgU (Fig. 3). PlptEF expression is upregulated in both nonmucoid and mucoid CF isolates (Fig. 3). Since lptF expression is dependent upon AlgU, and the lptEF promoter does not have an AlgU consensus sequence, there are two possible mechanisms for AlgU-dependent expression of lptF. Either AlgU drives transcription of LptF directly or it does so indirectly through expression of another transcription factor. Ultimately, LptF expression depends on the alginate master regulator AlgU.
To further characterize LptF, we generated an unmarked deletion mutant for downstream analysis. PAO1lptF was assayed for survival against killing by hydrogen peroxide and hypochlorite. Our data suggest that LptF has a role in resistance against hypochlorite; however, the deletion of lptF causes increased resistance to hydrogen peroxide. This difference suggests that LptF protection is specific for certain niches or environments. In the CF lung, P. aeruginosa forms biofilms (Singh et al., 2000), and colonization of the CF lung occurs first by nonmucoid strains (Burns et al., 2001). These early colonizing strains then establish an immunostimulatory phase of infection (Feldman et al., 1998), resulting in increased inflammation. Mucoid biofilm conversion occurs due to mutations in the anti-sigma factor mucA (Martin et al., 1993). Furthermore, with conversion to mucoidy comes upregulation of the stimulatory lipotoxins. Lipotoxins such as LptF stimulate inflammatory responses through TLR2 (Firoved et al., 2004). Motile strains with flagella activate TLR5 recognition (Zhang et al., 2005). Therefore, immune responses due to the presence of P. aeruginosa occur starting with the initial infection and continue through the rest of the CF patient's life due to inability to eradicate P. aeruginosa from the CF lung (Costerton, 2001).
Our data show that deletion of rpoN, which controls expression of flagella and pili (Ishimoto & Lory, 1989; Totten et al., 1990), severely attenuated adhesion to A549 epithelial cells. Flagella and pili are both required for early biofilm formation (O'Toole & Kolter, 1998). PAO1lptF, like PAO1, is motile (data not shown), and adheres to A549 cells to a lesser extent than PAO1. This suggests that LptF is likely to be recognized independently by epithelial cells, which may allow P. aeruginosa to attach to the tissue surface. Alternatively, the loss of LptF could result in blockage of transport of extracellular factors necessary to adhere to epithelial cells. PAO1kinB : : aacC1 adhered to A549 cells less than PAO1. Although PAO1kinB : : aacC1 produces alginate, there are other factors, such as repression of motility factors by AlgU (Baynham et al., 2006; Tart et al., 2006), that could affect adherence.
Lipotoxins such as LptF likely not only cause the inflammatory response and detrimental tissue damage in the CF lung, but also protect P. aeruginosa and preserve the biofilm. MudPIT proteomic analysis of the mucoid kinB mutant suggests that AlgU-dependent LptF is the major lipotoxin expressed in the mucoid strain proteome (Table 2). Unlike most of the other lipotoxins, LptF is an outer membrane protein (Fig. 2). We also observed that lptF expression was upregulated in CF isolates (Fig. 3), and LptF may have roles in protection (Table 3) and adhesion to lung epithelia (Fig. 4). Since LptF is highly expressed in mucoid strains that cause chronic infection, it will be interesting to use synthetic peptides to further analyse the activation of the specific inflammatory response to LptF. Recently, azithromycin has been shown to downregulate expression of lipotoxins LptF, LptE, LptD, SlyB, OsmE and PA1323 (Skindersoe et al., 2008). Also, other macrolides have been shown to alter biofilms (Wozniak & Keyser, 2004). Therefore, therapeutic treatments with azithromycin may be able to lessen the potential respiratory tract damage caused by P. aeruginosa lipotoxins such as LptF.
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ACKNOWLEDGEMENTS |
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Edited by: P. Cornelis
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Received 8 November 2008;
revised 20 January 2009;
accepted 21 January 2009.
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