Article
pubs.acs.org/jpr
Proteome Profiles of Outer Membrane Vesicles and Extracellular
Matrix of Pseudomonas aeruginosa Biofilms
Narciso Couto,†,⊥ Sarah R. Schooling,‡,§ John R. Dutcher,§ and Jill Barber*,†,∥
†
Michael Barber Centre for Mass Spectrometry, Manchester Institute for Biotechnology, University of Manchester, Princess Road,
Manchester, M1 7DN, U.K.
‡
Department of Molecular and Cellular Biology, College of Biological Science, University of Guelph, Guelph, ON N1G 2W1, Canada
§
Department of Physics, University of Guelph, Guelph, ON N1G 2W1, Canada
∥
Manchester Pharmacy School, University of Manchester, Stopford Building, Oxford Road, Manchester, M13 9PT, U.K.
S Supporting Information
*
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 physicochemical characteristics of these subproteomes, 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 analyzed 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 analyzing 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
■
INTRODUCTION
recognize 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
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 Matrix
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 signaling molecules, with various
functions, are also found in the matrix.11,12
Biofilm Lifestyle
P. aeruginosa is capable of adopting either a planktonic freeswimming 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
© 2015 American Chemical Society
Received: April 13, 2015
Published: August 25, 2015
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Journal of Proteome Research
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 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
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
dialyzed 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 microbicinchoninic acid (BCA)
protein assay kit (Pierce Biotechnology, IL, USA).
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
solubilization. These OMVs and matrix protein extracts were
used in all subsequent proteomic analysis.
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.
■
Two-Dimensional Gel Electrophoresis
Reagents for two-dimensional gel electrophoresis were
purchased from Bio-Rad (Hemel Hempstead, U.K.). Twodimensional (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 OMVenriched or whole matrix fractions were used in these
experiments. In the first dimension, isoelectric focusing (IEF)
was performed using IPG strips (11 cm, nonlinear pH 3−10).
In the second dimension, a 14% SDS polyacrylamide gel
electrophoresis (PAGE) was performed.
MATERIALS AND METHODS
Two-Dimensional LC−MS/MS
Materials
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 of
protein/trypsin (50:1) at 37 °C. 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 fractionated on an Ultimate 3000
(Dionex, Surrey, U.K.) with a 200 × 2.1 mm, 5 μm, 300 Å,
polysulfethyl A column (Poly LC, MD, USA). A linear gradient
was used to perform the elution over 40 min 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
The majority of materials and solvents used throughout this
work were of the highest quality available and were purchased
from Sigma-Aldrich (Poole, U.K.), unless otherwise stated.
Biofilm Growth, Isolation, and Purification of Membrane
Vesicles
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 h, 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.
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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 two
unique peptides with a peptide score above 20 were considered
true identifiers.
The exponentially modified protein abundance index
(emPAI) was applied to obtain semiquantitative 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
capillary LC system (Dionex, Surrey, U.K.) online connected to
a quadrupole time-of-flight (Q-ToF) Global mass spectrometer
(Waters, Manchester, U.K.) via a distal-coated fused silica
PicoTip emitter using a capillary voltage of 2.0−2.8 kV into a
Z-spray 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 s.
■
In-Gel Digestion and LC−MS/MS
RESULTS
Two different subproteomes of the P. aeruginosa PAO1 biofilm
(whole matrix and enriched outer membrane vesicles) were
independently analyzed 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 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 gel-based approach, protein separation was
achieved by 2D-gel electrophoresis, which was followed by ingel 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 subcellular location and the functional
role of each of the proteins detected. The information in these
databases is still incomplete and potentially biased in favor of
highly studied proteins; however, together they provide a
powerful tool for data organization based upon cell location or
function. An estimation of protein abundance was inferred
based on emPAI values.
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
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, U.K.) 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 as follows: dry
gas temperature was set to 150 °C, dry gas was set to 6 L min−1,
and the scan mode was set to standard enhanced with an m/z
range of 200−3000 for MS and ultrascan 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 s with isolation window of 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 to
300% of the set value.
Database Searching
Proteome Profile of OMVs and Whole Matrix
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 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 settings were used with data acquired on the
Amazon mass spectrometer, using trypsin as the enzyme,
allowing for two missed cleavages and assuming only the
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 peptides from each
protein and protein annotation are shown in Supporting
Information S2 and S3, respectively, for the OMVs and the
whole matrix. Two-dimensional 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
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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
Table 1. Number of Proteins Identified in Proteomic Studies
on P. aeruginosa Exoproteome in Both Planktonic and
Biofilm Cultures
OMV,
biofilm
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).
matrix,
biofilm
OMVs,
Planktonic
matrix,
Planktonic
338
145
204
76
207
178
327
194
ref
24
25
26a
27
present
work
a
Note that ref 26 refers to a different strain of P. aeruginosa from the
remaining studies.
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 subproteomes were compared, there was also
less overlap than might be expected (Figure 1B). Forty-eight
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.
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.
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 onedimensional 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 TonBdependent 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 h after
inoculation. In our experiments, biofilms were generated on
TSA agar plates and harvested after 24 h. Age at the time of
harvesting (with the potential for cell death and the
accumulation of cell debris) may account for the fact that a
high number of OMV proteins identified by Toyofuku et al.
OMVs as a Component of the Whole Matrix
Based on the number of identified proteins in the OMVenriched fraction, which were also detected in the matrix
exoproteome (Figure 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, 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 subproteome 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.
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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
PA3082
gbt
alginate export family protein
PA0291
oprE
PA5171
arcA
anaerobically induced outer membrane porin
OprE precursor
arginine deiminase
PA0958
oprD
PA3734
PA4270
PA4269
PA5112
rpoB
rpoC
estA
PA4221
fptA
PA2398
PA1092
fpvA
fliC
PA3186
oprB
PA4385
groEL
PA4710
phuR
PA4423
PA0427
oprM
PA1777
oprF
protein name
basic amino acid, basic peptide and imipenem
outer membrane porin OprD precursor
chlorophyllase family protein
DNA-directed RNA polymerase beta chain
DNA-directed RNA polymerase beta* chain
esterase estA
Fe(III)-pyochelin outer membrane receptor
precursor
ferripyoverdine receptor
flagellin type B
glucose/carbohydrate outer membrane porin
OprB precursor
GroEL protein
heme/hemoglobin uptake outer membrane
receptor PhuR precursor
LppC lipofamily protein
major intrinsic multiple antibiotic resistance
efflux outer membrane protein OprM
precursor
major porin and structural outer membrane
porin OprF precursor
PA1011
NlpB/DapX lipofamily protein
PA1288
OmpA-like transmembrane domain protein
PA4067
oprG
OmpW family protein
PA0595
ostA
PA2760
oprQ
organic solvent tolerance protein OstA
precursor
outer membrane porin, OprD family protein
PA3800
bamB
PA2939
PA1178
oprH
PA0622
PA0041
PA4675
PA3790
oprC
PA2394
PA4168
PA0972
pvdN
fpvB
tolB
PA0434
PA4525
PA4974
PA3923
PA5100
pilA
hutU
outer membrane protein assembly factor
BamB
peptidase M28 family protein
PhoP/Q and low Mg2+ inducible outer
membrane protein H1 precursor
probable bacteriophage protein
probable hemagglutinin
probable TonB-dependent receptor
putative copper transport outer membrane
porin OprC
PvdN
second ferric pyoverdine receptor FpvB
TolB protein
TonB-dependent siderophore receptor family
protein
type 4 fimbrial precursor PilA
type I secretion outer membrane, TolC family
protein
uncharacterized protein
urocanate hydratase
cellular locationa
OMVs (1); OM
(3)
OM (1); OMVs
(1); OM (3)
Cy (1); OMVs (1);
Cy (3)
OM (1); OMVs
(1); OM (3)
CM (3)
OMVs (1); Cy (3)
OMVs (1); Cy (3)
OM (1); OMVs
(1); OM (3)
OM (1); OM (3)
OM (1); OM (3)
Pe (1); OMVs (1);
Ex (3)
OM (1); OMVs
(1); OM (3)
OMVs (1); Cy (2);
Cy (3)
OM (1); OM (3)
OMVs (1); Pe (1);
CM (3)
OM (1); OMVs
(1); OM (3)
OM (1); OMVs
(1); Pe (1); OM
(3)
OMVs (1); OM
(3)
OMVs (1); OM
(2); OM (3)
OM (1); OMVs
(1); OM (3)
OMVs (1); OM
(3)
OM (1); OMVs
(1); OM (3)
OMVs (1); OM
(3)
Ex (1); Ex (3)
OM (1); OMVs
(1); OM (3)
OMVs (1); Un (3)
Ex (1); OM (3)
OMVs (1); OM
(2); OM (3)
OM (1); OMVs
(1); OM (3)
Un (3)
OM (2); OM (3)
OMVs (1); Pe (2);
Pe (3)
OM (2); OM (3)
OMVs (1); Ex (3)
OM (1); OMVs
(1); OM (3)
OM (3)
Cy (3)
4211
cellular function
amino acid biosynthesis and metabolism
membrane proteins; transport of small molecules
amino acid biosynthesis and metabolism
transport of small molecules
hypothetical, unclassified, unknown
transcription, RNA processing and degradation
transcription, RNA processing and degradation
secreted factors (toxins, enzymes, alginate); fatty acid and phospholipid
metabolism; protein secretion/export apparatus; motility and
attachment
transport of small molecules
transport of small molecules
motility and attachment
transport of small molecules
chaperones and heat shock proteins
transport of small molecules
hypothetical, unclassified, unknown
antibiotic resistance and susceptibility; membrane proteins; transport of
small molecules
membrane proteins; transport of small molecules
hypothetical, unclassified, unknown
membrane proteins; transport of small molecules
membrane proteins
adaptation, protection
transport of small molecules; motility and attachment; antibiotic
resistance and susceptibility
hypothetical, unclassified, unknown
secreted factors (toxins, enzymes, alginate)
membrane proteins; adaptation, protection; transport of small
molecules
related to phage, transposon, or plasmid
secreted factors (toxins, enzymes, alginate)
transport of small molecules
transport of small molecules
adaptation, protection
transport of small molecules
transport of small molecules
membrane proteins
motility and attachment
protein secretion/export apparatus
hypothetical, unclassified, unknown
amino acid biosynthesis and metabolism
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Table 2. continued
a
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.
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.
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
We also compared our OMVs and those of Toyofuku et al.27
with the planktonic OMVs reported by Choi et al.24 Twenty-six
proteins were common to all three studies. Results are
summarized in Table 3.
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.
Proteome Profile of OMVs and Whole Matrix According to
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).
Sixty-nine percent 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.
While 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
analyzed here (Table S3). Cytoplasmic proteins were not
expected; nevertheless, five (class 1) and eight (class 2)
cytoplasmic proteins were confidently identified (Table S4). In
total, five 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 seven identified proteins were experimentally observed by
others to be secreted by P. aeruginosa, of which only PasP and
LasA are also annotated as OMV proteins. These are indicated
in Table S6.
Hydrophobicity, pI, and MW
Because of the differences between the gel-based and gel-free
approaches, the physicochemical properties that 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 behavior 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 affect 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
behavior 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.
Semiquantitative 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
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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
PAO1a
locus
number
gene
name
PA3082
PA0291
gbt
oprE
PA5171
PA0958
arcA
oprD
PA4270
PA4269
PA5112
PA1092
PA3186
rpoB
rpoC
estA
fliC
oprB
PA4385
PA4423
PA0427
groEL
PA1777
oprF
PA1011
PA1288
PA4067
oprG
PA0595
ostA
PA2760
PA3800
PA1178
oprQ
bamB
oprH
PA0622
PA4675
PA3790
oprC
PA0972
PA4525
PA4974
oprM
tolB
pilA
protein name
alginate export family protein
anaerobically induced outer membrane porin
OprE precursor
arginine deiminase
basic amino acid, basic peptide and imipenem
outer membrane porin OprD precursor
DNA-directed RNA polymerase beta chain
DNA-directed RNA polymerase beta* chain
esterase estA
flagellin type B
glucose/carbohydrate outer membrane porin
OprB precursor
GroEL protein
LppC lipofamily protein
major intrinsic multiple antibiotic resistance
efflux outer membrane protein OprM
precursor
major porin and structural outer membrane
porin OprF precursor
NlpB/DapX lipofamily protein
OmpA-like transmembrane domain protein
OmpW family protein
ref
25,
28
28
28
25
25,
28
28
25,
28
25
organic solvent tolerance protein OstA
precursor
outer membrane porin, OprD family protein
outer membrane protein assembly factor BamB
PhoP/Q and low Mg2+ inducible outer
25,
membrane protein H1 precursor
28
probable bacteriophage protein
probable TonB-dependent receptor
putative copper transport outer membrane
25
porin OprC
TolB protein
type 4 fimbrial precursor PilA
25
type I secretion outer membrane, TolC family
protein
a
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 ref.
matrix proteins according to functional categories (26 in total
based on the P. aeruginosa database) is shown in Figure S3.
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.
Forty 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 microenvironment 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
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 and by P pili
or filamentous phage. Note in 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.
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
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Figure 3. Two-dimensional gels of the OMV and matrix proteomes. Examples of the same protein distributed in different regions on the 2D gels
from OMVs (left) and matrix (right). 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).
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 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, 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 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 and pili machinery:
PilA,F,Q,V,Y1), which are also recognized 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,51−54 as well as two receptors for
Fe(III) dicitrate.55 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
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pagL
oprQ
oprO
oprH
oprC
tolB
oprD
fptA
opdO
PA4661
PA2760
PA3280
PA1053
PA1178
PA3790
PA0972
PA0958
PA4221
PA2113
PA4514
4215
hupB
estA
aaaA
lptE
PA5112
PA0328
PA0622
PA3988
tagQ1
oprF
PA1804
PA3734
PA0070
PA1777
1.08
0.92
1.1
1.15
1.21
1.32
1.28
1.22
1.39
1.33
osmE
1.65
1.64
PA4876
PA4675
oprB
PA3038
PA3186
1.79
1.7
1.53
oprE
PA3674
PA0291
1.84
1.95
2.14
2.04
2.04
2.51
2.57
2.83
4.25
3.01
4.31
4.94
5.17
EmPAI
PA4423
oprG
PA4067
PA1288
gene
name
locus
number
arginine-specific autotransporter of Pseudomonas
aeruginosa, AaaA
probable bacteriophage protein
LPS-assembly lipoprotein LptE
esterase EstA
chlorophyllase family protein
glycine zipper 2TM domain protein
major porin and structural outer membrane porin OprF
precursor
DNA-binding protein HU
osmotically inducible lipoprotein OsmE
probable TonB-dependent receptor
type III secretion system lipochaperone family protein
anaerobically induced outer membrane porin OprE
precursor
outer membrane porin, OprD family protein
glucose/carbohydrate outer membrane porin OprB
precursor
LppC lipofamily protein
outer membrane protein OprG precursor
probable outer membrane protein precursor
basic amino acid, basic peptide and imipenem outer
membrane porin OprD precursor
Fe(III)-pyochelin outer membrane receptor precursor
pyroglutamate porin OpdO
probable outer membrane receptor for iron transport
TolB protein
pyrophosphate-specific outer membrane porin OprO
precursor
glycine zipper 2TM domain protein
PhoP/Q and low Mg2+ inducible outer membrane
protein H1 precursor
putative copper transport outer membrane porin OprC
OprQ
lipid A 3-O-deacylase
protein name
OMVs (1); OM (3)
OM (1); OMVs (1); OM
(3)
OM (1); OMVs (1); OM
(3)
OMVs (1); Pe (2); Pe
(3)
OM (1); OMVs (1); OM
(3)
OM (1); OM (3)
OM (2); OM (3)
OMVs (1); OM (2); OM
(3)
OMVs (1); OM (2); OM
(3)
OM (1); OMVs (1); OM
(3)
OMVs (1); Un (3)
OM (1); OMVs (1); OM
(3)
OM (2); OM (3)
OM (1); OMVs (1); OM
(3)
OMVs (1); Pe (1); CM
(3)
OMVs (1); Un (3)
OMVs (1); OM (2); OM
(3)
CM (3)
OMVs (1); Un (3)
OM (1); OMVs (1); Pe
(1); OM (3)
Cy (1); OMVs (1); Cy
(3)
OM (1); OMVs (1); OM
(3)
OMVs (1); OM (1); OM
(3)
OMVs (1); Un (3)
OM (1); OMVs (1); Un
(3)
OM (1); OMVs (1); Un
(3)
OM (1); OMVs (1); OM
(3)
OM (1); OM (3)
subcellular locationb
functional class
related to phage, transposon, or plasmid
hypothetical, unclassified, unknown
secreted factors (toxins, enzymes, alginate); fatty acid and phospholipid metabolism; protein
secretion/export apparatus; motility and attachment
hypothetical, unclassified, unknown; amino acid biosynthesis and metabolism
DNA replication, recombination, modification and repair
hypothetical, unclassified, unknown
membrane proteins; protein secretion/export apparatus
membrane proteins; transport of small molecules
membrane proteins; adaptation, protection
transport of small molecules
hypothetical, unclassified, unknown
transport of small molecules
transport of small molecules
hypothetical, unclassified, unknown
membrane proteins; transport of small molecules
membrane proteins
membrane proteins; transport of small molecules
transport of small molecules
transport of small molecules; membrane proteins
transport of small molecules
transport of small molecules
transport of small molecules
transport of small molecules
membrane proteins
membrane proteins; adaptation, protection; transport of small molecules
transport of small molecules
transport of small molecules; motility and attachment; antibiotic resistance and susceptibility
hypothetical, unclassified, unknown
Table 4. Top 30 Most Abundant Proteins in the OMVs Enriched Fraction from P. aeruginosa Biofilmsa
ref
27
27
27
27
27
25, 27,
28
27
27
25, 27,
28
27
27
25, 27,
28
27, 28
27
27
27
27, 28
27
25, 27
27
25, 28
27
27
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PA0623
PA1969
functional class
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
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
protein,67 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 are 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
a
Other work in which these proteins were found is indicated. bOMVs 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 are identified in ref 27 from OMVs in biofilm; others are from OMVs in planktonic population of P.
aeruginosa PAO1.
OMVs (1); Un (3)
OMVs (1); Un (3)
probable bacteriophage protein
peptidase inhibitor I78 family protein
0.86
0.86
subcellular locationb
protein name
EmPAI
gene
name
locus
number
Table 4. continued
related to phage, transposon, or plasmid
hypothetical, unclassified, unknown
27
ref
Journal of Proteome Research
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,
and 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
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Table 5. Top 30 Most Abundant Proteins in the Whole Matrix from P. aeruginosa Biofilmsa
locus
number
gene
name
EmPAI
protein name
PA0423
pasP
12.05
pasp
PA1092
fliC
10.19
flagellin type B
PA5100
PA5240
hutU
trxA
5.93
5.7
urocanase
thioredoxin
PA4922
PA1342
azu
5.26
4.62
PA2250
PA5171
lpdV
arcA
3.92
3.92
azurin
probable binding protein component of ABC
transporter
lipoamide dehydrogenase-Val
arginine deiminase
PA1074
braC
3.61
PA5339
PA3029
PA1337
PA3383
moaB2
ansB
phnD
3.23
2.9
2.72
2.68
PA1587
lpdG
2.6
branched-chain amino acid transport protein
BraC
endoribonuclease L-PSP, putative
molybdopterin biosynthetic protein B2
glutaminase-asparaginase
binding protein component of ABC
phosphonate transporter
lipoamide dehydrogenase-glc
PA4661
pagL
2.53
lipid A 3-O-deacylase
PA1159
PA3280
oprO
2.4
2.12
PA0852
cbpD
2.06
cold-shock DNA-binding domain protein
pyrophosphate-specific outer membrane porin
OprO precursor
chitin binding domain protein
PA0745
2.05
PA3836
PA0888
aotJ
1.96
1.93
PA5505
PA4370
icmP
1.93
1.92
PA0958
oprD
1.87
enoyl-CoA hydratase/isomerase family
protein
periplasmic binding domain protein
arginine/ornithine binding protein AotJ
PA3790
oprC
1.68
PA5369
pstS
1.66
PA5172
arcB
1.64
lipo, YaeC family protein
insulin-cleaving metalloproteinase outer
membrane protein precursor
basic amino acid, basic peptide and imipenem
outer membrane porin OprD precursor
probable binding protein component of ABC
sugar transporter
putative copper transport outer membrane
porin OprC
phosphate ABC transporter, periplasmic
phosphate-binding protein, PstS
ornithine carbamoyltransferase
PA5091
PA5098
hutG
hutH
1.45
1.41
formimidoylglutamase
histidine ammonia-lyase
PA3190
1.85
subcellular
locationb
Ex (1); OMVs
(1); Un (3)
Pe (1); OMVs
(1); Ex (3)
Cy (3)
Cy (3)
Pe (1); Pe (3)
Pe (2); Pe (3)
Cy (2); Cy (3)
Cy (1); OMVs
(1); Cy (3)
Pe (1); OMVs
(1); Pe (3)
Cy (3)
Cy (3)
Pe (3)
Pe (3)
functional class
ref
secreted factors (toxins, enzymes, alginate)
27
motility and attachment
26, 27,
43, 71
26, 27
26
amino acid biosynthesis and metabolism
translation, post-translational modification, degradation;
nucleotide biosynthesis and metabolism; energy metabolism
energy metabolism
transport of small molecules
amino acid biosynthesis and metabolism; energy metabolism
amino acid biosynthesis and metabolism
26, 27
27
26, 27
26
transport of small molecules
hypothetical, unclassified, unknown
biosynthesis of cofactors, prosthetic groups and carriers
amino acid biosynthesis and metabolism
transport of small molecules
OMVs (1); Cy
(2); Cy (3)
OM (1); OMVs
(1); Un (3)
Cy (2); Cy (3)
OM (1); OM
(3)
Ex (1); Ex (3)
amino acid biosynthesis and metabolism; energy metabolism
Cy (3)
putative enzymes
Pe (1); Un (3)
Pe (1); OMVs
(1); Pe (3)
CM (3)
OM (1); OMVs
(1); OM (3)
OM (1); OMVs
(1); OM (3)
Pe (3)
hypothetical, unclassified, unknown
transport of small molecules
hypothetical, unclassified, unknown
26, 27
26
26, 27,
71
transcriptional regulators; adaptation, protection
transport of small molecules
secreted factors (toxins, enzymes, alginate)
26, 27,
91
26
membrane proteins; transport of small molecules
membrane proteins
transport of small molecules
26, 27
transport of small molecules
26
OM (1); OMVs
(1); OM (3)
Pe (1); CM (3)
transport of small molecules
26, 27
Cy (1); OMVs
(1); Cy (3)
Cy (3)
Cy (1); Cy (3)
amino acid biosynthesis and metabolism
transport of small molecules
26, 27
amino acid biosynthesis and metabolism
amino acid biosynthesis and metabolism
a
Other work in which these proteins were found is indicated. bOMVs 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
proven location in other organisms, and 3 means computationally predicated. Proteins identified in ref 27 are also related to biofilm matrix, while
other references refer to proteins identified from supernatant cultures of P. aeruginosa PAO1, except ref 26, which refers to the matrix of supernatant
cultures of P aeruginosa PA14.
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.74
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
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
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oxygen 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.78 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.79
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.2,80,81
Redox Homeostasis. 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.82−84 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.85
In general, it still unclear how these proteins reach the
extracellular space; no signal sequences can be seen.84 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. 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.86
Like iron, copper is needed for the catalytic centers 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.87 Copper needs are also demonstrated by the high expression of OprC, a specific copper
transport porin.
Molybdenum is also required in the catalytic center and
under anaerobic conditions; nitrate reduction is dependent on
molybdate availability.88 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,
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 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 that were incorporated
into the groups assigned on the y axis.
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 a periplasmic protein typically involved in the
deamidation of asparagine and glutamine, yielding aspartic
and glutamic acids and ammonia. In E. coli75 and Salmonella
enterica,76 this enzyme is expressed during anaerobiosis. In
Shigella f lexneri, the enzyme is required for adhesion and
virulence.76,77 It is well-known that these metabolic mechanisms are activated under limited oxygen accessibility. A
correlation between our proteome data and anaerobic/
microaerobic behavior 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 low
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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 (gray 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.
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.41,97
In the very few published descriptions of the biofilm matrix
proteome, the occurrence of metabolic pathways in this
extracellular environment has never been specifically 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.98
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 spatiotemporal proximity has
implications for the processes that occur within biofilm
communities.
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.89−91
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.92 It is known that
LasB,92 ChiC, and CbpD are expressed in biofilms of clinical
isolates and are transcriptionally regulated and dependent on
quorum-sensing.12,90
Ribosyltransferases are involved in cell signaling and, for
example, queuine tRNA-ribosyltransferase was identified in the
matrix. In bacteria, they can be involved in the control of
anaerobic and aerobic metabolism, virulence, and antioxidant
defense.93 Deaminases can have DNA repair functions or act as
toxins;94 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 defense.95,96 Thus, this
macromolecular complex may represent a future target for
antibacterial developments.
Most of the behaviors described above are transcriptionally
regulated and dependent on quorum-sensing. This is in accord
with our understanding of biofilm-related 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
■
CONCLUSIONS
Combining two proteomics approaches (gel-based and gelfree), 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 localized in
the matrix can act in concert, serving to process materials, since
the matrix proteome potentially enables typical intracellular
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■
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 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 nontraditional location. Specific knowledge of these
mechanisms will help us to intervene in membrane vesicles
function in vivo.
■
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ASSOCIATED CONTENT
S Supporting Information
*
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jproteome.5b00312.
Full experimental procedures; coomassie stained 2D gel
from OMVs proteins from P. aeruginosa PAO1 biofilms;
coomassie stained 2D gel from the whole matrix proteins
from P. aeruginosa PAO1 biofilms; functional categories
of both OMVs and whole matrix proteins; most
hydrophilic (negative GRAVY values) and hydrophobic
(positive GRAVY values) proteins from both OMVs and
matrix proteomes of P. aeruginosaPAO1 biofilms;
periplasmic proteins identified in the OMVs; extracellular
proteins identified in the OMVs; cytoplasmic proteins
identified in the OMVs; cytoplasmic membrane proteins
identified in the OMVs; and extracellular proteins
identified in the whole matrix (PDF)
Peptides identified in both gel-based and gel-free
proteomics approaches (XLSX)
Proteins identified in both gel-based and gel-free
proteomics approaches (XLSX)
■
Article
AUTHOR INFORMATION
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, U.K.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by funds to J.R.D. from AFMnetNCE 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. N.C. would like to thank RSC/EPSRC Analytical
Chemistry Trust Fund of Royal Society of Chemistry, U.K.,
for financial support of his Ph.D. studies.
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DOI: 10.1021/acs.jproteome.5b00312
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