Identification of Proteins Associated with the Pseudomonas

Article pubs.acs.org/jpr

Identification of Proteins Associated with the Pseudomonas aeruginosa Biofilm Extracellular Matrix Masanori Toyofuku,*,‡,† Bernd Roschitzki,§ Katharina Riedel,∥ and Leo Eberl*,‡ ‡

Institute of Plant Biology, Department of Microbiology, University of Zurich, Zurich, Switzerland Functional Genomics Center, University and ETH Zurich, Zurich, Switzerland ∥ Institute of Microbiology, Ernst-Moritz-Arndt-University of Greifswald, Greifswald, Germany §

S Supporting Information *

ABSTRACT: Biofilms are surface-associated bacteria that are embedded in a matrix of self-produced polymeric substances (EPSs). The EPS is composed of nucleic acids, polysaccharides, lipids, and proteins. While polysaccharide components have been well studied, the protein content of the matrix is largely unknown. Here we conducted a comprehensive proteomic study to identify proteins associated with the biofilm matrix of Pseudomonas aeruginosa PAO1 (the matrix proteome). This analysis revealed that approximately 30% of the identified matrix proteins were outer membrane proteins, which are also typically found in outer membrane vesicles (OMVs). Electron microscopic inspection confirmed the presence of large amounts of OMVs within the biofilm matrix, supporting previous notions that OMVs are abundant constituents of P. aeruginosa biofilms. Our results demonstrate that while some proteins associated with the P. aeruginosa matrix are derived from secreted proteins and lysed cells, the large majority of the matrix proteins originate from OMVs. Furthermore, we demonstrate that the protein content of planktonic and biofilm OMVs is surprisingly different and may reflect the different physiological states of planktonic and sessile cells. KEYWORDS: Biofilm, Extracellular matrix, Membrane vesicle, P. aeruginosa

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from chronically infected CF patients are mucoid variants.9 While alginate is the major exopolysaccharide in mucoid strains, Psl and Pel exopolysaccharides are critical for biofilm formation on abiotic surfaces.10−12 Carbohydrate analysis indicated that Psl is a mannose- and galactose-rich polysaccharide while Pel is a glucose-rich polysaccharide.10,13 Psl was recently shown to form a helical structure on the P. aeruginosa cell surface.14 These exopolysaccharides are thought to work as a structural scaffold and protect the cells from antimicrobial substances. More recently, extracellular DNA was shown to constitute an important component of the P. aeruginosa biofilm matrix.15,16 While proteins are considered to be important matrix components of P. aeruginosa biofilms,12,17 very little information on their identity and function is currently available. Interestingly, a recent study identified a biofilm matrixassociated protein, CdrA, which was suggested to cross-link the Psl polysaccharides and/or tether Psl to the bacterial cell wall, providing biofilm stability.17 The objective of this study was to identify P. aeruginosa matrix-associated proteins. Many of the identified proteins were found to be associated with OMVs. OMVs are spherical bilayered phospholipids with an average diameter of 20−200 nm, which are secreted in the environment by a variety of Gram-negative bacteria (for a review, see refs 18−20). OMVs

INTRODUCTION Many microbes exist as aggregates or as surface-associated communities, commonly referred to as biofilms, in their natural habitats.1 A key step in biofilm formation is the production of extracellular polymeric substances (EPSs), also known as the matrix. The biofilm matrix completely embeds the cells and thus provides protection against harmful conditions.2 The matrix was for a long time thought to consist mainly of polysaccharides; however, recent work has shown that, in addition to polysaccharides, significant amounts of proteins, nucleic acids, and lipids are present in the biofilm matrix.3 For example, amyloid proteins such as curlie fimbriae in Escherichia coli and TasA in Bacillus subtilis have been shown to be important components of the biofilm matrices formed by these organisms.4,5 In fact, proteins are sometimes even more abundant than polysaccharides in natural cell aggregates, such as in activated sludge flocks.6−8 Although proteins have been detected in the matrices of various biofilms, knowledge of the identity of these proteins is scarce. Pseudomonas aeruginosa has become an important model organism for studying bacterial biofilm formation. This bacterium is an opportunistic human pathogen that can cause life-threatening infections in patients with a compromised immune system. P. aeruginosa produces at least three exopolysaccharides; alginate, Psl, and Pel, all of which have been implicated in biofilm development under particular circumstances. The predominant morphotypes of isolates © 2012 American Chemical Society

Received: April 27, 2012 Published: August 21, 2012 4906

dx.doi.org/10.1021/pr300395j | J. Proteome Res. 2012, 11, 4906−4915

Journal of Proteome Research

Article

4 °C, and the supernatant was filtered through a 0.2 μm pore size filter (Filtropur S plus 0.2, SARSTEDT). The supernatant was ultracentrifuged for 2 h at 150,000g, 4 °C, and the pellet was resuspended in 10 mM HEPES (pH 6.8)/0.85% NaCl (w/v).

typically consist of outer membrane lipids, lipopolysaccharide (LPS), proteins, and DNA. They have important biological functions and were implicated in pathogenesis, cell−cell communication, nutrient acquisition, horizontal gene transfer, and competition with other organisms.19 OMVs are also found in the biofilms matrix while their biological function is not clear.21 The results of this study reinforce the idea that vesicles are a major constituent of P. aeruginosa biofilms, and the identification of proteins associated with biofilm-derived OMVs provides important information of their putative biological function.

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OMV Purification

OMVs were purified using the Optiprep protocol.26 Pellets of ultracentrifuged OMVs were suspended in 1 mL of 45% (v/v) Optiprep in 10 mM HEPES/0.85% NaCl which contained EDTA-free protease inhibitor (Roche) according to the manufacture’s conditions. The OMV suspension was transferred to the bottom of a tube and an Optiprep gradient was layered with 2 mL of 40%, 35%, 30%, and 25% and 1 mL of 20%. Gradients were ultracentrifuged at 100,000g, for 3 h and 1 mL fractions were removed from the top. The protein concentration of each fraction was measured by the method of Bradford27 to identify the fractions containing OMVs.

MATERIALS AND METHODS

Bacterial Strains and Culture Conditions

Biofilms were cultured on polycarbonate membrane filters (25 mm diameter, 0.2 μm pore, Whatman) placed on Luria− Bertani (LB) plates with 2% glycerol (v/v).22 To this end, 10 μL of overnight planktonic cultures grown in LB medium and diluted to an optical density at 600 nm (OD600) of 0.1 were spotted on the membrane. The bacteria were incubated at 37 °C for 48 h. Planktonic cultures for OMV isolation were grown in LB medium supplemented with 2% glycerol (v/v) at 37 °C for 16 h. Overnight planktonic cultures grown in LB medium were inoculated to an (OD600) of 0.01.

Sample Preparations for Liquid Chromatography Coupled to Tandem Mass Spectrometry (LC-MS/MS)

Two biological replicates were analyzed for each protein fraction. The matrix fraction was sonicated to fragment the DNA. The matrix fraction, cell lysates, and purified OMVs were concentrated by using a Vivaspin 500 centrifugal concentrator column with a 10 kDa cutoff. Ten micrograms of protein was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% acrylamide gel.28 Proteins were visualized by staining with colloidal Coomassie blue29 (Supporting Information Figure S1). Each lane was cut into 10 pieces, and the gel pieces were destained with 50% methanol in 25 mM ammonium bicarbonate (pH 7.8). The gel pieces were washed with acetonitrile and 50 mM ammonium bicarbonate (pH 7.8) several times. In gel, digestion of the proteins was performed following the protocol of Shevchenko et al.30 with a slight modification. Proteins were reduced with DTT (10 mM in 50 mM ammonium bicarbonate (pH 7.8)) at 60 °C for 30 min, washed with acetonitrile twice, alkylated by incubating with iodoacetamide (50 mM in 50 mM ammonium bicarbonate (pH 7.8)) for 1 h in the dark at room temperature, again washed with acetonitrile and 50 mM ammonium bicarbonate (pH 7.8) several times, and finally dried at room temperature. Trypsin digestion was carried out overnight using sequencing grade modified trypsin (Promega, Madison, WI) in 25 mM ammonium bicarbonate (pH 7.8), at 30 °C. The resulting peptides were extracted from the gel by incubating the gel at room temperature with acetonitrile 1% formic acid and 10% formic acid. Pooled peptide samples were concentrated in a Speedvac at 30 °C and desalted with C18 ZipTip (Millipore). Prior to analysis, the samples were resuspended in 15 μL of 2% acetonitrile/0.2% formic acid.

Protein Preparations

Colony biofilms were scraped from the membrane filter with a spatula and resuspended in 0.9% NaCl on ice (4-colony biofilms per 10 mL of 0.9% NaCl). To keep contamination with cellular proteins as low as possible, we avoided chemical treatment as well as sonication of the cells and only vortexed the suspension for 2 min. After centrifugation for 40 min at 15,000g, 4 °C, the supernatant was filtered through a 0.2 μm pore size filter (Filtropur S plus 0.2, SARSTEDT) to eliminate all bacterial cells from the supernatant. The cell pellet was washed 3 times with 0.9% NaCl and was used for protein analysis of biofilm cells. The EPS was precipitated by adding 3 times the volume of 100% ethanol to the filtered supernatant. Following storage at −20 °C overnight, the matrix was collected by centrifugation for 30 min at 15,000g, 4 °C.23 The EPS pellet was resuspended in 2 mL of 50 mM Tris-HCl (pH 7.5) containing EDTA-free protease inhibitor (Roche) according to the manufacters’s instructions. For Western blotting, the EPS fraction was further purified by phenol purification in order to eliminate carbohydrates.24 Bacterial cell pellets were suspended in 1 mL of the same buffer mentioned above and were homogenized by ultrasonication on ice. The cell lysate was filtered using a 0.2 μm pore size filter (Filtropur S plus 0.2, SARSTEDT) to eliminate intact cells. Isolation of OMVs

To extract OMVs from biofilms, a previously reported method was adapted.21 Biofilms grown on membrane filters were collected and resuspended in 0.9% NaCl on ice. The biofilm suspension was vortexed for 2 min and then centrifuged for 40 min at 15,000g, 4 °C. The supernatant was filtered through a 0.2 μm pore size filter (Filtropur S plus 0.2, SARSTEDT) and ultracentrifuged for 2 h at 150,000g, 4 °C. The pellet containing OMVs was dissolved in 10 mM HEPES (pH 6.8)/0.85% NaCl (w/v) and observed by transmission electron microscopy (TEM) or was further purified. OMVs from liquid cultures were extracted by a modified previously described method.25 To this end, cell culture was centrifuged for 40 min at 15,000g,

Protein Identification

Resuspended peptides were injected into an Eksigent-nanoHPLC system (Eksigent Technologies, Dublin, CA, USA) by an autosampler. Peptides were separated on a reverse-phase tip column (75−80 mm) packed with C18 material (Magic C18 200A AQ, 3 mm, Bischoff, Leonberg, Germany). The column was equilibrated with 97% solvent A (1% acetonitrile; 0.2% formic acid in water) and 3% solvent B (80% acetonitrile; 0.2% formic acid in water). Peptides from the biofilm matrix or cells were eluted using the following gradient: 0−50 min, 3−30% B; 50−58 min, 30−50% B; 58−60 min, 45−97% B; 60−67 min, 4907



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Table 1. Top 20 Abundant Proteins Present in the Biofilm Matrix present ina protein Fe(III)-pyochelin outer membrane receptor precursor ferripyoverdine receptor putative copper transport outer membrane porin OprC precursor catalase probable hemagglutinin probable TonB-dependent receptor probable aminopeptidase ornithine carbamoyltransferase, catabolic Haem/Hemoglobin uptake outer membrane receptor PhuR precursor organic solvent tolerance protein OstA precursor arginine deiminase Haem uptake outer membrane receptor HasR precursor hypothetical protein probable bacteriophage protein probable outer membrane protein precursor esterase EstA hypothetical protein hypothetical protein anaerobically induced outer membrane porin OprE precursor basic amino acid, basic peptide, and imipenem outer membrane porin OprD precursor a

PA number

normalized spectrum count

subcellular localization

b_OMVs

p_OMVs

PA4221 PA2398 PA3790 PA4236 PA0041 PA4675 PA2939 PA5172 PA4710 PA0595 PA5171 PA3408 PA0434 PA0622 PA2760 PA5112 PA0572 PA0328 PA0291 PA0958

289 234 229 184 127 124 105 103 97 91 90 88 81 69 69 68 64 60 59 55

outer membrane outer membrane outer membrane unknown outer membrane outer membrane extracellular cytoplasmic outer membrane outer membrane cytoplasmic outer membrane outer membrane unknown outer membrane outer membrane outer membrane outer membrane outer membrane outer membrane

+ + + + + + + + + + + + + + + + − − + +

+ − + + + − − + − + + − − + + − + − + +

A protein was counted as present (+) when it was found in both replicates.

97% B; 67−70 min, 97−3% B; 70−80 min, 3% B at a flow rate of 0.2 mL/min. Peptides from OMVs were eluted with the following gradient: 0−30 min, 3−10% B; 30−50 min, 10−40% B; 50−58 min, 40−55% B; 58−60 min, 97% B; 60−67 min, 97% B; 67−70 min, 97−3% B; 70−80 min, 3% at a flow rate of 0.2 mL/min. Mass analysis was done using a LTQ-ICR-FT Ultra (Thermo Scientific, Bremen, Germany) in the mass range of m/z 300−2000 and a target value of 1 × 106 ions. Peak lists were generated using MASCOT Distiller software 2.1.1 (Matrix Science) and searched against a database containing entries from P. aeruginosa PAO1 and potential contaminants such as keratin and yeast proteins (http://fgcz-s-021.uzh.ch/FASTA/) using the MASCOT search algorithm. The following settings were used in the search: maximum number of missed cleavages, one; peptide mass tolerance, ±5 ppm; fragment mass tolerance, ±0.6 Da. Carboxyamidomethylation of cysteine was set as fixed modification, and oxidation of methionine was selected as variable modification. Raw data have been deposited at the PRIDE database (http://www.ebi.ac.uk/pride)31 under accession numbers 22024−22027. The data were converted using the PRIDE Converter32 Scaffold (Proteome Software Inc., Version 3.00.03), which was used to visualize and validate MS/MS-based peptide protein identification. The minimum peptide probability was set to 95%, and the minimum protein probability was set to 99%. At least two unique peptides were required for the protein identification. The list of peptides used to identify the reported proteins, their sequence and peptide charge state, as well as the protein identification probability are provided in Supporting Information Table S1. The false discovery rate was estimated as lower than 1% by searching a database containing reverse sequences of the target proteins.33 Semiquantitative analyses of protein abundances were performed on the basis of the normalized spectrum count by the Scaffold software. Subcellular localization of identified

proteins was analyzed by using PSORTb (version 3.0.2) (http://www.psort.org/psortb/index.html).34 Western Blotting

Proteins were loaded to 10% SDS-PAGE gels and transferred on a polyvinylidene difluoride (PVDF) membrane by electroblotting. The membrane was incubated overnight in 3% (w/v) BSA in Trisbuffered saline (TBS) (10 mM Tris-HCl [pH 7.5], 50 mM NaCl) at 4 °C. The membrane was washed twice with TBS-T buffer (20 mM Tris-HCl [pH 7.5], 500 mM NaCl, 0.05% [v/v] Tween 20, 0.2% [v/v] Triton X100) and washed once in TBS. This was followed by incubation for 1 h in the primary antibody raised against OstA at a dilution of 1:2,000 in binding buffer (1.5% [w/v] BSA in 50% [v/v] TBS). After washing the membrane twice in TBS-T buffer and once in TBS buffer, the membrane was incubated with a secondary goat antirabbit antibody conjugated with alkaline phosphatase (Sigma-Aldrich) at a dilution of 1:10,000 in binding buffer. The membrane was washed four times in TBS-T and exposed to the substrates (nitro-blue tetrazolium chloride/ 5-bromo-4-chloro-3′-indolylphosphatase p-toluidine salt (Roche)) in reaction buffer (100 mM Tris-HCl [pH 9.5], 100 mM NaCl, 5 mM MgCl2) until bands appeared. After the reaction, the membrane was rinsed well with water and then dried between sheets of filter paper. Microscopy

For electron microscopic inspection, 6 μL of OMV samples was loaded on carbon-coated copper grids (300 windows) for 1 min and stained with 2% uranyl acetate for 10 s 3 times and finally washed in water for 10 s by floating the grids face down on the drops of uranyl acetate or water. A Philips CM100 transmission electron microscope was used for inspection of samples.

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RESULTS

Determination of the P. aeruginosa PAO1 Matrix Proteome

To identify proteins that are associated with the biofilm matrix and the biofilm cells, we used a colony biofilm model where 4908



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P. aeruginosa PAO1 was cultured on a membrane filter placed on a nutrient agar plate.22 The biofilm matrix was separated from the cells as described in detail in the Materials and Methods section. In order to identify the set of proteins associated either with the matrix material or the biofilm cells, we investigated respective purified fractions by a combination of 1D-SDS-PAGE and an LTQ-ICR-FT Ultra mass spectrometer. Two independent replicates for each sample were analyzed, and proteins that were found in both biofilm matrix replicates are shown in Supporting Information Table S2 and the top 20 abundant proteins are shown in Table 1. For comparison, the whole-cell proteome of the biofilm cells (b_cells) was also determined. A list of all the proteins identified is provided in Supporting Information Table S3. A protein was designated as “present” when it was found in both replicates, while it was defined as “absent” when it was not found at all. 178 proteins were present in the matrix preparation, and 764 proteins were present in the b_cells. 108 proteins were present in both the b_cells and the matrix sample. Although this may, at least in part, be due to lysed cells in the biofilm, it is noteworthy that 45 proteins were found exclusively in the matrix fraction. These proteins are highlighted in Supporting Information Table S2. As anticipated, among these 45 proteins was CdrA (PA4625), a protein recently demonstrated to be a structural component of the biofilm matrix,17 suggesting that the protocol for the matrix preparation worked well. In addition, the presence of the histone-like DNA binding protein PA1804 in the matrix proteome supports the idea that DNA is one of the major constituents of the biofilm matrix.15

suggesting that they have been specifically enriched. Recently, it has been demonstrated that Gram-negative bacteria produce

Figure 2. Subcellular distribution of the identified proteins: b_Cells, biofilm cells; matrix, biofilm extracellular matrix; b_OMVs, biofilm derived OMVs; p_OMVs, planktonic culture derived OMVs.

sphere shaped outer membrane vesicles (OMVs) which contain biologically active proteins.19 Proteomic profiling of OMVs derived from E. coli showed that they were greatly enriched for outer membrane proteins,35 which is reminiscent of our matrix proteome. These results let us hypothesize that the outer membrane proteins identified in the biofilm matrix were mainly derived from OMVs, supporting previous notions that OMVs are an integral part of the P. aeruginosa biofilm matrix.21,36 Identification of Biofilm OMV Proteins

To investigate whether OMVs are indeed present in the biofilm matrix, the purified matrix was observed by transmission electron microscopy (TEM). Many OMVs-like structures were observed as well as fiber-like structures (Supporting Information Figure S2). OMVs were further fractionated from the biofilm matrix by ultracentrifugation and were examined by TEM (Figure 4A). OMVs-like structures could be observed in the sample, confirming results of previous studies21 and supporting the idea that proteins associated with OMVs are major constituents of the matrix proteome. The matrix OMVs were further purified by density gradient ultracentrifugation and inspected by TEM (Figure 4B). Since OMVs were collected from a particular density gradient, these OMVs showed a more homogeneous size distribution relative to the crude OMV preparation (Figure 4A, B). Also proteins present in purified biofilm OMVs (b_OMVs) were identified by 1D-SDS-PAGE combined with nano-LC-ESI-MS/MS. The proteins that were identified in two independent replicates are listed in Supporting Information Table S3. When compared with the matrix proteome, 53 proteins were in common between the matrix and the b_OMV protein sets, suggesting that approximately 30% of the matrix proteins are associated with OMVs. Moreover, the top 10 abundant proteins of the

Figure 1. Venn diagram of proteins overlapping between different biological samples: b_Cells, Biofilm cells; Matrix, biofilm extracellular matrix; b_OMVs, biofilm derived OMVs; p_OMVs, planktonic culture derived OMVs. A protein was counted as present when it was found in both replicates and as absent when it was not found in any of the replicates.

The putative subcellular localization of the 45 matrix-specific proteins is shown in Figure 2C. We found it interesting that, while only a few secreted proteins could be identified, many of the proteins are likely localized in the outer membrane (Figure 2B, C; Table 1; and Supporting Information Table S2), 4909



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Figure 3. Detection of OstA. Immunoblotting of OstA in biofilm cells, biofilm OMVs, biofilm extracellular matrix, planktonic OMVs, and planktonic cells. Ten micrograms of protein was separated on a 10% acrylamide SDS gel. Figure 4. Transmission electron microscopy of OMVs. The upper pictures show OMVs collected from biofilms, and the lower pictures show OMVs collected from planktonic cultures. Crude OMVs indicate OMVs collected from the pellet of the supernatant after ultracentrifugation (A, C), and purified OMVs indicate OMVs further purified by Optiprep density gradient centrifugation (B, D). Arrows show representative OMVs. Scale bars, 200 nm.

matrix (Table 1) were also found to be present in the b_OMVs, suggesting that proteins associated with OMVs are major constituents of the matrix proteome. Interestingly, 9 proteins were identified in the b_OMV samples that were not found in the matrix proteome. This may be due to the fact that the protein identification rate is generally increasing with decreasing complexity of the sample. The matrix-associated protein CdrA was not found in the b_OMVs sample, suggesting that the OMVs have been successfully separated from the matrix material. As anticipated, outer membrane proteins were highly enriched in the OMVs (36.8% of all identified proteins) while they only represented 4.7% of the biofilm cell proteins (Figure 2A, D). To validate our results, we performed Western blots using antibodies directed against OstA (LptD/Imp), an essential outer-membrane protein required for outer-membrane biogenesis.37 In agreement with the proteome analysis, OstA was detected in purified matrix material as well as in b_OMVs (Figure 3), suggesting that the high proportion of outer membrane proteins in the biofilm matrix is due to the presence of large amounts of OMVs. Several strong bands in addition to the 104 kDa OstA molecular weight band were detected from the b_cells and the matrix fraction. These bands are most likely unspecific proteolytic degradation products of OstA. The rapid degradation of OstA in biofilms also explains why the OstA signal is weak in the b_OMV fraction.

The proteins identified in both vesicle types are listed in Table 2. Some of these proteins appear to constitute the core proteome of OMVs. Of these, 51.8% were cytoplasmic and 24% were outer membrane proteins. As observed from the Western blot analysis, OstA was among the proteins identified in the p_OMVs (Figure 3). The OstA immunoblot signal in Figure 3 was weaker in the b_OMVs compared to the p_OMVs, possibly because of proteolytic degradation of OstA in the biofilm, as discussed above. Further evidence for degradation of OstA in b_OMVs samples was obtained from the MS analysis, as OstA peptides were identified from several pieces of the SDS-PAGE gel including sections containing proteins with sizes much smaller than 104 kDa. Similar results were also obtained with b_cells and EPS samples where OstA peptides were also identified in several gel pieces. By contrast, in the p_OMVs sample, OstA peptides were exclusively identified in the gel section that contained proteins in the 104 kDa range (data not shown). It has been suggested that OMVs could possibly deliver virulence factors to host cells,39−42 and we therefore searched the proteomes of OMVs for virulence factors. While p_OMVs contained LasA (PA1871), LasB (PA3724), and alkaline protease (PA1249), b_OMVs did not contain these virulence factors. Interestingly, while absent in b_OMVs, these enzymes together with protease IV (PA4175) were present in the biofilm matrix fraction, suggesting that these virulence factors may be at least loosely associated with other components of the biofilm matrix. Eighteen proteins (24% of the b_OMVs proteome) were found to be unique for b_OMVs when compared with p_OMVs, and of these, 4 proteins (22%) were outer membrane receptor proteins related to iron acquisition (FpvA (PA2398), HasR (PA3408), FpvB (PA4168), and PhuR (PA4710)). FpvA, HasR, and PhuR were among the 15 most abundant proteins of the b_OMVs proteins. FptA (PA4221) was also identified in p_OMVs but was approximately 5.7-fold less abundant than in b_OMVs. Collectively, the proteomic analysis of the two OMV types suggested that b_OMVs may carry fewer virulence factors than p_OMVs.

Comparison of the Proteomes of Biofilm and Planktonic OMVs

Previous work has provided evidence that planktonic and biofilm OMVs are different, e.g. in LPS content.21 To investigate this issue in better detail, we determined the proteome of planktonic OMVs (p_OMVs), which were purified from a stationary phase liquid culture (Figure 4C, D). The average size of the purified planktonic OMVs was similar to the one of biofilm OMVs (Figure 4B, D). The proteins of planktonic OMVs identified are shown in Supporting Information Table S3. Approximately 2.5-fold more (194 versus 76) proteins were found in p_OMVs relative to b_OMVs. The proteome of p_OMVs contained many cytoplasmic proteins that were not found in b_OMVs. The presence of cytoplasmic proteins in p_OMVs has also been observed in a recent study by Choi et al.38 Some of these proteins could originate from cell lysis. However, only a few cytoplasmic membrane proteins were identified when compared to the whole cell proteome (Figure 2), suggesting that most of the cell debris has been eliminated by the purification procedure. 54 proteins were found in both planktonic and biofilm OMVs (Figure 1B).

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DISCUSSION Proteins have been suggested to play vital roles in the function of the biofilm matrix, and it is therefore surprising that 4910



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Table 2. Proteins Present in Both b_OMVs and p_OMVs PA no.

PA no.

protein

a

protein

Cytoplasmic Membrane PA3734 hypothetical protein Periplasmic PA0972 TolB protein Outer Membrane PA0041 probable hemagglutinin PA0291 anaerobically induced outer membrane porin OprE precursor PA0427 major intrinsic multiple antibiotic resistance efflux outer membrane protein OprM precursor PA0595 organic solvent tolerance protein OstA precursor PA0958 basic amino acid, basic peptide, and imipenem outer membrane porin OprD precursor PA1178 PhoP/Q and low Mg2+ inducible outer membrane protein H1 precursor PA1288 probable outer membrane protein precursor PA1777 major porin and structural outer membrane porin OprF precursor PA2495 multidrug efflux outer membrane protein OprN precursor PA2760 probable outer membrane protein precursor PA3790 putative copper transport outer membrane porin OprC precursor PA3800 conserved hypothetical protein PA4221 Fe(III)-pyochelin outer membrane receptor precursor Extracellular PA0620 probable bacteriophage protein PA0852 chitin-binding protein CbpD precursor PA1080 flagellar hook protein FlgE PA1086 flagellar hook-associated protein 1 FlgK PA1087 flagellar hook-associated protein type 3 FlgL PA1092 flagellin type B PA1094 flagellar capping protein FliD Unknown PA0622 probable bacteriophage protein PA2624 isocitrate dehydrogenase PA4236 catalase

Cytoplasmic PA0266 4-aminobutyrate aminotransferase PA0401 noncatalytic dihydroorotase-like protein PA0432 S-adenosyl-L-homocysteine hydrolase PA0546 methionine adenosyltransferase PA0792 propionate catabolic protein PrpD PA1580 citrate synthase PA1588 succinyl-CoA synthetase β chain PA2250 lipoamide dehydrogenase-Val PA2991 soluble pyridine nucleotide transhydrogenase PA3013 fatty-acid oxidation complex β-subunit PA3014 fatty-acid oxidation complex α-subunit PA3247 hypothetical protein PA3570 methylmalonate-semialdehyde dehydrogenase PA3584 glycerol-3-phosphate dehydrogenase PA4238 DNA-directed RNA polymerase α chain PA4269 DNA-directed RNA polymerase β* chain PA4270 DNA-directed RNA polymerase β chain PA4385 GroEL protein PA4470 fumarate hydratase PA4694 ketol-acid reductoisomerase PA4740 polyribonucleotide nucleotidyltransferase PA5013 branched-chain amino acid transferase PA5100 urocanase PA5171 arginine deiminase PA5172 ornithine carbamoyltransferase, catabolic PA5243 δ-aminolevulinic acid dehydratase PA5554 ATP synthase β chain PA5556 ATP synthase α chain Cytoplasmic Membrane PA2494 resistance-nodulation-cell division (RND) multidrug efflux transporter MexF

Proteins that were present in both b_OMVs and p_OMVs are listed according to their subcellular localiation.

greatly enriched in these OMVs. 53% of the predicted OM proteins of the matrix proteome were also identified in the proteome of b_OMVs, suggesting that a large proportion of the matrix proteins are associated with OMVs. Our data provide evidence that the matrix proteome consists of secreted proteins, proteins derived from cell debris, and proteins associated with OMVs. It is tempting to speculate that some of these proteins may either have important enzymatic functions in the matrix or are structural components for adherence to the surface. As expected, several exoenzymes involved in macromolecule degradation were detected in the biofilm matrix. These are PasP (PA0423), alkaline protease (PA1249), Chitinase (PA2300), probable aminopeptidase (PA2939), LasA (PA1871), LasB (PA3724), and Protease IV (PA4175). Most of these enzymes are well characterized virulence factors49 and could play a role in providing the cells with nutrients during infection by degrading host tissue. Another virulence related protein, RahU (PA0122), was identified in the matrix proteome. Although the exact function of RahU is unclear, it has been demonstrated that this protein is involved in biofilm formation in the presence of host derived eukaryotic phospholipids.50 Most of these proteins, except PA2939, were not found in the b_OMVs proteome, implying that they are associated with other matrix components such as polysaccharides, DNA, or proteins. Such an association is reasonable, as the interaction of enzymes with polysaccharides

information on the identity of the matrix-associated proteins is scarce. In this study we have identified the proteins associated with the matrix of P. aeruginosa biofilms. In addition to the expected extracellular proteins, a large number of membrane and cytoplasmic proteins were identified in the matrix preparation (Figure 2B). Membrane proteins and cytoplasmic proteins have also been found in the matrices of several microorganisms and even in environmental biofilms.43−47 However, the origin of these proteins in the matrix materials remained obscure. Cell death has been observed during biofilm formation, and it is thought to be part of the biofilm developmental process,48 suggesting that cell lysis could be one of the reasons why intracellular proteins are present in the biofilm matrix. Our data support the idea of limited cell death within biofilms, as 45 cytoplasmic proteins identified in the proteome of biofilm cells were also present in the biofilm matrix but were not found in OMVs. Another reason for the presence of intracellular proteins in the matrix could be due to OMVs. OMVs have been shown to be an important matrix component of P. aeruginosa biofilms.21 In agreement with these reports, we observed OMVs in the biofilm matrix using TEM (Figure 4 and Supporting Information Figure S2). Our proteome analysis revealed that 30% of the proteins identified in the biofilm matrix were also present in OMVs isolated from the matrix. In agreement with previous studies,35,38 we observed that OM proteins were 4911



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was shown to increase their stability.2 It is likely that the presence of these enzymes in the biofilm matrix is an important line of defense strategy against macrophages, predators, and other microbes. PA0041 is a putative hemagglutinin that is similar to the FhaB filamentous hemagglutinin in Bordetella pertussis.51 FhaB is a surface associated adhesion protein that has a crucial role in host-cell binding and infection. The presence of this putative hemagglutinin in the P. aeruginosa biofilm matrix suggests that this protein may be important for host−pathogen interaction. PA0041 is unusually large (362 kDa) and may therefore belong to the family of large surface proteins. Many of these proteins have been shown to be required for biofilm formation in phylogenetically diverse bacteria.52 Another protein that could be related to biofilm maintenance is PA2959, which is predicted to be an Mg-dependent Dnase. DNA is one of the important matrix components, and it has been demonstrated that Dnase treatment of biofilms causes its disintegration.15 It is therefore possible that PA2959 could be required for biofilm dispersal by degrading the matrix DNA. A considerable number of proteins related to oxidative stress were also found in the matrix fraction. These are catalase HPII (KatE, PA2147), a probable peroxidase (PA3529) and KatA (PA4236). Interestingly, KatA has also been found in the culture supernatant of a stationary-phase culture of P. aeruginosa53 and was shown to be important for biofilm susceptibility to hydrogen peroxide.54,55 The resistance of KatA against various proteases could be the reason why this enzyme is often found in the supernatant.53 In our study, KatA was also found in OMVs, indicating that KatA may be released from the cells by OMVs. The presence of antioxidant proteins in the biofilm matrix could be important for survival during infection, as phagocytic cells attack infecting microbes by producing H2O2.56 Whether the intracellular proteins identified in the biofilm matrix as well as OMVs have any biological function remains to be investigated. However, reports have accumulated that suggest a role of intracellular proteins outside of the cell. Such proteins that display two distinct functions are known as moonlighting proteins.57 For example, the elongation factor Tu, which was also found in the present study, has not only been demonstrated to be on the surface of P. aeruginosa cells but also to be important in the host-pathogen interaction.58 Likewise, in lactobacilli the elongation factor Tu as well as the chaperonin protein complex GroEL have been identified on the cell surface and were shown to promote adhesion to mucin and human epithelial cells.59,60 These intracellular proteins play important roles in the interaction with the host, and it is tempting to speculate that some of the intracellular proteins identified in the biofilm matrix may have yet unknown functions in pathogenesis. We identified matrix-associated proteins that were found neither in biofilm cells nor in OMVs (Supporting Information Tables S2 and S3). One of these matrix-specific proteins was CdrA (PA4625). CdrA is a recently characterized protein that was shown to link cells to the Psl exopolysaccharide.17 The expression of CdrA, which is secreted by its partner secretion system CdrB, was demonstrated to be controlled by c-di-GMP.17 The identification of CdrA as a matrix-specific protein in our study is indicative that our matrix purification protocol worked well. Another matrix-specific protein identified is the DNA-binding protein PA1804. DNA binding proteins have also been identified in the matrices of E. coli and Haemophilus inf luenzae, and they are among the most abundant proteins of a mine drainage microbial community biofilm.43,44,46 Within cells, DNA-binding proteins alter the architecture of the DNA, thereby

affecting gene transcription.61 Given that extracellular DNA is an important structural component of P. aeruginosa biofilms,15,16 it may not be too surprising that DNA-binding proteins are present in the biofilm matrix. Whether these proteins affect the function of extracellular DNA, e.g. the chelation of cations,62 remains to be elucidated. Most studies on OMVs have investigated OMVs derived from planktonic cultures. We were therefore interested if there are differences in the proteomes of biofilm and planktonic OMVs. 54 proteins were found in both planktonic and biofilm OMVs (Figure 1B, Table 2), for instance, OstA, an outer membrane β-barrel protein, that is required for outer-membrane biogenesis. This protein has previously been shown to be associated with planktonic OMVs of P. aeruginosa and E. coli.35,38 Eighteen and 113 proteins were found to be unique for biofilm and planktonic OMVs, respectively. Hence, the protein content of planktonic and biofilm OMVs was surprisingly different, and we speculate that this may reflect the different physiological states of planktonic and sessile cells. This is in agreement with previous studies that demonstrated that the protein profiles of OMVs differ between growth stages and culture conditions.26,63 Recent studies have demonstrated that OMVs derived from planktonic P. aeruginosa cultures play a role in pathogenesis.39,40,64 In the present study, only few virulence factors were identified in biofilm OMVs. For example, LasA, LasB, and alkaline protease were shown to be associated with p_OMVs while none of these virulence factors could be detected in the b_OMVs. This result supports the idea that the biofilm mode of growth is responsible for chronic and persistent infections rather than acute infections, as is the case with planktonic bacteria.65,66 One of the intriguing findings of this study was that b_OMVs contained many proteins related to iron acquisition. Iron is not only essential for growth but at high concentrations also promotes transition from the planktonic to the sessile state of growth.67,68 Under iron-limited conditions, biofilm formation of P. aeruginosa has been shown to be inhibited69,70 and factors required for iron acquisition are differentially up-regulated in biofilm cells.71 This may provide a simple explanation for the presence of iron acquisition proteins in b_OMVs. In summary, we have shown that the proteins associated with the P. aeruginosa matrix are derived from secreted proteins, from lysed cells, and to a large degree from OMVs. Further work will be required to analyze the role of these proteins in the physiology of biofilm cells.

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ASSOCIATED CONTENT

S Supporting Information *

Representative image of SDS-PAGE of each extract (10 μg of protein was separated on 12% acrylamide SDS gel: b_Cells, biofilm cells; matrix, biofilm extracellular matrix; b_OMVs, biofilm OMVs; p_OMVs, planktonic OMVs) and transmission electron microscopy of the biofilm matrix, with OMVs-like structures indicated by white arrows and fiber-like structures typical of EPS indicated by gray arrows; tables of peptides used to identify proteins; tables of proteins present in the biofilm matrix; and tables of identified proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*L.E.: phone, 41-44-634-8220; fax, 41-44-634-8204; e-mail, [email protected]. M.T. Graduate School of Life and 4912



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(14) Ma, L.; Conover, M.; Lu, H.; Parsek, M. R.; Bayles, K.; Wozniak, D. J. Assembly and development of the Pseudomonas aeruginosa biofilm matrix. PLoS Pathog. 2009, 5 (3), e1000354. (15) Whitchurch, C. B.; Tolker-Nielsen, T.; Ragas, P. C.; Mattick, J. S. Extracellular DNA required for bacterial biofilm formation. Science 2002, 295 (5559), 1487. (16) Allesen-Holm, M.; Barken, K. B.; Yang, L.; Klausen, M.; Webb, J. S.; Kjelleberg, S.; Molin, S.; Givskov, M.; Tolker-Nielsen, T. A characterization of DNA release in Pseudomonas aeruginosa cultures and biofilms. Mol. Microbiol. 2006, 59 (4), 1114−1128. (17) Borlee, B. R.; Goldman, A. D.; Murakami, K.; Samudrala, R.; Wozniak, D. J.; Parsek, M. R. Pseudomonas aeruginosa uses a cyclic-diGMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol. Microbiol. 2010, 75 (4), 827−842. (18) Tashiro, Y.; Uchiyama, H.; Nomura, N. Multifunctional membrane vesicles in Pseudomonas aeruginosa. Environ. Microbiol. 2012, 14 (6), 1349−1362. (19) Kulp, A.; Kuehn, M. J. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu. Rev. Microbiol. 2010, 64, 163−184. (20) Mashburn-Warren, L. M.; Whiteley, M. Special delivery: vesicle trafficking in prokaryotes. Mol. Microbiol. 2006, 61 (4), 839−846. (21) Schooling, S. R.; Beveridge, T. J. Membrane vesicles: an overlooked component of the matrices of biofilms. J. Bacteriol. 2006, 188 (16), 5945−5957. (22) Rani, S. A.; Pitts, B.; Beyenal, H.; Veluchamy, R. A.; Lewandowski, Z.; Davison, W. M.; Buckingham-Meyer, K.; Stewart, P. S. Spatial patterns of DNA replication, protein synthesis, and oxygen concentration within bacterial biofilms reveal diverse physiological states. J. Bacteriol. 2007, 189 (11), 4223−4233. (23) Wingender, J.; Strathmann, M.; Rode, A.; Leis, A.; Flemming, H. C. Isolation and biochemical characterization of extracellular polymeric substances from Pseudomonas aeruginosa. Methods Enzymol. 2001, 336, 302−314. (24) Hurkman, W. J.; Tanaka, C. K. Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 1986, 81 (3), 802−806. (25) Tashiro, Y.; Sakai, R.; Toyofuku, M.; Sawada, I.; NakajimaKambe, T.; Uchiyama, H.; Nomura, N. Outer membrane machinery and alginate synthesis regulators control membrane vesicle production in Pseudomonas aeruginosa. J. Bacteriol. 2009, 191 (24), 7509−7519. (26) Tashiro, Y.; Ichikawa, S.; Shimizu, M.; Toyofuku, M.; Takaya, N.; Nakajima-Kambe, T.; Uchiyama, H.; Nomura, N. Variation of physiochemical properties and cell association activity of membrane vesicles with growth phase in Pseudomonas aeruginosa. Appl. Environ. Microbiol. 2010, 76 (11), 3732−3739. (27) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248−254. (28) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227 (5259), 680−685. (29) Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9 (6), 255−262. (30) Shevchenko, A.; Wilm, M.; Vorm, O.; Mann, M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal. Chem. 1996, 68 (5), 850−858. (31) Vizcaíno, J. A.; Côté, R.; Reisinger, F.; Barsnes, H.; Foster, J. M.; Rameseder, J.; Hermjakob, H.; Martens, L. The Proteomics Identifications database: 2010 update. Nucleic Acids Res. 2010, 38 (Database issue), D736−42. (32) Barsnes, H.; Vizcaíno, J. A.; Eidhammer, I.; Martens, L. PRIDE Converter: making proteomics data-sharing easy. Nat. Biotechnol. 2009, 27 (7), 598−599.

Environmental Sciences, University of Tsukuba, Tsukuba, Japan; phone, 81-29-853-7810; fax, 81-29-853-6627; e-mail, [email protected]. Present Address †

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Colin Manoil for providing us P. aeruginosa PAO1. We thank John A. Robinson and Katja Zerbe for kindly providing us the OstA antibody. We thank the proteomics team of Functional Genomic Center Zurich, especially Simon Barkow-Oesterreicher and Jonas Grossmann, for technical support. We thank Andreas Kaech and Therese Bruggmann of the Center for Microscopy and Image Analysis (University of Zurich) for helping us with TEM analysis. We also thank Mike Givskov, Yosuke Tashiro, and Gabriella Pessi for scientific discussions. We thank Alex Grunau and Stefanie Heller for technical support. M.T. was supported by the Uehara memorial foundation.

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