Proteome Profiles of Outer Membrane Vesicles and Extracellular

Aug 25, 2015 - Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G 2W1, Canada. § Depar...
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Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms Narciso Couto, Sarah R. Schooling, John R Dutcher, and Jill Barber J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.5b00312 • Publication Date (Web): 25 Aug 2015 Downloaded from http://pubs.acs.org on September 1, 2015

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Journal of Proteome Research is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Proteome Profiles of Outer Membrane Vesicles and Extracellular Matrix of Pseudomonas aeruginosa Biofilms

Narciso Couto1¥, Sarah R. Schooling2,3, John R. Dutcher3, Jill Barber1,4*

1

Michael Barber Centre for Mass Spectrometry, Manchester Institute for

Biotechnology, Princess Road, University of Manchester, Manchester, M1 7DN, UK. 2

Department of Molecular and Cellular Biology, College of Biological Science,

University of Guelph, Guelph, ON, N1G 2W1, Canada. 3

Department of Physics, University of Guelph, Guelph, ON, N1G 2W1, Canada.

4

Manchester Pharmacy School, University of Manchester, Stopford Building, Oxford

Road, Manchester, M13 9PT, UK.

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Abstract In the present work, two different proteomic platforms, gel-based and gel-free, were used to map the matrix and outer membrane vesicle exoproteomes of Pseudomonas aeruginosa PAO1 biofilms. These two proteomic strategies allowed us a confident identification of 207 and 327 proteins from enriched outer membrane vesicles and whole matrix isolated from biofilms. Because of the physico-chemical characteristics of these sub-proteomes, the two strategies showed complementarity, and thus the most comprehensive analysis of P. aeruginosa exoproteome to date was achieved. Under our conditions, outer membrane vesicles contribute approximately 20% of the whole matrix proteome, demonstrating that membrane vesicles are an important component of the matrix. The proteomic profiles were analysed in terms of their biological context, namely a biofilm. Accordingly relevant metabolic processes involved in cellular adaptation to the biofilm lifestyle as well as those related to P. aeruginosa virulence capabilities were a key feature of the analyses. The diversity of the matrix proteome corroborates the idea of high heterogeneity within the biofilm; cells can display different levels of metabolism and can adapt to local microenvironments making this proteomic analysis challenging. In addition to analysing our own primary data, we extend the analysis to published data by other groups in order to deepen our understanding of the complexity inherent within biofilm populations.

Keywords: Pseudomonas aeruginosa, biofilms, matrix, outer membrane vesicles, proteome

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Introduction Overview Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium, able to use a wide range of carbon sources and to grow in harsh environmental conditions including high temperature, high salt concentrations and reduced oxygen.1,

2

The

metabolic versatility of P. aeruginosa is in large part responsible for its ecological success. This bacterium is an opportunistic pathogen; in humans with compromised immune systems, it may cause severe and debilitating infections with lethal outcomes.3 The most susceptible individuals are patients with cystic fibrosis, in whom it causes severe respiratory infections and, because of its high resistance to antibiotics,4, 5 is almost impossible to eradicate.6

Biofilm lifestyle P. aeruginosa is capable of adopting either a planktonic free-swimming or a sedentary community lifestyle, commonly referred to as a biofilm. Biofilms are dynamic, complex and heterogeneous community structures, which are inherently resistant to antibiotics and biocides. They are the cause of many chronic infections. In the body, the immune system is able to recognise antigens produced by the bacteria in biofilms and to initiate the correct immune response and produce antibodies, but these are ineffective against biofilms and often lead to further complications in the form of unwanted immune reactions such as inflammation and tissue damage.3

The biofilm matrix

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Biofilm architecture and integrity is maintained by interactions of the bacterial population within a complex hydrogel matrix referred to as the extracellular polymeric matrix. Components of the matrix are responsible for adhesion to solid surfaces and for the cohesive structure of the biofilm itself. In addition to polysaccharides, the biofilm matrix also contains DNA, RNA and proteins.7-9 Proteins are abundant in the matrix and include proteins with auxiliary functions such as adhesin factors (type IV pili, flagella and fimbriae) and secreted factors that negate antibacterial agents; they also provide a barrier to phagocytes.10 Many small signalling molecules, with various functions, are also found in the matrix.11, 12 Like many Gram-negative bacteria, P. aeruginosa blebs vesicles from the outer membrane of the cell during both planktonic and sessile growth, so that the matrix also contains outer membrane vesicles.7-13 Membrane vesicles are spherical and between 50 and 250 nm in diameter, which allows them to pass through sterile filters.14 Because the outer membrane (OM) is the site of vesicle genesis, the main components of these vesicles include proteins and lipids of the outer membrane, and periplasmic material, which is pinched off into the vesicle during blebbing. More surprisingly, cytoplasmic proteins and genetic material such as DNA and RNA have also been reported in association with these structures. OMVs play an important role in pathogenicity, as a delivery system, although other secondary roles have also been proposed.8, 13 Although the biofilm has a major cellular protective role, its inhibition also represents a target for drug action. When iron is lacking, biofilm development is impaired.15-17 P. aeruginosa biofilm control and disruption can be achieved by siderophore-antibiotic conjugates.18 Attempts have been made to use the iron uptake system as a target in vaccine development.19, 20 OMVs derived from Neisseria meningitidis bacteria have

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been successfully used in vaccine trials against meningococcal disease and multicomponent OMV vaccines comprising antigens from more than one pathogen have also been developed.21, 22

Proteomic analysis of the biofilm matrix The decoding of the P. aeruginosa genome has allowed proteomic analysis of this bacterium under different conditions.23 Previous work has been published on the proteomes of both whole cells and OMVs in the planktonic population.24-26 In addition, there is a single publication describing a one-dimensional gel-LC-MS/MS analysis of the P. aeruginosa biofilm exoproteome.27 The present work describes two different,

complementary,

two-dimensional

strategies

for

understanding

the

exoproteome of the biofilm matrix and associated OMVs. The combined approach allows a deeper understanding of this complex environment, of significant importance to understanding biofilm-related infectious disease processes and for the treatment of the debilitating infections which blight the lives of individuals, such as those with cystic fibrosis.

Materials and Methods Materials The majority of materials and solvents used throughout this work were of the highest quality available and were purchased form Sigma Aldrich (Poole, UK), unless otherwise stated.

Biofilm growth, isolation and purification of membrane vesicles

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Experiments involving biofilm growth, isolation and purification of membrane vesicles were executed according to Schooling et al.7,

8

P. aeruginosa PAO1 biofilms were

grown using the agar plate model (TSA, Becton Dickinson and Company, Oxford, England). An overnight inoculum of grown cells in Tryptic Soy Broth (TSB), at 37 °C and 125 rpm, was swirled over the surface of freshly prepared agar plates. The excess liquid was removed and the plate incubated at 37 °C for 24 hours which is the equivalent to the early stationary phase as measured by the biomass generated. Isolation of the matrix and outer membrane vesicles from the biofilm were performed as described in Supporting Information E1. Briefly, biofilms were scraped from the surface and cell-free matrix was achieved by centrifugation and filtration. The absence of cells was verified by transmission electron microscopy (TEM) and by plating triplicate 100 µL aliquots onto TSA plates and incubating 18 h at 37 °C. A brief description of sample preparation for transmission electron microscopy analysis is supplied in Supporting Information E2. When the matrix was processed for OMV isolation, the purified outer membrane vesicle fraction was obtained by ultracentrifugation followed by isopycnic density gradient centrifugation. Whole matrix material was only extensively dialysed to remove pigments and low molecular weight components. TEM was also used as a quality control to assess the efficient removal of OMVs from the matrix and fractionation from other particulates was achieved. Biological replicates were prepared in triplicate and, for each biological replicate both the matrix and the OMVs were derived from a single technical replicate. The protein content of the isolated fractions was quantified, in triplicates, using a micro-bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, IL, USA).

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Delipidation of samples Delipidation of samples was based on described methods,28,

29

with some

modifications, and the experimental procedure is described in Supporting Information E3. After this procedure, protein content was estimated using the BCA assay (Pierce Biotechnology, IL, USA). Assays were supplemented with 0.01 % (w/v) sodium dodecyl sulfate (SDS) to help protein solubilisation. These OMVs and matrix protein extracts were used in all subsequent proteomic analysis.

Two-dimensional (2D) gel electrophoresis Reagents for two-dimensional gel electrophoresis were purchased from Bio-Rad (Hemel Hempstead, UK). 2D gel electrophoresis was performed according to Nouwens et al.30 and a detailed description is supplied in Supporting Information E4. Triplicate biological replicates were assessed. Briefly, 200 µg of protein extracts from the OMV-enriched or whole matrix fractions were used in these experiments. In the first dimension, isoelectric focussing (IEF) was performed using IPG strips (11 cm, non-linear pH 3-10). In the second dimension, a 14% SDS polyacrylamide gel electrophoresis (PAGE) was performed.

2D LC-MS/MS Proteins extracted from OMVs and matrix (200 µg of each sample) were precipitated overnight in acetone at 4 °C and the pellet resuspended in 50 mM ammonium bicarbonate. Disulfide bonds were reduced with DL-dithiothreitol (DTT) and alkylated with iodoacetamide, prior to tryptic digestion with a ratio protein: trypsin (50:1) at 37 ºC. Following digestion, peptides were dried in vacuo, resuspended in strong cation exchange (SCX) buffer A (10 mM KH2PO4, pH 2.8, 20% (v/v) CH3CN) and off-line

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fractionated on an Ultimate 3000 (Dionex, Surrey, UK) with a 200 × 2.1 mm, 5 µm, 300 Å, polysulfoethyl A column (Poly LC, MD, USA). A linear gradient was used to perform the elution over 40 minutes from 0% to 40% solvent B (10 mM KH2PO4, 500 mM KCl, pH 2.8, 20% (v/v) CH3CN). Twenty fractions were manually collected at 1 min intervals throughout the gradient, dried in vacuo to remove organic solvent and stored at -20 °C. The second (reverse-phase) dimension of the chromatographic separation was performed on an Ultimate 3000 capillary LC system (Dionex, Surrey, UK) on-line connected to a quadrupole time-of-flight (Q-ToF) Global mass spectrometer (Waters, Manchester, UK) via a distal-coated fused silica PicoTip emitter using a capillary voltage of 2.0-2.8 kV into a Z-sprayTM ion source. Reverse-phase specifications are provided in Supporting Information E5. Product ion spectra were acquired in an automatic acquisition mode where the MS survey scan was performed over the range m/z 400-1800 and MS/MS survey scan mode over the range m/z 50-1800. The three most intense peaks on each MS survey scan were chosen for collision induced dissociation (CID) and MS/MS acquisition. Collision cell offset voltages were optimized and applied according peptide m/z values and charge state. A collision energy profile was generated for doubly, triply and quadruply charged species (25-50 V) in the m/z range of 50-1600 units. Active exclusion was set at 2 spectra and released after 30 seconds.

In-gel digestion and LC-MS/MS After 2D gel electrophoresis, 43 and 83 gel spots were excised from representative gels of the OMV-enriched or whole matrix fractions respectively, cut into small cubes and transferred into a microcentrifuge tube and in-gel digestion was performed as

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previously described.31, 32 The in-gel digestion experimental procedure is described in the Supporting Information E6. After in-gel digestion, an Ultimate 3000 capillary LC system (Dionex, Surrey, UK) interfaced to an Amazon ion trap mass spectrometer (Bruker, Bremen, Germany) was used to perform reverse-phase chromatography and MS/MS acquisition. Reverse-phase separation was performed as described in the Supporting Information E5. On the ion trap mass spectrometer, the electrospray was achieved via a distal-coated fused silica Picotip emitter using a capillary voltage of 1.7-2.2 kV. The typical settings for the mass spectrometer were: dry gas temperature was set to 150 °C, dry gas was set to 6 L min-1, the scan mode was set to standard enhanced with an m/z range of 200-3000 for MS and ultra-scan for MS/MS acquisitions. Ions were accumulated in the trap until the ion charge count (ICC) reached 200,000 with a maximum accumulation time of 200 ms. AutoMS(n) was selected where the top three more intense peaks are chosen for collision induced dissociation (CID) with a total ion count (TIC) absolute threshold of 25,000 and a relative threshold of 5% of the base peak. Precursor m/z values were dynamically excluded after 2 spectra for 60 seconds with isolation window 4 m/z units. CID was performed in the presence of helium and MS/MS fragmentation amplitude was set at 1.0 V ramped from 30-300% of the set value.

Database searching All

database

searches

were

submitted

to

an

in-house

Mascot

search

(http://msct.smith.man.ac.uk/mascot/home/html). A P. aeruginosa .fasta file was uploaded from UniProt (http://www.uniprot.org/, January 2015) and used to perform the search, using the following specifications. For data acquired on a Q-ToF mass

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spectrometer: the enzyme was specified as trypsin, one missed cleavage was allowed, fixed modification: carbamidomethyl on cysteines, variable modifications: deamidation of asparagine and glutamine, pyroglutamate formation on N-terminus glutamate and glutamine and oxidation of methionine, peptide tolerance: 200 ppm, MS/MS tolerance: 0.5 Da. Similar setting were used with data acquired on the Amazon mass spectrometer, using trypsin as the enzyme, allowing for two missed cleavages and assuming, only the peptide tolerance and MS/MS tolerance were changed to 300 ppm and 0.6 Da respectively. Duplicate technical analyses were carried out. A decoy database was also used to increase confidence in the results and only results fitting a false discovery rate (FDR) of 1% and proteins containing 2 unique peptides with a peptide score above 20 were considered true identifiers. The exponentially modified protein abundance index (emPAI) was applied to obtain semi-quantitative information.33 EmPAI offers approximate, label-free, relative quantification of the proteins in a mixture based on protein coverage by the peptide matches in a database search result. Assignment of predicted protein isoelectric point (pI), mean Kyte-Doolittle hydropathicity (GRAVY score), cellular location and functional class was performed according to the designation in the Pseudomonas genome project (http://www.pseudomonas.com/, January 2015).34, 35

Results

Two different sub-proteomes of the P. aeruginosa PAO1 biofilm (whole matrix and enriched outer membrane vesicles) were independently analysed using two different proteomic workflows (gel-based and gel-free). In the gel-free approach, samples were treated with trypsin and the resulting tryptic peptides were fractionated by two-

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dimensional (2D) liquid chromatography. A stand-alone strong cation exchange (SCX) step was followed by LC-MS/MS in which the LC was (as usual) a reverse phase chromatography and the MS/MS was performed on an ESI Q-ToF. In the gelbased approach, protein separation was achieved by 2D-gel electrophoresis, which was followed by in-gel trypsinolysis and fractionation of the tryptic peptides by RF liquid chromatography prior to MS/MS using an ion-trap mass spectrometer for data acquisition. Mascot (using the P. aeruginosa database) was used for protein recognition using peptide false discovery rate of less than 1 % and the presence of at least two unique peptides to confirm the presence of any protein. The UniProt database and the specialist P. aeruginosa database, the Pseudomonas genome project (http://www.pseudomonas.com/),34,

35

were used together to provide

information on the predicted isoelectric point (pI), the hydrophobicity using the grand average of hydropathicity index (GRAVY), the sub-cellular location and the functional role of each of the proteins detected. The information in these databases is still incomplete and potentially biased in favour of highly studied proteins; however, together they provide a powerful tool for data organisation based upon cell location or function. An estimation of protein abundance was inferred based on emPAI values.

Proteome profile of OMVs and whole matrix Our proteomics strategies, gel-free and gel-based, allowed the identification of 128 and 141 proteins respectively in the OMV enriched fraction from P. aeruginosa biofilms. Similarly, 227 and 259 proteins were identified from the whole matrix (including OMVs). The combination of both strategies allowed the identification of 207 and 327 proteins in the purified OMVs and matrix biofilms (Figure 1). Identified

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peptides from each protein and protein annotation are shown in Supporting Information S2 and S3 respectively for the OMVs and the whole matrix. 2D-gel images are shown in Figures S1 and S2 for OMVs and matrix respectively. Figure 1A shows immediately that the two workflows led to the identification of different proteins. The overlap was surprisingly small; just 62 out of 207 proteins from the OMVs and 160 out of 326 proteins in the whole matrix fraction were identified by both methods. When the two sub-proteomes were compared, there was also less overlap than might be expected (Figure 1B). 48 proteins were common between OMVs and the matrix using the gel-free methodology while, with the gel-based approach, 49 were common. Altogether, 68 proteins were common between the OMV-enriched fraction and the whole matrix. Since the OMVs are contained within the matrix, all OMV proteins must be present in the matrix exoproteome. The presence of very abundant non-OMV proteins in the matrix meant that low abundance OMV proteins fell below the detection limit of the experiment.

Figure 1 goes here

OMVs as a component of the whole matrix Based on the number of identified proteins in the OMV-enriched fraction which were also detected in the matrix exoproteome (Fig 1B) and assuming that these proteins are exclusively derived from the OMVs, an estimation of the contribution of proteins by OMVs to the matrix could be made. In the gel-free approach, 48 out of 227 proteins detectable in the matrix were attributable to OMVs; therefore, the OMVs contribute about 21% of the whole matrix exoproteome. From the gel-based strategy,

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49 out of 259 proteins were attributable to OMVs, contributing 19% of the whole matrix. Accounting for all proteins from both proteomics approaches, 21% of the whole matrix sub-proteome is attributable to the presence of OMVs. The gel-based strategy relies upon selection of spots for analysis, whereas the gel-free strategy allows interrogation of the whole sample, so it is gratifying that the two estimates differ by only 2 %. We also quantified the total protein (using a BCA assay) of OMVs and total matrix, and this indicated that OMVs quantitatively contributed approximately 15% (w/w) of the total matrix proteome. Overall, our results suggest that OMVs contribute approximately one-fifth of the protein content of the matrix. This contrasts with Toyofuku et al.27 who estimated that OMVs account for approximately 30% of the total matrix proteome. Since vesiculation is dependent on the growth of the biofilm and environmental stressors, very close agreement between their results and the present study is not necessarily expected.

Comparison of our proteome profile with others We present here the most comprehensive proteomic analysis to date of the exoproteome of matrix and OMVs from biofilms of P. aeruginosa. Earlier work concentrated on the extracellular proteome and OMVs of P. aeruginosa planktonic populations (Table 1). Bauman et al.28 identified the most abundant proteins in planktonic OMVs while Choi et al.24 identified 338 planktonic OMV proteins, and their work was used to annotate the P. aeruginosa database.

Maredia assessed the

effect of ciprofloxacin on vesiculation in planktonic populations and 145 proteins were identified.25 From the culture supernatant26 of P. aeruginosa PA14, 205 proteins were identified.

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Table 1 goes here

Toyofuku et al.27 recently compared the proteome profiles of OMVs from both biofilm and planktonic cultures, and the extracellular matrix from biofilm. As shown in Table 1, in total 76 OMVs and 178 matrix proteins were identified from biofilms, rather more from planktonic cultures. Biofilm-derived OMVs are smaller than their planktonic counterparts and 1-D SDS-PAGE protein analysis indicated a less diverse protein profile for the biofilm OMVs, a result consistent with those of other groups.8 Although the annotated OMV proteins in the P. aeruginosa database derive from a planktonic population, 93 (out of 207 proteins) of our OMV proteins and 68 (out of 327 proteins) of our matrix proteins were common. Many of the annotated OMV proteins such as EF-G, DnaK, catalase and 50S ribosomal proteins L11, L21 and L25 might reasonably be present in the matrix as a result of cell lysis or leakage. Many outer membrane proteins that are expected to exist in the OMVs are not annotated in the P. aeruginosa database. These include highly abundant proteins such as porin P, ferripyoverdine receptor, Fe(3+)-pyochelin receptor and several TonB-dependent siderophore receptors. This database is curated by peer-generated contributions and, as discussed earlier, this is a limitation in data interpretation. Toyofuku et al.27 identified 76 OMVs proteins (found in two biological replicates). Only 37 are common to our work, as shown in Table 2. This is not necessarily unexpected, since biofilms vary over time and are particularly sensitive to environmental and nutritional conditions. In Toyofuku’s experiments,27 biofilms were grown on polycarbonate membrane filters on LB agar plates and harvested 48 hours after inoculation. In our experiments, biofilms were generated on TSA agar plates and harvested after 24 hours. Age at the time of harvesting (with the potential for cell

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death and the accumulation of cell debris) may account for the fact that a high number of OMV proteins identified by Toyofuku et al. were annotated as cytosolic (39 %). Our own OMVs underwent particularly rigorous purification as confirmed here by electron microscopy (Figure 2), and previous assessments during protocol development.14, 15

Figure 2 goes here

Table 2 goes here

We also compared our OMVs and those of Toyofuku et al.27 with the planktonic OMVs reported by Choi et al.24 26 proteins were common to all three studies. Results are summarised in Table 3.

Table 3 goes here

The small number of common proteins between the three studies may be attributed to several factors. Choi et al worked with planktonic cultures instead of biofilms, and growth and harvesting were different in each of the three studies. The MS dynamic range also contributes to variability – it is often the case that a poorly abundant protein falls below the detection limit for the instrumentation, resulting in a false negative result, because of the presence of other (abundant) proteins.

Hydrophobicity, pI and MW

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Because of the differences between the gel-based and gel-free approaches, the physico-chemical properties which affect protein solubility and separation such as hydrophobicity, isoelectric point (pI) and MW were investigated. MW, pI, and hydrophobicity are important parameters that affect proteins that are separated using 2D-gels. Most hydrophobic proteins have positive GRAVY values while most hydrophilic proteins have negative values. Predicted hydrophobicity values were extracted from the P. aeruginosa database and almost all identified proteins have a negative GRAVY value indicating that they are predominantly hydrophilic. In the OMVs enriched fraction and the whole matrix, only 16 out of 207 (8%) and 57 out of 327 (17%) have positive values and the most hydrophilic and hydrophobic proteins from both fractions are shown in Table S1. This was not expected for OMV proteins which are normally outer membrane associated, although hydrophilic behaviour of OMV proteins has also been observed by others.30, 36 Two factors may contribute to the anomalous hydrophilicity.

Domains of high

hydrophilicity located in the cytosol or facing the extracellular region will impact on GRAVY scores. In addition, it is worth noting that these predicted values do not account for post translational processing and modifications (such as phosphorylation and glycosylation) which can affect the observed MW and pI. This behaviour was also observed in the membrane proteome of P. aeruginosa.30 Glycosylation30 and phosphorylation are important post-translational modifications in the outer membrane and exoproteome .26 Figure 3 shows examples of the same protein distributed in different regions of the 2D gels for both OMVs and the matrix.

Figure 3 goes here

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Semi-quantitative evaluation of OMVs and matrix proteins Tables 4 and 5 show the 30 most abundant proteins in OMVs and in the matrix biofilms based on EmPAI values. The most abundant proteins in the OMV purified fraction were PagL, OprQ and OprO, while in the matrix the most abundant proteins were PasP, FliC and HutU. Toyofuku et al.27 reported that LasA, LasB and alkaline phosphatase were absent in the OMVs of biofilms though present in the planktonic counterpart. In our samples, the virulence factor LasB is abundant in the matrix, though absent in the OMVs; conversely, LasA is seen in OMVs but is not sufficiently abundant to be seen in the matrix. It appears that LasB is not efficiently entrapped in the OMVs but is released into the extracellular environment.

Tables 3 and 4 go here

Proteome profile of OMVs and whole matrix according cellular location The P. aeruginosa database incorporates protein cellular location information with three different levels of confidence: level 1 when localization has been experimentally observed, level 2 when high similarity with similar proteins in other organisms exists, and level 3 when location is computationally predicted. It should be noted that some of the identified proteins have a disputed cellular location and can exist in different parts of the cell or even externally, and a high percentage of proteins have unknown function. From our OMV enriched fraction, the majority of the identified proteins could be assigned to the category of OMVs (93 out of 207 proteins), outer membrane (30) and unknown location (39) (Figure 4A).

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Figure 4 goes here

69% of the OMV proteins observed here were also observed (or predicted) by others to exist in the OMVs or outer membrane of planktonic P. aeruginosa.24, 25, 27, 30 These results are consistent with the origin of OMVs from the outer membrane. Whilst outer membrane proteins were dominant in our OMV fraction, only 10 identified proteins (class 1 and 2) are annotated as periplasmic (Table S2). This is consistent with the average smaller lumen of biofilm OMVs compared with planktonic OMVs.15 In total, only 8 annotated extracellular proteins, classes 1 and 2, were found in the biofilm OMVs analysed here (Table S3). Cytoplasmic proteins were not expected, nevertheless 5 cytoplasmic proteins (class 1) and 8 (class 2) were confidently identified (Table S4). In total, 5 cytoplasmic membrane associated proteins, classes 1 and 2, were found in this fraction as shown in Table S5. Some controversy exists here but proteins such as DNA-binding protein HU, NirF, arginine deiminase and GroEL have been associated with OMVs by others. Moreover, the presence of DNA-binding HU and DNA-directed RNA polymerase in OMVs supports the presence of DNA in association with OMVs. From the entire matrix, 327 proteins were distributed according to cellular location as shown in Figure 4B. The majority of the observed proteins were predicted to be cytoplasmic although only 26 are annotated as such in the P. aeruginosa database. These observations are consistent with the extracellular matrix containing proteins derived from cell lysis and/or leakage of cytoplasmic content during the formation of outer membrane vesicles.

Surprisingly, from the whole matrix, only 7 identified

proteins were experimentally observed by others to be secreted by P. aeruginosa, of

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which only PasP and LasA are also annotated as OMV proteins. These are indicated in Table S6.

Proteome profile of the biofilm OMVs and matrix according to function Figure 5 shows the cellular distribution of the proteins identified here, and the distribution of identified OMVs and matrix proteins according to functional categories (26 in total based on the P. aeruginosa database) is shown in Figure S3.

Figure 5 goes here

Proteins involved in the transport of small molecules are particularly abundant in the OMV enriched fraction (Figure 5); the majority of these are (not surprisingly) membrane proteins. 40 outer membrane proteins including porins (OprC, OprH, OprQ, OprH, OprD, OprO, OprF and OprE) and receptors with different levels of specificity were identified among these small molecule transporters. Some of those proteins reflect adaptation to the biofilm micro-environment with its particular specificities (temperature, pH, oxygen, and nutrient availability) which typically differs from the planktonic population. Proteins that reflect this adaptation include: the anaerobically-induced outer membrane porin OprE, the organic solvent tolerant OstA; and the osmotically inducible lipoprotein OsmE. Motility and attachment are particularly important in biofilm development and maturation and proteins associated with motility, attachment and protection such as FimV, B-type flagellin FliC, Neisseria PilC beta-propeller, Type IV pilus secretion PilQ, fimbrial protein and protein PilW were identified; these proteins are required for biofilm formation and interaction with host cells. Since these proteins must traverse the periplasm and the outer

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membrane in order to carry out their normal functions, their detection in OMVs is not unexpected. P. aeruginosa is known for its high resistance to antibiotics and the outer membrane proteins OprM, OprD and OprQ, FemA and acyl-homoserine lactone acylase QuiP have been shown to be involved in antibiotic resistance. The abundance of PagL, a lipid A deacylase, and the esterase EstA is interesting. These proteins may be involved in remodelling of the biofilm matrix, perhaps to reduce its immunogenic potential (and therefore its recognition by the host).37-41 A large number of proteins are of hypothetical or unknown function, reflecting the fact that P. aeruginosa is a pathogen, not a laboratory test organism. It is not known how many of these proteins are essential, and whether there is potential for new drug targets to be found among them. The whole biofilm matrix also contains many proteins (67) classified as having unknown function, although some are predicted to have enzymatic function based on the presence of amino acid motifs. Proteins involved in iron regulation (also present in the OMV enriched fraction), enzymes from the glyoxylate shunt and arginine deiminase pathway usually involved in energy metabolism were also seen. These proteins are normally regarded simply as lysis products – the debris of cell death. The presence of whole metabolic pathways is interesting, however, and suggests that a functional role cannot be ruled out. Twenty two of the identified matrix proteins were categorized as putative enzymes, 21 proteins are involved in adaptation and protection, and seven secreted factors are known to exist extracellularly: elastase LasB, esterase EstA, PasP, chitin binding domain protein CbpD, chitinase ChiC, lactonizing lipase LipA and an aminopeptidase (PA2939) of the M28 family of metalloproteases.38 Some of these are known for their

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virulence capabilities and were also observed in the enriched OMV fraction. Secreted factors have important roles in survival; for example, LasB is involved in the inactivation of host tissues by degrading elastin and inactivating immune system components such as immunoglobulins. All together, these results reflect cell adaptation to the biofilm lifestyle and its implications in metabolism and its regulation.

Discussion The biofilm matrix is a complex and heterogeneous environment containing a wide range of cellular material derived from secretion and export processes, as well as, potentially, from cell leakage or lysis. We have examined the matrix and OMV subproteomes of P. aeruginosa in biofilms and Tables 3 and 4 describe the biological contexts of the most abundant proteins. Our results are consistent with the accepted view that OMVs are derived by segregation from the outer membrane and we here show that the OMVs represent a further and distinct division of the extracellular subproteome of the matrix, akin to that described in the extracellular supernatant associated with planktonic populations.

Outer membrane vesicles proteome The most abundant proteins in the OMVs were outer membrane associated proteins involved in transport of small molecules; P. aeruginosa expresses large numbers of abundant specific pores,23, 42 which play crucial roles in virulence, pathogenesis and adaptation. They include the porins (OprB,C,D,E,H,O,Q), as well as OstA (thought to protect against organic solvents and antibiotics) and OsmE (which is involved in cell envelope integrity). Adhesion components of the outer membrane (B-type flagellin

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and pili machinery: PilA,F,Q,V,Y1) which are also recognised as virulence factors, are (unsurprisingly) observed in the OMVs.43-47 Proteins involved in the uptake of iron are also highly expressed.48, 49 P. aeruginosa has more than 200 iron responsive genes including 24 outer membrane receptors. Iron availability is a limiting factor for the growth of P. aeruginosa,49,

50

and the

bacterium employs various very efficient strategies to capture iron. 48-50 In the OMVs (and also in the whole matrix) several iron receptors were highly expressed: FpvA, FpvB, FptA, FoxA, FiuA, PirA, and PhuR receptor, Fe(III) dicitrate.

55

51-54

as well as two receptors for

The list contains receptors for both Fe(II) and Fe (III). In addition,

enzymes involved in ferric storage such as bacterioferritin FtnA were identified.

56-58

Free iron is highly toxic, inducing reactive oxygen species through the Fenton reaction; therefore, these molecules also have a protective role.32 Outer membrane enzymes (lipases, peptidases and ribonucleases) involved in pathogenicity are, as expected, highly represented in the outer membrane vesicles. These include lipases PagL (the most abundant protein in the OMVs),37 LipA,59 EstA, peptidases AaaA, PepA, PasP, MucD, CtpA, Lon, IcmP and the M23 metaloprotease LasA.60-62 The virulence factors alkaline phosphatase PhoA and a probable phosphoserine phosphatase are secreted to scavenge phosphate when availability is limited.63 Others have found the exoproteome of P. aeruginosa to be highly phosphorylated.26, 63 Against this background of catabolic activity, we see, quite intriguingly, several proteins involved in enzymatic inhibition. These include the peptidase inhibitor I78 family protein, MliC, and Lcp (inhibitor of cysteine peptidase). It is possible that these proteins protect the cell wall from damage; they have also been reported to take part in the regulation of cellular homeostasis.64

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It is interesting to note that even in our simple biofilm model proteins involved in antibiotic resistance were expressed. These include the major intrinsic multiple antibiotic resistance efflux outer membrane protein OprM,65, 66 beta-lactamase family protein67 and colicin V, a bacteriocin secreted to kill other bacteria in the biofilm therefore reducing competition when essential nutrients are scarce.68 We expected to observe more prototype periplasmic proteins, however knowledge about this cellular location remains limited; in particular, dual locations are not always annotated. Some periplasmic putative virulent factors such as TolB, LppC, OprF, FliC, SoD, BON domain protein, GlpQ, MucD, AnsB, DctP, periplasmic solute binding family protein and QuiP were present in OMVs. Interestingly, some typical cytoplasmic proteins were found in OMVs. It is not clear whether these proteins, including arginine deiminase and urocanate hydratase, are targeted to the OMVs or whether their presence is caused by leakage. Arginine deiminase is known to be related to low-oxygenated environments and urocanate hydratase is typically involved in histidine metabolism which is known to be important in biofilm formation.69, 70 Overall, OMVs carry information about the cellular state and are full of enzymatically active proteins, typically involved in virulence and pathogenesis.71 Enzymatic activity is regulated by several processes involving transcriptional regulators, also expressed here, as well two component sensors which are known to regulate kinase/ phosphatases processes. Two component systems typically involve a sensor kinase and a response regulator, in which the C-terminal is typically a helix-turn-helix motive that can bind DNA. Two-component sensor NarX proteins (OMVs only) detect nitrate and are absolutely essential for nitrate reductase expression.72

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The matrix proteome as a snapshot of cellular state within the biofilm It is still not at all clear whether simple cell lysis makes a major contribution to the composition of the biofilm matrix. The presence of typical intracellular proteins, such as elongation factors (EF-Tu, EF-G and EF-Ts), chaperones (DnaK, GroEL, GroES) and cold shock protein CapB within the matrix is not a novel finding.24, 27 GroEL and EF-Tu are two of the most abundant proteins in the bacterial cell, and would be expected in the matrix if significant cell lysis occurs. Intriguingly, however, both are known to moonlight.73 EF-Tu has been found in the cell wall, membranes and secretome of several bacteria. GroEL is believed to have an extracellular function in pathogenic processes. Other typical cytoplasmic proteins also found associated with the matrix include the ribosomal proteins L3, L10, L21 and L22 and proteins involved in carbon and amino acid metabolism. Some of these proteins are known to be abundant in the cytoplasm and might be prime candidates for appearing in the matrix because of cell lysis.24 Cell lysis would be expected to yield whole ribosomes with equimolar proteins whereas secretion would be expected to yield quite different ratios; mass spectrometric methods to distinguish these possibilities have recently been developed.39 Metabolic enzymes known to be involved in cellular adaptation to the biofilm lifestyle were found in the matrix. These include enzymes of L-histidine degradation, arginine fermentation (arginine deiminase pathway), glyoxylate shunt (the Mg2+-requiring enzymes isocitrate lyase and malate synthase), cyanogenesis and denitrification. The importance of amino acid metabolism in biofilms and during infection by P. aeruginosa is well recorded in the published literature. The histidine degradation pathway also leads to the generation of ammonia, which has been argued to be important for the regulation of pH within biofilms. Glutaminase-asparaginase AnsB is

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a periplasmic protein typically involved in the deamidation of asparagine and glutamine, yielding aspartic and glutamic acid and ammonia. In E. coli74 and Salmonella enterica,75 this enzyme is expressed during anaerobiosis. In Shigella flexneri, the enzyme is required for adhesion and virulence.75, 76 It well known that these metabolic mechanisms are activated under limited oxygen accessibility. A correlation between our proteome data and anaerobic/microaerobic behaviour is discussed below.

Anaerobic environment within the biofilm matrix Several enzymes of the arginine deiminase pathway (ArcA, ArcB, AaaA, AotJ, ornithine AruBC, agmatine AguA and glutamate ArgC) were found in the matrix. The arginine fermentation and denitrification pathways in P. aeruginosa are known to be induced under low oxygen conditions and to a lesser extent when carbon and energy sources are depleted. Similarly, the glyoxylate shunt pathway is induced under lowoxygen conditions and both enzymes of this pathway (isocitrate lyase and malate synthetase) are abundant. Isocitrate lyase has recently been identified as a virulence determinant for infectivity by P. aeruginosa and it is constitutively up-regulated in clinical isolates from chronic infections.77 Cyanide production has been linked to isocitrate lyase activity, so the presence of cyanate hydratase cynS (which degrades cyanide to ammonia and carbon dioxide) in the matrix is not surprising.78 Representative enzymes of the denitrification processes (reduction of nitrate to nitrogen) were also found in the matrix, as well as enzymes that regulate nitrogen metabolism.2, 79, 80

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Oxygen levels in different regions of the biofilm vary and proteins involved in the maintenance of redox homeostasis appear alongside proteins involved in adaptation to oxygen-depleted environments. In our exoproteome, we identified proteins catalases KatA,B, superoxide dismutase SodB, thioredoxins, thioredoxin reductase TrxB1, glutathione reductase, thiol:disulfide interchange proteins DsbA,D, electron transfer flavoprotein-ubiquinone oxidoreductase and several enzymes involved in glutathione and cysteine biosynthesis. Superoxide dismutase and catalase activity have been measured in the extracellular exoproteome from several organisms.81-83 The presence of two cytochromes Cbb3 oxidase (CcoP1P2), known to have high affinity for oxygen, is consistent with variable oxygen content in the biofilm. An enzyme involved in the limiting step of oxidative decarboxylation in haem biosynthesis, the oxygen-dependent coproporphyrinogen-III oxidase HemF, was also identified in the matrix. This enzyme is overexpressed in oxygen limited environments and it has been postulated that, in the context of a biofilm, the role of this enzyme is not so much to synthesize haem as to scavenge oxygen, thereby protecting the biofilm from oxidative damage.84 In general, it still unclear how these proteins reach the extracellular space; no signal sequences can be seen.83 A strong possibility is that these proteins are supplied extracellularly through OMVs.

Role of metal ions on biofilm regulation The expressed exoproteome indicates limited oxygen availability in the biofilm and under these conditions ferrous iron is expected to dominate over ferric iron. Fe(II) can easily diffuse across the outer membrane and be transported into the cell whereas Fe(III) is known to have low solubility under physiological conditions.

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Siderophores secreted by P. aeruginosa are able to solubilize precipitated iron (III) and transport it into the cell where it is released by reduction to ferrous iron and enters metabolism. As previously shown, P. aeruginosa OMVs have abundant receptors for Fe(III). In addition, a ferrous iron transporter FeoA protein was identified in the whole matrix.85 Like iron, copper is needed for the catalytic centres of several enzymes (NirS, NosZ and azurin) involved in nitrate or nitrite reduction. Cells starved of copper were unable to convert nitrous oxide to nitrogen.86 Copper needs are also demonstrated by the high expression of OprC, a specific copper transport porin. Molybdenum is also required in the catalytic centre and under anaerobic conditions; nitrate reduction is dependent on molybdate availability.87 The molybdate-binding protein modA and molybdenum cofactor biosynthesis MoaB2 are present in the matrix proteome.

Hydrolytic activity and virulence in the biofilm matrix Hydrolytic enzymes such as proteases, phospholipases and nucleases, which degrade extracellular material, abound in the matrix.71 Some are also seen in the OMV enriched fraction. The extracellular proteins, elastase LasB, chitinase ChiC and chitin binding protein CbpD are observed in our matrix proteome and are secreted by the type II secretion system

88-90

. ChiC and CbpD are involved in chitin degradation,

but chitin is not available as a carbon source in these conditions. It has been suggested that these proteins may have a second role in the rearrangement of the extracellular matrix.91 It is known that LasB,91 ChiC and CbpD are expressed in biofilms of clinical isolates and are transcriptionally regulated and dependent on quorum-sensing.12, 89

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Ribosyltransferases are involved in cell signalling and, for example, queuine tRNAribosyltransferase was identified in the matrix. In bacteria, they can be involved in the control of anaerobic and aerobic metabolism, virulence, and antioxidant defence.92 Deaminases can have DNA repair functions or act as toxins;93 we identified cytosine deaminases PA5106 and PA3170. These catabolic processes in the extracellular environment have been reported as crucial for bacteria to survive in the presence of host organisms or during exposure to antibiotics and bactericides. MagB and MagD, which are regulated at the posttranscriptional level, with roles in virulence and pathogenicity deserve some attention. High structural similarities exist between MagD and the human 2-macroglobulin, a large-spectrum protease inhibitor with important roles in innate immunity.

Mimicry of the human protein allows

bacterial cells to evade recognition; MagD is thus essential for bacterial defence.94, 95 Thus, this macromolecular complex may represent a future target for antibacterial developments. Most of the behaviours described above are transcriptionally regulated and dependent on quorum-sensing. This is in accord with our understanding of biofilmrelated processes and the concept of biofilms as a protective mechanism for the preservation and continuity of life. Two component sensory systems also play a role in the control of biofilm formation via production of extracellular appendices such as flagella, type IV fimbriae and cup fimbriae at the surface. The chaperone-usher pathway (cup) fimbriae expression is controlled by regulatory proteins involving the global regulator ANR (arginine deaminase and nitrate reduction regulator) under anaerobic conditions.41, 96 In the very few published descriptions of the biofilm matrix proteome, the occurrence of metabolic pathways in this extracellular environment has never been specifically

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reported or discussed in any detail. There are, however, rare instances of excreted enzymes having specific metabolic function, one example being a secreted mammalian tyrosine kinase, which phosphorylates released proteins in response to stimuli.

97

Indeed, the description of catabolic processes within the biofilm matrix is

typically limited to descriptions of single enzymes. Yet, the biofilm is a special environment where cells and matrix are closely connected and this spatio-temporal proximity has implications for the processes that occur within biofilm communities.

Conclusions Combining two proteomics approaches (gel-based and gel-free), we obtain the most comprehensive proteome profile of both the OMV enriched fraction and the whole matrix extracted from P. aeruginosa PAO1 biofilms. Because of the characteristics of both proteomics strategies, complementary information was obtained. The proteome profile was investigated, as a snapshot of cellular metabolism and collectively the data presented here are an important contribution for the correlation between biochemistry data and proteomics data for the understanding of how the matrix works and contributes to biofilm populations. The biofilm matrix is a very complex and heterogeneous environment and many aspects of this lifestyle are poorly understood. It is interesting to speculate whether multiple enzymes localised in the matrix can act in concert, serving to process materials, since the matrix proteome potentially enables typical intracellular mechanisms to occur in the extracellular environment. As a result of cellular lysis, secretion or release through OMVs, these exoproteomes reveal how cells adapt to the biofilm lifestyle. Vesiculation confers a competitive advantage to bacterial communities, allowing the transfer of important small and large molecules including, for example, proteins responsible for antibiotic

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resistance. Membrane vesicle proteins have therefore been proposed as vaccine targets. The potential of metabolic pathways to occur in the extracellular environment of the biofilm, affecting biofilm development, presents a very exciting point. Future work is required to define whether cytoplasmic proteins are truly associated with OMVs and, more critically, whether these proteins are physiologically-relevant or functional within this non-traditional location. Specific knowledge of these mechanisms will help us to intervene in membrane vesicles function in vivo.

Associated content Supporting Information available: This material is available free of charge via Internet at http://pubs.acs.org. Supporting Information S1 is a Word document containing full experimental procedures, Figures S1-S3 and Tables S1-S6. Figure S1 – Coomassie stained 2D gel from OMVs proteins from P. aeruginosa PAO1 biofilms. Figure S2 – Coomassie stained 2D gel from the whole matrix proteins from P. aeruginosa PAO1 biofilms. Figure S3 – Functional categories of both OMVs and whole matrix proteins. Table S1 – Most hydrophilic (negative GRAVY values) and hydrophobic (positive GRAVY values) proteins from both OMVs and matrix proteomes of P. aeruginosa PAO1 biofilms. Table S2 – Periplasmic proteins identified in the OMVs. Table S3 – Extracellular proteins identified in the OMVs. Table S4 –

Cytoplasmic proteins

identified in the OMVs. Table S5 – Cytoplasmic membrane proteins identified in the OMVs. Table S6 – Extracellular proteins identified in the whole matrix. Supporting Information S2 and S3 are Excel documents providing peptides and proteins identified in both gel-based and gel free proteomics approaches.

Authors Information

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Corresponding author *

Tel: 44-161-275-2369. E-mail: [email protected].

Present address ¥

ChELSI Institute, Department of Chemical and Biological Engineering, University of

Sheffield, Mappin Street, Sheffield, S1 3JD, UK Notes The authors declare no competing financial interest.

Acknowledgements This work was supported by funds to J.R.D. from AFMnet-NCE and NSERC. J.R.D. acknowledges support from the Canada Research Chairs (CRC) program. TEM of samples was performed at the NSERC Guelph Regional Integrated Imaging Facility (GRIIF), which is partially funded by a NSERC-MFA grant. Narciso Couto would like to thank RSC/EPSRC Analytical Chemistry Trust Fund of Royal Society of Chemistry, UK, for financial support of his PhD studies.

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95. Borth, W. Alpha 2-macroglobulin, a multifunctional binding protein with targeting characteristics. FASEB J. 1992, 6, 3345-3353. 96. Sampedro, I.; Parales, R. E.; Krell, T.; Hill, J. E. Pseudomonas chemotaxis. FEMS Microbiol. Rev. 2015, 39, 17-46. 97. Bordoli, M. R.; Yum, J.; Breitkopf, S. B.; Thon, J. N.; Italiano, J. E.; Xiao, J.; Worby, C.; Wong, S.-K.; Lin, G.; Edenius, M. A secreted tyrosine kinase acts in the extracellular environment. Cell 2014, 158, 1033-1044.

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Tables

Table 1 – Number of proteins identified in proteomic studies on P. aeruginosa exoproteome in both planktonic and biofilm cultures. Note that reference 26 refers to a different strain of P. aeruginosa from the remaining studies.

OMV, Biofilm

Matrix, Biofilm

OMVs, Planktonic

Matrix, Planktonic

338

24

145

25 204

76

178

207

327

Ref.

194

26 27 Present work

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Table 2 – Common proteins between our work (37 out of 207) and Toyofuku et al. (37 out of 76) on OMVs from P. aeruginosa PAO1 biofilms. Locus number

Gene name

Protein name

Cellular location*

Cellular function

PA3082

gbt

Alginate export family protein

OMVs (1); OM (3)

Amino acid biosynthesis and metabolism

PA0291

oprE

Anaerobically-induced outer membrane porin

OM (1); OMVs (1); OM (3)

Membrane proteins;

Arginine deiminase

Cy (1); OMVs (1); Cy (3)

Amino acid biosynthesis and metabolism

Basic amino acid, basic peptide and

OM (1); OMVs (1); OM (3)

Transport of small molecules

Chlorophyllase family protein

CM (3)

Hypothetical, unclassified, unknown

OprE precursor PA5171

arcA

PA0958

oprD

Transport of small molecules

imipenem outer membrane porin OprD precursor PA3734 PA4270

rpoB

DNA-directed RNA polymerase beta chain

OMVs (1); Cy (3)

Transcription, RNA processing and degradation

PA4269

rpoC

DNA-directed RNA polymerase beta* chain

OMVs (1); Cy (3)

Transcription, RNA processing and degradation

PA5112

estA

Esterase estA

OM (1); OMVs (1); OM (3)

Secreted Factors (toxins, enzymes, alginate); Fatty acid and phospholipid metabolism; Protein secretion/export apparatus; Motility & Attachment

PA4221

fptA

Fe(III)-pyochelin outer membrane receptor

OM (1); OM (3)

Transport of small molecules

precursor PA2398

fpvA

Ferripyoverdine receptor

OM (1); OM (3)

Transport of small molecules

PA1092

fliC

Flagellin type B

Pe (1); OMVs (1); Ex (3)

Motility & Attachment

PA3186

oprB

Glucose/carbohydrate outer membrane porin

OM (1); OMVs (1); OM (3)

Transport of small molecules

OprB precursor PA4385

groEL

GroEL protein

OMVs (1); Cy (2); Cy (3)

Chaperones & heat shock proteins

PA4710

phuR

Heme/Hemoglobin uptake outer membrane

OM (1); OM (3)

Transport of small molecules

LppC lipofamily protein

OMVs (1); Pe (1); CM (3)

Hypothetical, unclassified, unknown

Major intrinsic multiple antibiotic resistance

OM (1); OMVs (1); OM (3)

Antibiotic resistance and susceptibility;

receptor PhuR precursor PA4423 PA0427

oprM

efflux outer membrane protein OprM

Membrane proteins;

precursor PA1777

oprF

Transport of small molecules

Major porin and structural outer membrane

OM (1); OMVs (1); Pe (1);

Membrane proteins;

porin OprF precursor

OM (3)

Transport of small molecules

PA1011

NlpB/DapX lipofamily protein

OMVs (1); OM (3)

Hypothetical, unclassified, unknown

PA1288

OmpA-like transmembrane domain protein

OMVs (1); OM (2); OM (3)

Membrane proteins; Transport of small molecules

PA4067

oprG

OmpW family protein

OM (1); OMVs (1); OM (3)

Membrane proteins

PA0595

ostA

Organic solvent tolerance protein OstA

OMVs (1); OM (3)

Adaptation, Protection

OM (1); OMVs (1); OM (3)

Transport of small molecules;

precursor PA2760

oprQ

Outer membrane porin, OprD family protein

Motility & Attachment; Antibiotic resistance and susceptibility PA3800

bamB

Outer membrane protein assembly factor

OMVs (1); OM (3)

Hypothetical, unclassified, unknown

Peptidase M28 family protein

Ex (1); Ex (3)

Secreted Factors (toxins, enzymes, alginate)

PhoP/Q and low Mg2+ inducible outer

OM (1); OMVs (1); OM (3)

Membrane proteins;

BamB PA2939 PA1178

oprH

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membrane protein H1 precursor

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Adaptation, Protection; Transport of small molecules

PA0622

Probable bacteriophage protein

OMVs (1); Un (3)

Related to phage, transposon, or plasmid

PA0041

Probable hemagglutinin

Ex (1); OM (3)

Secreted Factors (toxins, enzymes, alginate)

PA4675

Probable TonB-dependent receptor

OMVs (1); OM (2); OM (3)

Transport of small molecules

PA3790

oprC

Putative copper transport outer membrane

OM (1); OMVs (1); OM (3)

Transport of small molecules

PA2394

pvdN

PvdN

Un (3)

Adaptation, Protection

PA4168

fpvB

Second ferric pyoverdine receptor FpvB

OM (2); OM (3)

Transport of small molecules

PA0972

tolB

TolB protein

OMVs (1); Pe (2); Pe (3)

Transport of small molecules

TonB-dependent siderophore receptor family

OM (2); OM (3)

Membrane proteins

Type 4 fimbrial precursor PilA

OMVs (1); Ex (3)

Motility & Attachment

Type I secretion outer membrane , TolC

OM (1); OMVs (1); OM (3)

Protein secretion/export apparatus

Uncharacterized protein

OM (3)

Hypothetical, unclassified, unknown

Urocanate hydratase

Cy (3)

Amino acid biosynthesis and metabolism

porin OprC

PA0434

protein PA4525

pilA

PA4974

family protein PA3923 PA5100

hutU

* - OMVs stands for outer membrane vesicles, OM stands for outer membrane, Pe stands for periplasmic, Cy stands for cytosolic, Ex stands for extracellular and Un stands for unknown. Numbers 1, 2, and 3 stands for level of confidence, where 1 means experimentally proven location in P. aeruginosa PAO1, 2 means experimentally proved location in other organisms and 3 means computationally predicated.

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Table 3 - Common proteins between our work, Choi et al.24, and Toyofuku et al.27 on OMVs from P. aeruginosa PAO1. The majority of these proteins are highly abundant in all three studies. Other work in which these proteins were identified is indicated in the column headed Reference. Locus number

Gene name

Protein name

PA3082

gbt

Alginate export family protein

PA0291

oprE

Anaerobically-induced outer membrane porin OprE precursor

PA5171

arcA

Arginine deiminase

PA0958

oprD

Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor

PA4270

rpoB

DNA-directed RNA polymerase beta chain

PA4269

rpoC

DNA-directed RNA polymerase beta* chain

PA5112

estA

Esterase estA

PA1092

fliC

Flagellin type B

PA3186

oprB

Glucose/carbohydrate outer membrane porin OprB precursor

PA4385

groEL

GroEL protein

PA0427

oprM

Major intrinsic multiple antibiotic resistance efflux outer membrane protein OprM precursor

PA1777

oprF

Major porin and structural outer membrane porin OprF precursor

PA4423

Reference

25, 28

28

28

25

LppC lipofamily protein

25, 28

PA1011

NlpB/DapX lipofamily protein

PA1288

OmpA-like transmembrane domain protein

28

PA4067

oprG

OmpW family protein

25, 28

PA0595

ostA

Organic solvent tolerance protein OstA precursor

25

PA2760

oprQ

Outer membrane porin, OprD family protein

PA3800

bamB

Outer membrane protein assembly factor BamB

PA1178

oprH

PhoP/Q and low Mg2+ inducible outer membrane protein H1 precursor

PA0622

Probable bacteriophage protein

PA4675

Probable TonB-dependent receptor

PA3790

oprC

Putative copper transport outer membrane porin OprC

PA0972

tolB

TolB protein

PA4525

pilA

PA4974

25, 28

Type 4 fimbrial precursor PilA

25

25

Type I secretion outer membrane , TolC family protein

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Table 4 - Top 30 most abundant proteins in the OMVs enriched fraction from P. aeruginosa biofilms. Other work in which these proteins were found is indicated. Locus

Gene

number

name

EmPAI

Protein name

Subcellular location*

Functional class

Ref

PA4661

pagL

5.17

Lipid A 3-O-deacylase

OM (1); OMVs (1); Un (3)

Hypothetical, unclassified, unknown

27

PA2760

oprQ

4.94

OprQ

OM (1); OMVs (1); OM (3)

Transport of small molecules;

27

Motility & Attachment; Antibiotic resistance and susceptibility PA3280

oprO

4.31

Pyrophosphate-specific outer membrane porin

OM (1); OM (3)

Transport of small molecules

OprO precursor PA1053 PA1178

oprH

4.25

Glycine zipper 2TM domain protein

OMVs (1); OM (3)

Membrane proteins

27

3.01

PhoP/Q and low Mg2+ inducible outer membrane

OM (1); OMVs (1); OM (3)

Membrane proteins;

25, 28

protein H1 precursor

Adaptation, Protection; Transport of small molecules

PA3790

oprC

2.83

Putative copper transport outer membrane porin

OM (1); OMVs (1); OM (3)

Transport of small molecules

25, 27

OprC PA0972

tolB

2.57

TolB protein

OMVs (1); Pe (2); Pe (3)

Transport of small molecules

27

PA0958

oprD

2.51

Basic amino acid, basic peptide and imipenem

OM (1); OMVs (1); OM (3)

Transport of small molecules

27, 28

OM (1); OM (3)

Transport of small molecules

27

OM (2); OM (3)

Transport of small molecules;

27

outer membrane porin OprD precursor PA4221

fptA

2.14

Fe(III)-pyochelin outer membrane receptor precursor

PA2113

opdO

2.04

Pyroglutamate porin OpdO

Membrane proteins PA4514

2.04

Probable outer membrane receptor for iron

OMVs (1); OM (2); OM (3)

Transport of small molecules

27

OMVs (1); OM (2); OM (3)

Membrane proteins;

27, 28

transport PA1288

1.95

Probable outer membrane protein precursor

Transport of small molecules PA4067

oprG

PA3674

1.84

Outer membrane protein OprG precursor

OM (1); OMVs (1); OM (3)

Membrane proteins

1.79

Type III secretion system lipochaperone family

OMVs (1); Un (3)

Hypothetical, unclassified, unknown

OM (1); OMVs (1); OM (3)

Membrane proteins;

25, 27, 28

protein PA0291

oprE

1.7

Anaerobically-induced outer membrane porin OprE precursor

PA3038 PA3186

oprB

25, 27, 28

Transport of small molecules

1.65

Outer membrane porin, OprD family protein

OM (2); OM (3)

Transport of small molecules

27

1.64

Glucose/carbohydrate outer membrane porin

OM (1); OMVs (1); OM (3)

Transport of small molecules

27

27

OprB precursor PA4423

1.53

LppC lipofamily protein

OMVs (1); Pe (1); CM (3)

Hypothetical, unclassified, unknown

1.39

Osmotically inducible lipoprotein OsmE

OMVs (1); Un (3)

Membrane proteins;

PA4675

1.33

Probable TonB-dependent receptor

OMVs (1); OM (2); OM (3)

Transport of small molecules

27

PA3734

1.32

Chlorophyllase family protein

CM (3)

Hypothetical, unclassified, unknown

27

1.28

Glycine zipper 2TM domain protein

OMVs (1); Un (3)

Membrane proteins;

27

PA4876

osmE

Adaptation, Protection

PA0070

tagQ1

Protein secretion/export apparatus PA1777

oprF

1.22

Major porin and structural outer membrane porin

OM (1); OMVs (1); Pe (1); OM (3)

OprF precursor

Membrane proteins;

25, 27, 28

Transport of small molecules

PA1804

hupB

1.21

DNA-binding protein HU

Cy (1); OMVs (1); Cy (3)

PA5112

estA

1.15

Esterase EstA

OM (1); OMVs (1); OM (3)

DNA replication, recombination, modification and repair Secreted Factors (toxins, enzymes, alginate);

27

Fatty acid and phospholipid metabolism; Protein secretion/export apparatus; Motility & Attachment PA0328

aaaA

1.1

Arginine-specific autotransporter of

OMVs (1); OM (1); OM (3)

Pseudomonas aeruginosa, AaaA PA0622

Hypothetical, unclassified, unknown;

1.08

Probable bacteriophage protein

OMVs (1); Un (3)

Related to phage, transposon, or plasmid

0.92

LPS-assembly lipoprotein LptE

OM (1); OMVs (1); Un (3)

Hypothetical, unclassified, unknown

PA0623

0.86

Probable bacteriophage protein

OMVs (1); Un (3)

Related to phage, transposon, or plasmid

PA1969

0.86

Peptidase inhibitor I78 family protein

OMVs (1); Un (3)

Hypothetical, unclassified, unknown

PA3988

lptE

27

Amino acid biosynthesis and metabolism 27

27

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* - OMVs stands for outer membrane vesicles, OM stands for outer membrane, CM stands for cytoplasmic, Pe stands for periplasmic, Cy stands for cytosolic, Ex stands for extracellular and Un stands for unknown. Numbers 1, 2, and 3 stands for level of confidence, where 1 means experimentally proven location in P. aeruginosa PAO1, 2 means experimentally proved location in other organisms and 3 means computationally predicated. Proteins identified in reference 27 from OMVs in biofilm, others are from OMVs in planktonic population of P. aeruginosa PAO1.

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Table 5 - Top 30 most abundant proteins in the whole matrix from P. aeruginosa biofilms. Other work in which these proteins were found is indicated. Locus number

Gene name

EmPAI

Protein name

Subcellular location

Functional class

PA0423

pasP

12.05

PasP

Ex (1); OMVs (1); Un (3)

Secreted Factors (toxins, enzymes, alginate)

PA1092

fliC

10.19

Flagellin type B

Pe (1); OMVs (1); Ex (3)

Motility & Attachment

PA5100

hutU

5.93

Urocanase

Cy (3)

Amino acid biosynthesis and metabolism

PA5240

trxA

5.7

Thioredoxin

Cy (3)

Translation, post-translational modification, degradation; Nucleotide biosynthesis and metabolism; Energy metabolism

PA4922

azu

5.26

Azurin

Pe (1); Pe (3)

Energy metabolism

4.62

Probable binding protein component of ABC transporter

Pe (2); Pe (3)

Transport of small molecules

PA1342 PA2250

lpdV

3.92

Lipoamide dehydrogenase-Val

Cy (2); Cy (3)

Amino acid biosynthesis and metabolism; Energy metabolism

PA5171

arcA

3.92

Arginine deiminase

Cy (1); OMVs (1); Cy (3)

Amino acid biosynthesis and metabolism

PA1074

braC

3.61

Branched-chain amino acid transport protein BraC

Pe (1); OMVs (1); Pe (3)

Transport of small molecules

3.23

Endoribonuclease L-PSP, putative

Cy (3)

Hypothetical, unclassified, unknown

PA5339 PA3029

moaB2

2.9

Molybdopterin biosynthetic protein B2

Cy (3)

Biosynthesis of cofactors, prosthetic groups and carriers

PA1337

ansB

2.72

Glutaminase-asparaginase

Pe (3)

Amino acid biosynthesis and metabolism

PA3383

phnD

2.68

Binding protein component of ABC phosphonate transporter

Pe (3)

Transport of small molecules

PA1587

lpdG

2.6

Lipoamide dehydrogenase-glc

OMVs (1); Cy (2); Cy (3)

Amino acid biosynthesis and metabolism; Energy metabolism

PA4661

pagL

2.53

Lipid A 3-O-deacylase

OM (1); OMVs (1); Un (3)

Hypothetical, unclassified, unknown

2.4

Cold-shock' DNA-binding domain protein

Cy (2); Cy (3)

Transcriptional regulators; Adaptation, Protection

PA1159 PA3280

oprO

2.12

Pyrophosphate-specific outer membrane porin OprO precursor

OM (1); OM (3)

Transport of small molecules

PA0852

cbpD

2.06

Chitin binding domain protein

Ex (1); Ex (3)

Secreted Factors (toxins, enzymes, alginate)

PA0745

2.05

Enoyl-CoA hydratase/isomerase family protein

Cy (3)

Putative enzymes

PA3836

1.96

Periplasmic binding domain protein

Pe (1); Un (3)

Hypothetical, unclassified, unknown

1.93

Arginine/ornithine binding protein AotJ

Pe (1); OMVs (1); Pe (3)

Transport of small molecules

1.93

Lipo, YaeC family protein

CM (3)

Membrane proteins; Transport of small molecules

1.92

Insulin-cleaving metalloproteinase outer membrane protein precursor

OM (1); OMVs (1); OM (3)

Membrane proteins

1.87

Basic amino acid, basic peptide and imipenem outer membrane porin OprD precursor

OM (1); OMVs (1); OM (3)

Transport of small molecules

1.85

Probable binding protein component of ABC sugar transporter

Pe (3)

Transport of small molecules

PA0888

aotJ

PA5505 PA4370 PA0958

icmP oprD

PA3190 PA3790

oprC

1.68

Putative copper transport outer membrane porin OprC

OM (1); OMVs (1); OM (3)

Transport of small molecules

PA5369

pstS

1.66

Phosphate ABC transporter, periplasmic phosphatebinding protein, PstS

Pe (1); CM (3)

Transport of small molecules

PA5172

arcB

1.64

Ornithine carbamoyltransferase

Cy (1); OMVs (1); Cy (3)

Amino acid biosynthesis and metabolism

PA5091

hutG

1.45

Formimidoylglutamase

Cy (3)

Amino acid biosynthesis and metabolism

PA5098

hutH

1.41

Histidine ammonia-lyase

Cy (1); Cy (3)

Amino acid biosynthesis and metabolism

Ref. 27 26, 27, 43,71 26, 27 26

26, 27 27 26, 27 26

26, 27 26

26, 27,71

26, 27, 90

26

26, 27 26 26, 27

26, 27

* - OMVs stands for outer membrane vesicles, OM stands for outer membrane, CM stands for cytoplasmic membrane, Pe stands for periplasmic, Cy stands for cytosolic, Ex stands for extracellular and Un stands for unknown. Numbers 1, 2, and 3 stands for level of confidence, where 1 means experimentally proven location in P. aeruginosa PAO1, 2 means experimentally proved location in other organisms and 3 means computationally predicated. Proteins identified in reference 27, are also related to biofilm matrix while other references refer to proteins identified from supernatant cultures of P. aeruginosa PAO1 except reference 26 which refers to the matrix of supernatant cultures of P aeruginosa PA14.

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Figure legends Figure 1 – Venn diagram showing the overlap between the two different proteomic approaches (gel-free and gel-based) used for OMVs and matrix exoproteome analysis of P. aeruginosa biofilms. Overlapping between the OMV and matrix proteomes derived from gel-based and gel-free approaches (A). Overlapping between the OMV and matrix proteomes derived from gel-free, gel based and the combined OMVs and matrix (gel-free and gel-based) (B). Figure 2 - Micrographs of negatively stained samples of P. aeruginosa PAO1 disrupted biofilm (A) and steps during the isolation protocol: (B) matrix, (C) crude OMV pellet, and (D) OMVs purified by isopycnic density gradient centrifugation. Arrows without annotation indicate OMVs, arrows accompanied by F denote flagella, by P pili or filamentous phage. Note in these Figures A-C the presence of OMVs, flagella and pili, the latter of which are absent in the purified OMVs (D) used in this study. Bars, 200 nm. Figure 3 – 2D gels of the OMV and matrix proteomes. Examples of the same protein distributed in different regions on the 2D gels from OMVs (A) and matrix (B). Predicted molecular mass and pI are indicated between brackets after protein name (MW; pI). In gel A, three proteins are indicated, E stands for EstA (69.6; 4.46), O stands for OprE (49.7; 9.06) and L for lipid A deacylase (18.4; 6.25). In gel B, 5 proteins are assigned. L stands for lipid A deacylase and E for EstA as before and A stands for azurin (16.0; 6.93), C stands for chitin binding protein (41.9; 6.85) and B stands for LasB (53.7; 6.74). Figure 4 – Subcellular distribution of the identified proteins in the OMV enriched fraction (A) and the whole matrix (B) of P. aeruginosa PAO1 biofilms. With the exception of the unknowns, only proteins belonging to class 1 are included. The 45 ACS Paragon Plus Environment

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same protein can also be incorporated in more than one category as happens with the OMVs which are also frequently annotated as outer membrane or unknown. The x axis represents the number of proteins which were incorporated into the groups assigned on the y axis. Figure 5 – OMV and matrix identified proteins distributed according to functional categories. The most representative (14 out of 26) functional categories for both OMV enriched proteins (grey bars) and matrix (black bars) are shown. The x axis represents the number of proteins belonging to the functional category indicated on the y axis.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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For TOC only

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