The bacterial strains used in this study, P. aeruginosa strains 383 and 2192, are non-mucoid and mucoid, respectively. These strains were isolated 2 days apart from the sputum of the same CF patient (Hanna et al., 2000). Bacterial cultures were grown overnight in Luria Bertani (LB) medium at 37°C in shaking (200 rpm), diluted to 1:100 in 30 ml of fresh media, and grown to mid-log phase, to an optical density (OD₆₀₀) of 0.5.

Total proteins were extracted from strains 383 and 2192 using Tris–EDTA buffer (10 mM Tris; pH 8.3, 5 mM EDTA). Briefly, 10 ml of each bacterial culture of 0.5 (OD₆₀₀) were harvested by centrifugation and cell pellets were resuspended in 1.0 ml of Tris–EDTA buffer containing 50 μl of cocktail protease inhibitors (Sigma-Aldrich, St. Louis, MO, USA), and 1 μl (5000 units) of benzonase (Sigma). The cells were disrupted by sonication on ice four to five times for 30 s with a micro-tip probe, then incubated at room temperature for 1 h, followed by centrifugation at 6000 × g for 15 min at 4°C. Supernatant solution was transferred to a microfuge tube and the protein concentration was estimated by Bradford method using Bio-Rad protein assay reagent (Bio-Rad, Hercules, CA, USA). Approximately 1 mg of protein was precipitated with 10% trichloroacetic acid (TCA) at 4°C for 1 h and centrifuged at 20,800 × g for 10 min, followed by washing three times with cold acetone. Analytical replicate samples from each strain were used for iTRAQ labeling [Applied Biosystems Inc. (ABI), Foster City, CA, USA].

One analytical separation was performed with replicate samples of each strain (383 and 2192). Samples were reduced, digested, labeled, and combined per ABI’s standard protocol. Pooled samples were subjected to MS/MS for peptide/protein identification after strong cation exchange (SCX) chromatographic fractionation. iTRAQ reagents are isobaric in nature and thus yield similar m/z for the same peptide for the first stage of MS, when labeled with either 114, 115, 116, or 117 tags. Briefly, 100 μg of proteins from each strain were resuspended in dissolution buffer (0.5 M triethyl ammonium bicarbonate, TEAB), and then reduced, alkylated, digested, and labeled according to ABI’s standard protocol. Briefly, 20 μl of dissolution buffer, 1 μl of denaturing solution (2% SDS), and 2 μl of the reducing agent (Tris 2-carboxyethyl phosphine) were added to each protein-containing tube and incubated for 1 h at 60°C. At the end of this reduction process, proteins were brought to room temperature and the samples were alkylated by addition of 1 μl of cysteine blocking reagent [200 mM methyl methanethiosulfonate (MTT) in isopropanol] and incubation for 10 min at room temperature. The reduced and alkylated protein samples were digested with sequencing-grade modified porcine trypsin that was reconstituted with Milli Q water; 5 mg trypsin was added to each tube and incubated overnight at 37°C. The peptide samples were labeled as follows: 383-A peptides were labeled with 114, 2192-A with 115, 383-B with 116, and 2192-B with 117. Each labeling was done in 70 μl of ethanol at room temperature for 1 h (according to manufacturer’s instructions). Equivalent aliquots (77 μg) of each labeled peptide solution were pooled in a fresh tube and then dried completely in a vacuum centrifuge. These pooled, tryptic-labeled peptide samples were desalted as per manufacturer’s protocol prior to SCX chromatographic fractionation. The entire sample was reconstituted in buffer A [10 mM KH₂PO₄, pH 2.93, 25% acetonitrile (ACN)] and loaded onto a Pharmacia Mono S, 5 mm × 50 mm (Pharmacia, Uppsala, Sweden) column, and peptides were eluted with a linear gradient of 0–100% buffer B (10 mM KH₂PO₄, pH 2.73, 25% ACN, and 1 M KCl) over 20 min. For the MS/MS analysis, four fractions (8–11) were selected based on the UV peptide elution profile recorded at absorbance of OD₂₈₀, and dried samples were stored at −80°C (Figure S1 in Supplementary Material).

Each SCX fraction was reconstituted in loading buffer (98:2, water/ACN, 0.1% acetic acid) and peptides were separated by reverse phased chromatography. Two microliter of each fraction were loaded onto an in-house LC column packed with 7.5 cm of Gemini 5 μm packing material; the column was washed for 10 min with 1% acetic acid, and the sample was introduced into a QStar mass spectrometer (ABI) via a Tempo nano-LC pumping system. The LC gradient moved from 98% solvent A (0.1 M acetic acid) to 80% solvent B (a mixture of 90% ACN, 10% solvent A) over 120 min, with a flow rate of 250 nL/min. The QStar Elite was run with 3300 V electrospray voltage and a curtain gas setting of 17. The scans were set to collect one MS scan followed by the product ion scans of the three most intense ions from the MS scan.

During MS/MS analysis, peptides are fragmented with appropriate collision energy, and generate a product that corresponded to reporter ions from 114, 115, 116, and 117 m/z and peptide fragment ions, from which amino acid sequences were deduced. This MS/MS spectrum provides the data for protein identification and quantification. The resulting data were analyzed using ProteinPilot software 4.0 (ABI) with the Paragon search engine to search the Pseudomonas protein database of strain PAO1³. The default search settings used for quantitative processing and protein identification allowed for trypsin cleavage sites lysine and arginine, and the cysteine residues were blocked and alkylated with methyl methanethiosulfonate as described in the iTRAQ protocol. Protein detection threshold was set at 95% peptide confidence level. All four of the data files were concatenated into a single file for analysis and quantitation. Peptide quantification was carried out using ProteinPilot, which computes the area under the peaks at 114, 115, 116, and 117 Da, corresponding to the four iTRAQ reagents. The ratio between proteins was calculated by using each dataset as a denominator and shown in File S1 in Supplementary Material.

For 2-DE proteome analysis, total cellular proteins were extracted from strains 383 and 2192 by using Chaotropic Membrane Extraction Reagent 2 (Sigma; 5.0 M Urea, 2.0 M thiourea 40 mM Trizma^™ base, 2.0% CHAPS, and 2.0% SB3-10, pH 10.1), followed by reduction and alkylation procedures, as previously described (Herbert et al., 1998; Molloy et al., 1998). Briefly, 10 ml of 0.5 (OD₆₀₀) cultures were harvested by centrifugation and cell pellets were resuspended in 2.0 ml of Chaotropic Membrane Extraction Reagent 2 containing 100 μl of protease inhibitors (Sigma), 1 μl of benzonase (Sigma), and 80 μl of tributylphosphine (TBP; Sigma) to get a final concentration of 5 mM in the total volume. Cell suspensions were disrupted by sonication with a micro-tip probe for 4–5 min on ice, followed by centrifugation at 6000 × g for 15 min at 4°C for supernatant collection, and removal of unbroken cells. Further, the reduced proteins were alkylated by adding 23 μl of iodoacetamide (Sigma) to a final concentration of 15 mM. This mixture was incubated for 90 min at room temperature in the dark. Protein concentration was determined by the Bradford method, and sample aliquots were stored at −20°C until use. Triton X-114 extractions were performed as described previously (Bordier, 1981), with slight modifications. Two ml bacterial pellets from 0.5 OD₆₀₀ cultures were collected from strains 383 and 2192. Proteins were extracted by solubilizing in 2 ml of 1.7% Triton X-114 (Sigma) in 1× phosphate buffered saline (pH 7.4; PBS) with 100 μl of protease inhibitors (Sigma) by gentle rocking at 4°C for 2 h. Insoluble cell debris was removed by centrifugation at 6000 × g for 15 min at 4°C and the Triton X-114 solubilized proteins were collected by transferring supernatants to fresh microcentrifuge tubes, adding PBS to reduce the concentration of Triton X-114 to 1%, and incubating at 30°C for 3 min. The resulting cloudy solutions were partitioned into aqueous and detergent phases by centrifugation at 300 × g for 3 min at room temperature. The upper aqueous phase was transferred to a fresh tube and re-extracted three times following the above procedure. The lower partitioned detergent phase was pooled from each extract and washed with cold 1× PBS. Finally, total extract, aqueous partitioned, and detergent partitioned proteins were precipitated with cold acetone (1:10, v/v) overnight at −70°C. Acetone precipitated proteins were collected after centrifugation at 20,800 × g for 15 min at 4°C and protein samples were subjected to 2-DE gel electrophoresis. The protein samples were prepared for electrophoresis in Chaotropic Membrane Extraction Reagent 2 (Sigma) by reduction and alkylation procedures performed as described earlier. Protein concentrations of each sample were determined by the Bradford method and samples were stored at −20°C until use.

All protein preparations were subjected to 2-DE analysis by using PROTEAN^® IEF Cell (Bio-Rad). One hundred fifty microgram of proteins were solubilized in rehydration buffer (Bio-Rad) containing 9 M urea, 2% Triton X-100, 2% Pharmalyte pH 3–10 (Pharmacia), 2% β-mercaptoethanol, bromophenol blue, and TBP (20 mM) was added. Total proteins were applied on each immobilized pH gradient (IPG) strip (isoelectric point [pI] of 3–10 NL, 11 cm) from Bio-Rad, and active rehydration was carried out for 12–15 h under low-viscosity paraffin oil. Proteins were first separated by IEF with the following voltage/time profile: a linear step for 0–500 V for 15 min, 500–8000 V up to a total of 30,000 V/h. After completing the isoelectrofocusing (IEF), strips were incubated with equilibration buffer (3.7 g of Tris base, 150 ml of 10% SDS, 57.5 ml of glycerol, 7.71 g of DTT and 0.2 g of bromophenol blue, and made to 500 ml distilled H₂O) for 15–30 min. For the second dimension, proteins were resolved on 8–16% SDS-PAGE (Criterion gels) with Dodeca cell system from Bio-Rad at 100 V. Gels were stained with silver or with Bio-Safe Coomassie (Bio-Rad). Stained gels were scanned using HP scanner and images were stored as tagged image files (TIFF) files. All the cored spots from silver and Coomassie were characterized by either peptide mass mapping (PMM) or tandem mass spectrometry (MS/MS) for protein identification.

The selected individual protein spots were cut out of the stained gels and were submitted to the W. M. Keck Biomedical Mass Spectrometry Laboratory at the University of Virginia Biomedical Research Facility for analysis, as described elsewhere (Hanna et al., 2000). Data was analyzed by using the SEQUEST (ThermoFinnigan) search algorithm against Pseudomonas genome deposited with National Center for Biotechnology Information (NCBI).

Rabbit polyclonal antibodies were raised and purified against synthetic peptides CLASAPKPKDDEPQA for TssB1 (PA0083), CVDGDPAHSTETTKA for TssC1 (PA0084), KYIDKSTPNLMMAC for Hcp1 (PA0085; GenScript, Piscataway, NJ, USA).

Pseudomonas aeruginosa strains (383 and 2192) were grown on L agar overnight at 37°C. Cells were scraped of the plates and suspended in L broth. Starting cultures were matched to an OD₆₀₀ of 0.1 and then diluted 1:1 with L broth. Five milliliter of culture were rotated at 100 rpm 37°C for 3 h until the strains were at OD₆₀₀ of 0.5. Cultures were streaked onto L agar to ensure non-mucoid and mucoid phenotypes were maintained during log growth. Cell suspensions were matched and diluted in 2× Laemmli buffer. Samples were frozen at −80°C until separated on 12% polyacrylamide SDS-PAGE and then transferred to 0.2 μm nitrocellulose. Blocking and probing of membranes were performed with 5% skim milk in phosphate buffered saline. Polyclonal antibodies for T6SS proteins were diluted 1:500 and monoclonal antibodies to RpoA (Neoclone). Secondary antibodies with horseradish peroxidase were diluted 1:3000. Amersham Advanced ECL chemiluminescent substrate utilized for T6SS blots and Amersham ECL was used for RpoA blots. Amersham ECL hyper film was exposed for 1 min to probed membranes and developed.

Early P. aeruginosa isolates taken from the CF airway are generally non-mucoid and resemble environmental strains. Strains 383 and 2192 have been classified as isogenic (Hanna et al., 2000). Strain 383 is non-mucoid and thus represents an initially infecting isolate, while 2192 is mucoid and represents a chronically infecting isolate. To discover the proteomic differences in these two CF isolates, iTRAQ analysis and quantitation was performed. This approach is a “gel-free” mass spectrometry technique that uses isobaric amine-specific tags to compare the peptide intensities between samples and infer quantitative values for corresponding proteins. Two analytical replicates of protein preparations from each strain were subjected to digestion and iTRAQ labeling, followed by MS/MS analyses. Expression data were compiled by scoring of peptides with 99% confidence using ProteinPilot software version 4. Proteins were identified on the basis of having at least one distinct peptide with threshold of 1% false positive rate using the ProteinPilot search algorithm against all bacterial and non-redundant database entries deposited with NCBI. In this study we report the identification of 399 proteins (above 95% confidence) from 5690 peptides. The relative quantification data were obtained for 297 proteins between strains 383 and 2192. Of the quantified 297, 81 proteins showed significant p values in either one or both iTRAQ replicates (File S1 in Supplementary Material). In comparison with earlier studies of other P. aeruginosa strains (Guina et al., 2003; Wu et al., 2005), here we have observed similar numbers differentially expressed proteins.

Isobaric tags for relative and absolute quantitation analysis of the proteomes of 383 and 2192 identified 297 proteins differentially regulated (Table S1A and File S1 in Supplementary Material). The 297 quantified proteins can be grouped into three categories: (1) 67 proteins (23%) showed higher expression in the non-mucoid strain, 383, (2) 77 proteins (26%) in showed higher expression in the mucoid strain, 2192, and (3) 153 proteins (51%) showed no difference in relative expression between the non-mucoid and mucoid strains (Table S1A in Supplementary Material).

Bioinformatics analysis of the 297 proteins recognized from iTRAQ analysis allowed functional classifications as defined by the Pseudomonas Genome Database⁴ (Winsor et al., 2009; Table 1 and File S2 in Supplementary Material). The distribution of proteins observed showed representatives of almost all of the functional classes identified suggest iTRAQ analysis. However, this may represent a shallow sampling of the proteome and may also define the limits of detection. No proteins corresponding to the functional classes of antibiotic resistance and susceptibility, chemotaxis, or phage proteins were detected in the analysis, but proteins assigned to the other 23 functional classes were identified. Proteins representing 16% the amino acid biosynthesis and metabolism class were observed. Interestingly most of the proteins of this class were expressed at equal levels between the two strains. We would expect that a mucoid P. aeruginosa strain, such as 2192 would have increased expression of proteins corresponding to the cell wall/LPS/capsule class. iTRAQ analysis indicated four proteins of this class were upregulated in strain 2192 (Table 1). Furthermore the mucoid phenotype would place a demand on metabolism pathways to overproduce alginate. Eight proteins of the central intermediary metabolism class were upregulated in mucoid strain 2192 (Table 1). However, the protein class of energy metabolism has an equal number of increased and decreased proteins and 13 proteins equally expressed. If iTRAQ analysis properly represents the proteome, it would be expected that some classes of proteins would not be discordant. Seven proteins of the associated with DNA maintenance were equal expressed between the two strains (Table 1).

The bacterial membrane is the largest single structure of a bacterium. Moreover, homeostasis is maintained by upholding the integrity of this structure. In our iTRAQ analysis of the two CF isolates, we noted a total of 12 proteins (19.4% of the entire class), which correspond to the fatty acid and phospholipid metabolism class (Table 1). Since membrane integrity is paramount for bacteria, we would predict this class would be highly represented in a distribution of the total proteome. Another validation of the iTRAQ analysis was the identification of differential expression of transcriptional regulators and transcriptional machinery (Table 1). Transcriptional regulators are rarely expressed at high levels. Therefore, identification of these regulators via iTRAQ suggests the analysis was able to detect at least some proteins that may be of relatively low abundance. Conversely translational machinery would likely represent a high amount of the peptide concentration within a total protein lysate. iTRAQ analysis identified 36% of the PseudoCAP functional class of translation, post-translational modification and degradation. Seventy-one of the 197 proteins belonging to this class were identified. However, most (46) were equally expressed by the two strains (Table 1).

We have also compared the proteins detected by iTRAQ with the location of the genes on the chromosome. As anticipated by our microarray analysis (Rao et al., 2008), in some cases proteins expression seemed to correspond to genes organized in operons. A particularly prominent region of the chromosome from PA4239 to PA4274, which encodes most of the 50S and 30S ribosomal subunits, were identified in our iTRAQ analysis (Table S1B in Supplementary Material). In vitro growth analysis, as determined by ribosomal content, has previously suggested that P. aeruginosa isolated from chronically infected patients grow as much as three-fold slower than laboratory strains such as PAO1 (Yang et al., 2008). Here in our iTRAQ analysis, we observed similar down-regulation of ribosomal proteins in strain 2192 (Table S1A in Supplementary Material).

The high detection of ribosomal proteins in both samples likely decreases the depth of detection of the total proteome and is a limitation of the current study. In light of this, one way to increase the depth of iTRAQ analysis may be to deplete the amount of translation associated proteins in a sample. Fractionations that remove the translational machinery may result in more unique analysis of a proteome. This idea is reminiscent of the recent push to deplete rRNA from total RNA samples for RNA-seq analysis. With the majority of a total RNA sample being comprised of rRNA, removal of rRNA can provide more unique sequence analysis. Application of ribosomal depletion to total protein samples may increase the depth of analysis of a proteome and is currently being investigated in our laboratory.

Table 2 indicates selected proteins identified in iTRAQ analysis. Additional data representing the entire iTRAQ analysis is supplied in Files S1 and S2 in Supplementary Material. Since strain 2192 is mucoid, we would expect to see proteins such as those that play a role in alginate overproduction. Our proteomic analysis indicated that alginate motility regulator Z (AmrZ), AlgF, AlgC, and AlgD were upregulated in mucoid strain 2192 (Table 2). AmrZ, is a ribbon–helix–helix DNA-binding protein (Baynham et al., 2006) that can act as a transcriptional repressor (Tart et al., 2006) or enhancer of algD expression (Baynham et al., 1999). It was expected that AmrZ would be present as we had also observed its increased expression in our previous transcriptome analysis (Rao et al., 2008). AlgC is a phosphomannomutase enzyme which catalyzes the second step in the alginate pathway (Zielinski et al., 1991). AlgC converts mannose 6-phosphate into mannose 1-phosphate. Furthermore, AlgC is a phosphoglucomutase involved in LPS synthesis (Coyne et al., 1994). Here we observed AlgC significantly upregulated in strain 2192 (Table 2). AlgD, GDP-mannose 6-dehydrogenase, converts GDP-mannose into GDP-mannuronate which is the precursor of polymannuronic acid (Tatnell et al., 1994). Another alginate enzyme, AlgF was also upregulated by not identified as significant due to low number of peptide identifications (Table 2). AlgF is one of three proteins that function to acetylate O-2 and or O-3 of the mannuronic acid residues (Franklin and Ohman, 2002). Acetylation changes the physical and immunological properties of the alginate (Pier et al., 2001).

In addition to identifying σ²²-dependent alginate production proteins, outer-membrane protein F (PA1777) was also upregulated in strain 2192 (Table 2). Previous studies have shown that OprF is upregulated in mucoid strains (Hanna et al., 2000; Malhotra et al., 2000) and is directly controlled by σ²² (Firoved et al., 2002). Thus, our shotgun proteomic analysis was validated by the identification of some known alginate biosynthesis proteins and AlgU-dependent proteins such as OprF that were upregulated in the mucoid 2192 strain.

When P. aeruginosa overproduces alginate, there would be high demands on the central metabolism to produce metabolic intermediates and such to fuel the high energy demand for alginate overproduction. In Table 2, a number of proteins are shown that have direct roles in metabolism. Additionally, we noticed a number of hypothetical proteins upregulated in 2192 some of which are shown in Table 2.

Several heat shock proteins were downregulated in strain 2192 (Table 2). GroES, HtpG, and GroEL were significantly downregulated. Previous work has shown that CF patients produce antibodies to GroEL (Ulanova et al., 1997). Other recent work has shown that heat shock proteins such as GroES and HtpG are actually downregulated in biofilms (Patrauchan et al., 2007). It is interesting that heat shock proteins may be differentially expressed depending on the growth conditions such as planktonic versus biofilm modes of growth.

Many total transcriptome analyses have examined the effect of σ²² expression (Firoved et al., 2002, 2004a,1991; Firoved and Deretic, 2003; Wood et al., 2006; Wood and Ohman, 2009). However, here unlike other studies, we were also interested in proteins that have decreased expression in mucoid strains. In our iTRAQ analysis, three proteins which are components of one (HIS-I) of the three T6SS (Mougous et al., 2006) were downregulated in the mucoid strain. These proteins, TssB1 (PA0083), TssC1 (PA0084), Hcp1 (PA0085), have greater expression levels in non-mucoid strain 383 than in mucoid strain 2192 (Table 2). Hcp1 is secreted by the T6SS and has been found in CF sputum (Mougous et al., 2006). CF patients also have antibodies in their serum to Hcp1 (Mougous et al., 2006). Although the exact role of Hcp1 is not clear, it has been suggested that Hcp1 may form a channel through which macromolecules might pass and may be used for communication with the host (Mougous et al., 2006). Recently, the HSI-I system was shown to secrete Tse2 (PA2702) which is a toxin that arrests both prokaryotes and eukaryotes; however, Tse2 is targeted toward prokaryotes (Hood et al., 2010). We do not observe expression of Tse2 in either of our proteomic analysis or differential expression in transcriptomic analysis (Rao et al., 2008); however, tse2 is present in the 2192 genome⁵.

RetS is a hybrid sensor kinase that controls expression of the T6SS in P. aeruginosa. Without the retS, all of the HSI-I genes are highly expressed (Goodman et al., 2004; Mougous et al., 2006; Ventre et al., 2006; Figure 2). Comparison of our previous transcriptome analysis to the transcriptome analysis of ΔretS is shown in Figure 2. It seems than non-mucoid strain 383 has increased expression of most of the HIS-I operon and the HSI-II genes hsiA2, hsiC2, and hsiF1 (Figure 2). Furthermore, as mentioned, T6SS proteins TssB1, TssC1, Hcp1 were detected in iTRAQ analysis (Table 2). Also of interest, HSI-II genes were not affected by deletion of retS, but strain 383 has increased HSI-II expression of hsiA2, hsiC2, and hsiF2 (Figure 2). These data suggest that mucoidy may suppress expression of Type 6 secretion machinery in P. aeruginosa. To further corroborate this, examination of the literature revealed that inactivation of σ²² in a mucA mutant strain caused increased expression of HSI-I and HSI-II genes (Tart et al., 2005). In a recent study, extensive transcriptome analysis of early and late CF isolates revealed that, tssB1, tssC1, and hcp1 were upregulated in non-mucoid derivatives of mucoid CF strains (Rau et al., 2010). Collectively, our data along with others suggested an inverse relationship between the T6SS and the mucoid phenotype of P. aeruginosa. It has been previously theorized that the T6SS may be used to out compete other bacteria and increase the fitness of P. aeruginosa for infection of the CF lung (Hood et al., 2010). The addition of mucoid phenotype to this model paints a picture whereby early non-mucoid isolates compete to initiate infection and then mucoid phenotype emerges, after which chronic infection can be established, negating the necessity of T6SS.

One inherent drawback to the use of chromatography-based proteomics is potential bias due to association and elution of peptides during liquid separation. Theoretically, some peptides could lost due to interactions during separation. The advantage of 2-DE analysis is that it gives the size and pI of the isolated proteins, whereas only protein fragments are detected by iTRAQ. To visualize protein differences between the non-mucoid strain 383 and the mucoid strain 2192, independent biological samples for each strain, 383 and 2192, were separated and representative examples are shown in Figure S2 in Supplementary Material. Spots with differential expression were visualized (Figure S3 in Supplementary Material) and quantified for each strain. The predicted size and pI were compared to that of the full-length protein sequences (Table S2A in Supplementary Material). A protein spot on 2-DE can contain multiple co-migrating proteins (Table S2A in Supplementary Material). To determine the proteins that make up a 2-DE spot, either PMM or mass spectral analysis or both were performed (Table S2A in Supplementary Material). PMM indicates the proteins that are the most abundant in a cored spot (Table S2B in Supplementary Material). MS/MS does not suggest a protein is the most abundant but rather identifies peptides that may correspond to the any protein in the cored spot. As expected half of the protein spots analyzed contained multiple proteins and some proteins were identified in more than one location (Table S2A in Supplementary Material).

Total protein lysates, as well as membrane, and extracellular protein enriched preparations, were utilized for 2-DE analysis. For membrane/extracellular preparations, Triton X-114 treatments as indicated in Section “Materials and Methods” were also employed to enrich for detergent-soluble hydrophobic membrane proteins (Bordier, 1981). This method has been used in other bacterial proteomic studies (Sinha et al., 2005). 2-DE analysis revealed two outer-membrane proteins, OprH and OprF were differentially expressed between strains 383 and 2192 (Figure 3A). OprH and OprF spots were visible in the Triton X-114 enrichment as expected (Figure 3A). Previously, it has been shown that OprH protein is expressed in both mucoid and non-mucoid strains (Kelly et al., 1990; Hanna et al., 2000). Here, an OprH protein spot was observed in both strains but was visually more abundant in the mucoid 2192 strain than non-mucoid strain 383 (Figure 3A) however our densitometry did not corroborate this observation (Table 3).

One differential stained protein spot between mucoid strain 2192 and non-mucoid strain 383 (whole cell extract spot #39 and #53 Triton X-114 preparation) was a conserved hypothetical protein (PA0460; Figure 3B). Little to no information is available for PA0460 except that it has been shown to be five-fold upregulated when the sensor kinase phoQ is deleted from strain PAO1 (Gooderham et al., 2009). Based on our data presented here (Figure 3B), PA0460 may be a secreted protein, as it was predicted with signal peptide at N-terminal 21 amino acids followed by a cleavage site (data not shown). Spot #43 from mucoid strain (2192) whole cell extract identified PA0315, another conserved hypothetical protein (Figure 3B). PA0315 was upregulated in 2192 and our densitometry corroborated this observation. Recent data suggests PA0315 may be a homolog to a quorum sensing regulated protein of Burkholderia cepacia (Riedel et al., 2003).

Our previous transcriptome analysis had indicated a differential expression of T6SS proteins as described above. Furthermore, iTRAQ analysis revealed a significant decrease in Hcp1 in strain 2192. As expected, 2-DE gel electrophoresis confirmed our iTRAQ analysis (Table 2) that Hcp1 and TssB1 (PA0083) were increased in expression in 383 (Figure 3B and Table 3). However, more direct confirmation of this observation was necessary. For this, antibodies specific to TssB1 (PA0083), TssC1 (PA0084), and Hcp1 (PA0085) were used to quantify expression of the T6SS in CF strains 383 and 2192. In Figure 4, Western blot analysis is shown for TssB1, TssC1, and Hcp1. Antibodies specific for the alpha subunit of RNA polymerase (RpoA) were utilized as a loading control for the analysis. Western blot analysis confirmed iTRAQ and 2-DE and showed that TssB1 and Hcp1 were more highly expressed in the non-mucoid strain 383 than mucoid strain 2192 (Figure 4). Of interest antibodies for TssC1 (PA0084) suggested that TssC1 correlates with TssB1 and Hcp1 expression. These data suggest that strain 383 has increased T6SS protein expression than strain 2192 (Figure 4). Ultimately our data corroborate both the microarray and the two shotgun proteomic methods and show that the T6SS is differentially regulated in CF strains 383 and 2192.