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REGULAR ARTICLE
Proteome analysis reveals adaptation of Pseudomonas
aeruginosa to the cystic fibrosis lung environment
Dinesh Diraviam Sriramulu1, 2, Manfred Nimtz3and Ute Romling1
1Microbiology and Tumor Biology Center (MTC), Karolinska Institutet, Stockholm, Sweden
2Department of Cell Biology and Immunology, Gesellschaft für Biotechnologische Forschung,
Braunschweig, Germany
3Department of Structural Biology, Gesellschaft für Biotechnologische Forschung,
Braunschweig, Germany
Pseudomonas aeruginosa is known for the chronic lung colonization of cystic fibrosis (CF) patients
in addition to eye, ear and urinary tract infections. With the underlying disease CF patients are
predisposed to P. aeruginosa chronic lung infection, which leads to morbidity and mortality. In
this study, we compared the protein expression profile of a CF lung-adapted P. aeruginosa strain
C with that of the burn-wound isolate PAO. Differentially expressed proteins from the whole-cell,
membrane, periplasmic as well as extracellular fraction were identified. The whole-cell proteome
of strain C showed down-regulation of several proteins involved in amino acid metabolism, fatty
acid metabolism, energy metabolism and adaptation leading to a highly distinct proteome pat-
tern for strain C in comparison to PAO. Analysis of secreted proteins by strain C compared to
PAO revealed differential expression of virulence factors under non-inducing conditions. The
membrane proteome of strain C showed modulation of the expression of porins involved in
nutrient and antibiotic influx. The proteome of the periplasmic space of strain C showed reten-
tion of elastase despite that the equal amounts were secreted by strain C and PAO. Altogether,
our results elucidate adaptive strategies of P. aeruginosa towards the nutrient-rich CF lung habitat
during the course of chronic colonization.
Received: June 1, 2004
Revised: November 20, 2004
Accepted: December 27, 2004
Keywords:
Periplasmic proteome / Adaptation / Proteome minimalism / Cystic fibrosis / Pseudo-
monas aeruginosa
3712 Proteomics 2005, 5, 3712–3721
1 Introduction
Pseudomonas aeruginosa is a common environmental bacte-
rium possessing a rich gene pool, which enables it to colo-
nize multiple niches and to utilize a variety of compounds as
energy source. A large number of virulence factors from this
opportunistic pathogen contribute to the infection of burn
wounds, lungs, eyes and also the urinary tract. In addition to
acute lung infections manifested in patients in intensive care
units, P. aeruginosa chronic lung infection in cystic fibrosis
(CF) accounts for morbidity and mortality. Once taken resi-
dence in the lung, P. aeruginosa cannot be eradicated with
most aggressive antibiotic therapies [1]. CF isolates of P. aer-
uginosa converge towards a common phenotype such as lack
of flagella, amino acid auxotrophy, changes in the lipopoly-
saccharide molecule like the loss of O-antigen and modifica-
tion of lipid A, reduced secretion of virulence factors, the
mutator phenotype and mucoidy regardless of their genetic
background [2–7]. The genetic basis of such differences can
be point mutations in structural or regulatory genes like
Correspondence: Dr. Ute Romling, Box 280, Microbiology and
Tumor Biology Center, Karolinska Institutet, SE-17177 Stock-
holm, Sweden
E-mail: Ute.Romling@mtc.ki.se
Fax: 146-8-330-744
Abbreviations: CF, cystic fibrosis; TSB, trypticase soy broth; TSA,
trypticase soy agar; SOD, superoxide dismutase; ROS, reactive
oxygen species
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
DOI 10.1002/pmic.200401227
Proteomics 2005, 5, 3712–3721 Microbiology 3713
mutS,mucA and rpoN or gene knockouts accompanied by
large chromosomal inversions [7–10]. Aforementioned fea-
tures of CF lung isolates of P. aeruginosa distinct from their
environmental counterpart reflect the selective pressure
offered by the unusual niche of the CF lung to modulate
gene expression.
A decade-long epidemiological study revealed that a pre-
dominant lineage of P. aeruginosa, clone C was found in the
lungs of CF patients, in other diseases and in clinical and
nonclinical environments such as rivers, swimming pool
and drinking water throughout Europe [5, 11, 12]. In this
study we analyzed the proteome of a member of P. aerugi-
nosa clone C, designated as strain C, a CF lung isolate which
has a genotype [5] frequently occurring in CF patients of the
Medical School of Hannover. P. aeruginosa strain C was iso-
lated during the onset of the colonization process in the
CF lung. P. aeruginosa strain C has been reported to harbor
600 kb additional genetic material compared to the reference
strain P. aeruginosa PAO due to insertions which ranged
from 23 to 155 kb, resulting in a mosaic-like structure of the
chromosome [13]. Here we present a detailed comparison of
the strain C and PAO proteome, which gave an overview of
the overall protein expression pattern. A detailed protein
expression profile was established by fractionation proce-
dures, which revealed localization of membrane and secreted
proteins and proteins in the periplasmic space in a P. aeru-
ginosa strain that inhabits the unusual niche, the CF lung.
Interpreting the data obtained, we hypothesize that P. aeru-
ginosa strain C exerts ‘proteome minimalism’ by turning off
metabolic pathways not required in the nutrient-rich CF
lung habitat.
2 Materials and methods
2.1 Bacterial strains and growth conditions
Pseudomonas aeruginosa strains PAO1 (ATCC 15692) and C
were pre-cultured in trypticase soy broth (TSB) (1% Tryp-
tone, 0.5% sodium chloride) overnight at 377C with shaking.
The pre-culture was used to prepare a lawn culture on tryp-
ticase soy agar (TSA) (TSB containing 1.5% agar) and incu-
bated for 16 h at 377C.
2.2 Preparation and purification of whole-cell
proteins
All preparative steps were carried out at 47C unless otherwise
mentioned. Cultures were scraped from the plate, washed
twice with 0.01 MPBS, pH 7.5 and centrifuged (6000 6gfor
10 min. at 47C) to obtain the bacterial pellet. Whole-cell pro-
teins were extracted as described [14]. A 1 mL volume of cell
pellet was thoroughly dispersed in 2 mL of solubilization
buffer (7 Murea, 2 Mthiourea, 4% w/v CHAPS, 30 mMDTT,
2mMleupeptin, 1 mMPefaBloc SC, 20 mL1MTris (pH 9.5),
0.5% w/v Pharmalyte (pH 3–10)) and lysed step-wise using a
Branson probe sonicator (Danbury, CT, USA) on ice (40%
duty cycle; Microtip limit 5) for 30 s. Further sonications
were carried out for 3 620 s with an interval of 30 s which
ensured efficient lysis. The sample mixture was then cen-
trifuged at 50 000 6gfor 30 min at 107C. The supernatant
was separated carefully from the pellet and used immedi-
ately for phenol-acetone purification of proteins.
2.3 Phenol-acetone purification
The crude protein extract was purified by a procedure
described by Hancock and Nikaido [15]. One volume of buf-
fer-saturated phenol was added to the protein extract, vor-
texed vigorously, incubated at 707C for 10 min and snap-
cooled on ice. To this mixture, an equal volume of water was
added, vortexed vigorously and warmed and cooled as before.
After centrifugation at 4000 6gfor 10 min at 47C, the upper
aqueous phase was discarded and proteins in the lower
organic phase were precipitated using 5 volumes of ice-cold
acetone. The pure protein pellet was recovered after low-
speed centrifugation at 47C. The final protein pellet was
resuspended in an appropriate volume of solubilization buf-
fer and centrifuged at 10 000 6gfor 3 min to remove insol-
uble materials. Finally, the supernatant corresponding to
600 mg of protein was used immediately for IEF across the
linear pH range 4–7 after quantification using the Bio-Rad
protein assay kit (Bio-Rad).
2.4 Isolation of secretory proteins
Secreted proteins were isolated as described [16]. Bacteria
grown in 100 mL TSB until the stationary phase was reached
were centrifuged at 6000 6g for 15 min and the super-
natant was passed through a 0.22 mm pore size membrane
filter to remove bacterial cells. Deoxycholic acid (0.2 mg/mL)
was added and the mixture was incubated for 30 min. on ice.
Then TCA acid was added (6% w/v), incubated overnight at
47C to precipitate proteins and centrifuged at 18 000 6gfor
30 min. The pellet was resuspended in a small volume of
water and precipitated with 8 volumes of ice-cold acetone.
After incubation at 2207C for 2 h the mixture was cen-
trifuged at 3500 6gfor 20 min and the pellet was dried at
room temperature for 5 min. Dried pellet was dissolved
thoroughly in an appropriate volume of solubilization buffer
and centrifuged at 50 000 6gfor 40 min at 107C to remove
insoluble materials. The supernatant was treated with phe-
nol-acetone as described above. Four hundred micrograms of
protein was separated by 2-DE across the linear pH range 3–
10.
2.5 Isolation and purification of membrane proteins
A method described by Hancock and Nikaido [15] was fol-
lowed with modifications. Harvested bacterial pellet was
washed twice with 10 mMTris-Cl (pH 7.5) and resuspended
in 0.5 Msucrose buffer containing protease inhibitors
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3714 D. D. Sriramulu et al. Proteomics 2005, 5, 3712–3721
(1 tablet Complete Mini cocktail (Roche) in 30 mL solution).
The suspension was sonicated (Microtip limit 5; 40% duty
cycle) three times for 30 s on ice with an interval of 30 s each.
A discontinuous sucrose density gradient (0, 0.5, 1.0, 1.5 and
2.0 Msucrose) was prepared, the suspension was carefully
layered on the top of the gradient and centrifuged at
50 000 6gfor 1 h. Fractions at the interfaces of 0.5:1.0 M
(inner membrane) and 1.0:1.5 M(outer membrane) were
collected by successively sucking the liquid from the top and
pooled together. The pooled fractions were diluted with at
least 2 volumes of deionized water in order to achieve a
sucrose concentration below 20% and centrifuged at
50 000 6gfor 1 h to pellet the membranes.
The pellet was resuspended thoroughly in 1 MTr i s -C l
(pH 7.8) containing appropriate amount of Complete mini
protease inhibitor cocktail. To remove contaminants that inter-
fere with IEF, phenol-acetone purification was carried out as
described above. The resulting pellet was treated with
2 volumes of ether and the precipitated protein was resus-
pended in a modified solubilization buffer (7 Murea,
2Mthiourea, 2 mMtributyl phosphine, 1% amidosulfobe-
taine-14, 1tablet Complete Mini protease inhibitor cocktail
(Roche) per 5mL,20mL1MTris (pH 9.5), 0.5% w/v Pharma-
lyte (pH 3–10)). The protein preparation was centrifuged to
achieve a clear supernatant and used for IEF; 400 mgofprotein
was separated by 2-DE across the linear pH range 3–10.
2.6 Extraction of proteins from the periplasmic space
Proteins from the bacterial periplasm were isolated by the
chloroform shock procedure as described by Ames [17].
Briefly, harvested bacteria grown in 100 mL TSB until the
stationary phase was reached were washed, dispersed
homogenously in 0.5 mL chloroform and allowed to stay for
15 min at room temperature. 10 mL of 10 mMTris-Cl
(pH 8.0) was added and mixed thoroughly. The cells were
then removed by centrifugation at 10 000 6gfor 20 min to
obtain a clear supernatant. Proteins were extracted from the
resulting ‘chloroform shock fluid’ by following the procedure
described for the isolation of secretory proteins. Finally
400 mg of purified protein was separated by 2-DE across the
non-linear pH range 3–10.
2.7 2-DE
Isoelectric focussing was carried out using IPGphor
(Amersham Pharmacia) at 207C. IPG strips were rehydrated
for 4 h and the following scheme was used for focussing:
30 V constant for 10 h, 150 V gradient for 2 h, 300 V gradient
for 2 h, 600 V gradient for 2 h, 1500 V gradient for 4 h,
3500 V gradient for 8 h, 8000 V gradient for 3 h, and a final
8000 V constant for a total of 250 kVh. IPG strips were
reduced (6 Murea, 30% glycerol, 1% w/v DTT, 2% w/v SDS
in 0.05 MTris-Cl (pH 8.8)) for 12 min and free SH-groups
were blocked with iodoacetamide (6 Murea, 30% glycerol,
260 mMiodoacetamide, 2% w/vw/v SDS in 0.05 MTris-Cl
(pH 8.8) for 12 min. Then the strips were embedded on top
(using 0.5% agarose in running buffer colored with bromo-
phenol blue) of a 12–15% pore-gradient SDS-PAGE and run
in 24 mMTris, 0.2 Mglycine, 0.1% SDS at 6 V/cm at 107C
until the bromophenol blue dye front reached the end of the
gel. The gels were fixed in 40% ethanol and 10% acetic acid
mixture for at least 4 h to overnight, successively stained
overnight with CBB G-250 (Biomol, Hamburg, Germany) as
described by Neuhoff [18] and destained overnight in dis-
tilled water. After scanning (Umax Powerlook III) the gels,
protein spots, which showed at least three-fold different
intensities as evident from the comparison by using Phor-
etix 2D software (Nonlinear Dynamics, USA) and by visual
examination between comparative gels were excised and
processed for mass spectrometry or archived for future use.
All experiments were carried out at least twice, such as, pro-
tein extracts for the whole-cell proteome and sub-proteome
analyses were prepared from at least two independent bacte-
rial cultures and at least two gels were analyzed.
2.8 Protein identification and characterization
Preparation of protein spots for mass spectrometry was car-
ried out as described [19]. Briefly, a protein spot was excised
from the gel and chopped into tiny pieces. After washing
with deionized water the pieces were shrunk with 50 mL
ACN. The supernatant was discarded and the gel pieces were
reswollen in 50 mLof10m
MDTT in 0.1 MNH4HCO3to
reduce the disulfide bonds of the proteins. After incubation
at 567C for 30 min, 50 mL of ACN was added to shrink gel
pieces. The supernatant was removed and the gel pieces were
reswollen in 50 mLof55m
Miodoacetamide in
0.1 MNH4HCO3. After incubation for 20 min in the dark at
room temperature, the supernatant was removed completely.
Gel pieces were shrunk using ACN as before, dried briefly,
reswollen in trypsin solution (2 ng/mL trypsin in
0.05 MNH4HCO3) and incubated overnight at 377C for
digestion. After incubation 10–15 mL25m
MNH4HCO3was
added to the gel pieces and incubated at 377C for 15 min with
shaking. Then 50 mL ACN was added to the previous mixture
and incubated at 377C for 15 min with shaking. Supernatant
was collected and stored. To the gel pieces 40–50 mL 5% for-
mic acid was added and incubated at 377C for 15 min with
shaking. Then, 50 mL of ACN was added to the previous
mixture and incubated at 377C for 15 min with shaking. The
gel particles were spun down to collect the supernatant,
pooled with previously collected supernatant and dried com-
pletely in a SpeedVac (Eppendorf). Dried peptides were
recovered in 10 mL of 0.5% HCOOH in 5.0% methanol and
desalted using C18 ZipTip (Millipore). Elution of an aliquot of
peptides on MALDI target was done with an equal volume of
saturated solution of alpha-cyano-4-hydroxy cinnamic acid in
0.5% formic acid/65% methanol mixture. PMF maps of
trypsin-digested peptides generated by MALDI-Tof Reflex II
(Bruker-Franzen-Analytik) mass spectrometer were com-
pared to a database of all translated ORF from P. aeruginosa
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Proteomics 2005, 5, 3712–3721 Microbiology 3715
PAO1 (www.pseudomonas.com) using MS-Fit of Protein
Prospector package (http://falcon.ludwig.ucl.ac.uk/
ucsfhtml3.2/msfit.htm). A minimum of 25% sequence cov-
erage was considered sufficient to identify a protein with
confidence. If the sequence coverage was less than 25%, at
least three peptide-sequence tags were sequenced by ESI
MS/MS using Q-Tof-2 (Micromass) to identify the protein. In
some cases peptide sequencing of tryptic digests was done
without prior MALDI-Tof analysis. If unambiguous assign-
ment was not possible, N-terminal sequencing (Procise Pro-
tein Sequencer) was carried out with protein spots excised
from a PVDF-blotted 2-D gel and the sequences were
screened against protein databases using Fasta3 (www.ebi.
ac.uk/fasta33).
3 Results and discussion
3.1 Whole-cell proteome comparison of P. aeruginosa
PAO and C
2-D separation of whole-cell proteins revealed characteristic
protein expression profiles for the burn wound isolate
P. aeruginosa PAO (Fig. 1a) and the CF-lung isolate C
Figure 1. Whole-cell proteome of P. aeruginosa (a) PAO and
(b) strain C. Numbered arrows indicate the identity of protein
spots listed in Table 1.
(Fig. 1b). The proteome of strain C as visible in the pH range
4–7 showed a large number of protein spots with lower
intensity as compared to PAO. Such a unique proteome pat-
tern was not only shown by strain C (Fig. 1b) isolated during
the onset of the colonization process but also by a few other
CF isolates belonging to clone C, from the mid- and late-
phase of chronic lung infection (data not shown). Due to the
huge number of differences between 2-D whole-cell protein
gels from P. aeruginosa PAO and strain C some spots had to
be selected. Therefore protein spots, which could not be vis-
ualized in one of the compared gels and showed at least
three-fold different intensities that could be visually dis-
tinguished between strain C and PAO were chosen for fur-
ther analysis by PMF and/or by MS/MS and/or by sequenc-
ing the N-terminal region. A total of 192 proteins (see Table 1
and Supplementary table) belonging to different functional
categories were identified with the help of the available
whole genome sequence database (www.pseudomonas.
com).
3.2 Down-regulation of proteins involved in general
metabolic pathways in strain C
Enzymes involved in biosynthesis as well as degradation of
several amino acids were down-regulated in the strain C
proteome. Most prominently down-regulated were the
enzymes involved in the biosynthesis and metabolism of
branched-chain amino acids (leucine, isoleucine and valine
(LIV)), serine, threonine, phenylalanine, lysine, methionine,
glutamine and tryptophan (Table 1). Acetyl-Coenzyme A (A-
CoA) is a central metabolite and is used in several metabolic
processes like amino acid biosynthesis and fatty acid biosyn-
thesis. Three proteins of a multi-enzyme complex (pyruvate
dehydrogenase (AceE), lipoamide dehydrogenase-Val (LpdV)
and branched-chain alpha-keto acid dehydrogenase (BkdB))
that catalyzes the formation of A-CoA from pyruvate
(www.kegg.org) were found down-regulated in the strain C
proteome. AceE in complex with acetolactate synthase is also
required for the conversion of pyruvate to 2-hydroxyethyl
thiamine diphosphate, which enters into valine and iso-
leucine biosynthetic pathways. Ketol-acid reductoiosmerase
(IlvC), the second enzyme involved in two catalytic reactions
in the biosynthetic pathway leading to the formation of
valine and isoleucine was also down-regulated in the strain C
proteome. 2-Oxoisovalerate dehydrogenase, a complex en-
zyme with two subunits (BkdA1 and BkdA2) and beta-keto-
adipyl CoA thiolase (PcaF) involved in the initial step and
final steps, respectively, of the LIV degradation pathway were
down-regulated in the strain C proteome. Other differentially
expressed proteins involved in amino acid metabolism are
shown in Table 1. Amino acids were found at higher con-
centrations in the CF sputum compared to sputum of heal-
thy individuals and proposed to play a significant role in the
emergence and maintenance of P. aeruginosa mutants auxo-
trophic for amino acids during chronic colonization of the
lung [6, 20, 21]. Differential expression of proteins
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3716 D. D. Sriramulu et al. Proteomics 2005, 5, 3712–3721
Table 1. Identity of protein spots shown in Figures 1 through 4
Spot
no.
PA
no.
Function Gene Mass
(kDa)
pIExpression
level
Amino acid biosynthesis and metabolism
1 PA2250 Lipoamide dehydrogenase-Val lpdV 48.6 6.16 ;
2 PA5413 Low specificity L-threonine aldolase ltaA 38.2 5.18 ;
3 PA2248 2-Oxoisovalerate dehydrogenase-Beta subunit bkdA2 38.3 5.37 ;
4 PA0865 4-Hydroxyphenylpyruvate dioxygenase hpd 39.9 5.10 ;
5 PA2249 Branched-chain alpha-keto acid dehydrogenase bkdB 45.8 5.84 ;
6 PA5171 Arginine deiminase arcA 46.4 5.52 ;
7 PA5015 Pyruvate dehydrogenase aceE 99.6 5.56 ;
8 PA2247 2-Oxoisovalerate dehydrogenase-Alpha subunit bkdA1 45.3 5.67 ;
9 PA4938 Adenylosuccinate synthetase purA 46.8 5.70 ;
10 PA0432 S-adenosyl-L-homocysteine hydrolase sahH 51.4 5.71 ;
11 PA4759 Dihydrodipicolinate reductase dapB 28.3 5.74 ;
12 PA5172 Ornithine carbamoyl transferase, catabolic arcB 38.1 6.13 ;
13 PA0609 Anthranilate sythetase - component I trpE 54.9 5.00 ;
14 PA5119 Glutamine synthetase (glutamate-ammonia ligase) glnA 52.1 5.10 ;
15 PA0871 Pterin-4-alpha-carbinolamine dehydratase phhB 13.3 6.89 ;
16 PA0316 D-3-phosphoglycerate dehydrogenase serA 44.2 6.52 ;
17 PA0870 Aromatic amino acid aminotransferase phhC 43.2 6.24 ;
18 PA0447 Glutaryl-CoA dehydrogenase gcdH 43.3 6.12 ;
19 PA4694 Ketol-acid reductoisomerase ilvC 36.4 5.65 ;
Fatty acid biosynthesis and metabolism
20 PA4848 Biotin carboxylase accC 48.9 5.92 ;
21 PA1609 Beta-ketoacyl-ACP synthase I fabB 42.8 5.40 ;
22 PA3639 Acetyl coenzyme A carboxylase carboxyl transferase-alpha accA 35.0 5.15 ;
23 PA0228 Beta-ketoadipyl CoA thiolase pcaF 42.1 5.52 ;
Membrane proteins
24 PA1092 Flagellin type B fliC 49.2 5.40 *
\;
25 PA0958 Porin D precursor oprD 48.4 4.75 ;
26 PA2760 Probable outer membrane protein oprQ 46.8 5.51 ;
27 PA1777 Outer membrane protein OprF precursor oprF 37.6 4.98 :
28 PA4370 Insulin-cleavage metalloproteinase icmP 47.2 4.68 :
29 PA0766 Serine protease MucD precursor mucD 50.4 7.77 :
30 PA3262 Probable peptidyl-prolyl cis-trans isomerase FkbP-type 26.9 6.80 :
31 PA1041 Probable outer membrane protein 21.8 5.06 :
32 PA2853 Major OM lipoprotein precursor (murein-lipoprotein) oprI 8.80 8.51 :
33 PA1178 PhoP/Q & low Mg21inducible OMP H1 precursor oprH 21.6 9.43 :
Secreted proteins
34 PA5112 Lipase/esterase precursor estA 71.7 4.66 *
\;
35 PA1080 Flagellar hook protein flgE 48.3 4.42 *
\;
36 PA1092 Flagellin type B fliC 49.2 5.40 *
\;
37 PA2939 Probable aminopeptidase 59.3 4.97 ;
38 PA3724 Propeptide fragment of elastase lasB 18.0 5.90 :
39 PA4175 Protease IV prpL 26.0 8.70 :
40 PA3724 Elastase lasB 53.7 6.28 nc
Periplasmic proteins
41 PA1092 Flagellin type B fliC 49.2 5.40 *
\;
42 PA1074 Branched chain amino acid binding protein braC 39.8 5.80 ;
43 PA0301 Polyamine transport protein PotF3 potF3 40.1 5.51 ;
44 PA3250 Putative spermidine/putrescine substrate-binding protein 38.3 5.92 ;
45 PA4913 Probable binding component of ABC transporter 39.8 6.20 :
46 PA0852 Chitin binding protein precursor cbpD 42.0 6.60 ;
47 PA2442 Glycine cleavage system protein T2 gcvT2 39.9 5.95 *
\;
48 PA0300 Polyamine transport protein PotF2 potF2 40.6 6.97 ;
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Proteomics 2005, 5, 3712–3721 Microbiology 3717
Table 1. Continued
Spot
no.
PA
no.
Function Gene Mass
(kDa)
pIExpression
level
49 PA1493 Sulfate binding protein of ABC transporter cysP 36.5 7.76 ;
50 PA1946 Binding protein component precursor of ABC ribose transporter rbsB 33.9 7.78 :
51 PA3724 Elastase lasB 53.7 6.28 :
52 PA5489 Disulphide oxidoreductase/thiol:disulphide interchange protein dsbA 23.4 5.98 :
53 PA5153 Probable periplasmic binding protein 27.7 5.13 ;
54 PA2513 Anthranilate dioxygenase small subunit antB 19.4 6.65 *
\;
55 PA2614 Lipoprotein-specific chaperone lolA 23.1 5.75 :
56 PA2300 Chitinase chiC 53.0 5.23 *
57 PA4356 Xenobiotic reductase xenB 37.8 5.11 :
58 PA5018 Peptide methionine sulfoxide reductase msrA 23.7 5.20 :
59 PA0852 Chitin binding protein (cleavage product) cbpD 30.0 6.40 :
60 PA0888 Arginine/ornithine binding protein aotJ 28.0 6.43 ;
61 PA0139 Alkyl hydroperoxide reductase subunit C ahpC 20.6 5.89 ;
62 PA4468 Superoxide dismutase (manganese cofactored) sodM 22.5 5.81 ;
63 PA4366 Superoxide dismutase (iron cofactored) sodB 21.4 5.27 ;
64 PA4922 Azurin precursor azu 16.0 6.39 ;
Symbols represent differential expression of proteins in strain C compared to PAO
;down regulated
:up regulated
*
\Not found in the gel of strain C
*Spot hidden by an overlapping protein
nc, no change
involved in amino acid metabolism has also been reported
for P. aeruginosa during the course of biofilm formation [22].
In Bacillus subtilis, it has been shown that addition of
0.2% casamino acids in the medium had a pronounced effect
on the expression of proteins involved in amino acid metab-
olism [23]. Based on the global changes in amino acid me-
tabolism in strain C, we speculate that strain C, though a
prototroph, has developed partial auxotrophy during the
course of chronic lung infection. It has been known for a
long time that P. aeruginosa develops auxotrophy for various
amino acids in the CF lung [6].
Beta-ketoadipyl CoA thiolase (PcaF), required for the LIV
degradation, is also required for the first step in the fatty acid
biosynthesis and metabolism. In addition to PcaF, other
enzymes such as FabB and AccAB involved in the fatty acid
biosynthetic pathway were down-regulated by strain C. Fatty
acids are essential constituents of bacterial membrane phos-
pholipids and lipid A and broad-spectrum antimicrobials tar-
get fatty acid biosynthetic pathway enzymes [24]. Down-reg-
ulation of proteins involved in fatty acid biosynthesis might be
one of the adaptation strategies of strain C to the CF lung en-
vironment, where there is a constant antimicrobial pressure.
3.3 Membrane subproteome of P. aeruginosa strain C
Identification of 15 differentially expressed protein spots
from the membrane fraction revealed that there was no con-
tamination with cytoplasmic proteins (Fig. 2a and b and
Supplementary table). As a prominent spot in the 2-D gel,
the major nonspecific outer membrane porin (OprF) [25] was
up-regulated in strain C. OprF is a major multifunctional
outer membrane protein, which was shown to have a large
exclusion limit allowing molecules with molecular weight up
to 3000 which includes amino acids, di- and tri-saccharides to
pass into periplasmic space [25–27]. Recently, OprF was
shown to be significantly up-regulated by P. aeruginosa
growing in the CF lung [28]. OprH involved in the resistance
to polycationic antibiotic polymyxin B [29, 30] and the major
outer membrane lipoprotein OprI (spot 32 in Fig. 1a and b)
were also up-regulated, as was a peptidyl-prolyl-cis-trans
isomerase (PA3262) associated with outer membrane
involved in the folding of proteins. MucD, a serine protease
and a repressor of mucoid phenotype [31], was also up-regu-
lated by strain C. This observation is consistent with the non-
mucoid phenotype exhibited by strain C on a solid culture
medium (data not shown). About five protein spots (Table 1
and Supplementary table) identified as proteins of unknown
function were up-regulated in the strain C membrane sub-
proteome.
Conversely, OprD, a porin specific for antibiotic influx
[32] was expressed at a lower level by strain C. This observa-
tion seems considerable as the resistance mechanism to car-
bapenem antibiotics requires down-regulation of this porin
which serves as the primary route of entry [32, 33]. Moreover,
down-regulation or loss of OprD has been shown by P. aeru-
ginosa isolates derived from the CF lung environment [34].
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3718 D. D. Sriramulu et al. Proteomics 2005, 5, 3712–3721
Figure 2. Membrane sub-proteome of P. aeruginosa (a) PAO and
(b) strain C. Numbered arrows indicate the identity of protein
spots listed in Table 1.
3.4 Secretory proteome
Extracellular proteins were extracted from the stationary
phase rich medium culture of P. aeruginosa PAO and C
(Fig. 3a and b) and 13 protein spots (see Supplementary
table) were identified. Protease IV, an important corneal
virulence factor [35] was expressed in higher quantities by
strain C. This protease was shown to be a virulence factor
in corneal infection and it can digest biologically important
proteins such as immunoglobulin, complement compo-
nents, fibrinogen and plasminogen [36, 37]. Over-
expression and secretion of proteases by strain C might be
necessary to alleviate host immune factors, for example,
transferrin and lactoferrin [38]. Elastase is one of the major
virulence factors produced by P. aeruginosa [39, 40]. Both
strain C and PAO produced equal amount of the secreted
form of elastase.
Figure 3. Secretory proteome of P. aeruginosa (a) PAO and
(b) strain C. Numbered arrows indicate the identity of protein
spots listed in Table 1.
The cell surface appendage protein flagellin (FliC) and its
associated hook protein (FlgE) were found in the extra-
cellular fraction of PAO1 as the flagellum can easily be
detached from the cell especially in shaking cultures and is
not excluded by the filtration procedure [39]. However, FliC
was not detected in the secretory proteome of strain C, con-
sistent with strain C being non-motile (data not shown).
3.5 Proteome of the periplasmic space
Periplasm not only serves as a buffer zone that controls the
influx and efflux of nutrients, antibiotics and other compo-
nents but also serves as a milieu where a number of viru-
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3712–3721 Microbiology 3719
lence factors are processed for subsequent secretion [40–42].
Proteins in the periplasmic space were extracted from sta-
tionary phase cultures by the ‘chloroform shock’ procedure.
A large number of differentially expressed protein spots
could be observed from the periplasmic sub-proteome of
P. aeruginosa PAO and C (Fig. 4a and b). About 39 protein
spots (Table 1 and Supplementary table) were chosen for
further characterization. The predicted and reported peri-
plasmic location of the identified proteins established the
absence of contaminating proteins from other compart-
ments. Prominently visible protein spots from the P. aerugi-
nosa PAO periplasmic subproteome and down-regulated in
the strain C periplasmic subproteome were identified as fla-
gellin type B (FliC), branched-chain amino acid binding
protein (BraC), arginine/ornithine binding protein (AotJ),
superoxide dismutase (SodB) and azurin precursor (Azu).
ATP-binding cassette transport systems in Gram-nega-
tive bacteria are basically composed of a substrate-binding
protein located in the periplasm and a membrane-bound
complex which consists of a permease located in the inner
Figure 4. Sub-proteome of the periplasmic space of P. aerugi-
nosa (a) PAO and (b) strain C. Numbered arrows indicate the
identity of protein spots listed in Table 1.
membrane and a ATP-binding protein associated with inner
membrane. Seven proteins (spots 42, 43, 44, 48, 49, 53 and
60 in Table 1) down-regulated in strain C were identified as
the substrate-binding component of ATP-binding cassette
transport systems of amino acids, amines, salts and sugar
(www.pseudomonas.com). Only two periplasmic substrate-
binding proteins were found up-regulated, one of them
being the binding protein component of ribose transporter
(RbsB).
In the P. aeruginosa PAO genome, there exist the well-
characterized high-affinity branched-chain amino acid (LIV)
ABC transporter, BraCDEFG [43]. The periplasmic binding
component BraC (PA1074), is down-regulated in strain C.
Surprisingly, a paralogous protein encoded by PA4913 which
shows 42.7% identity to BraC (PA1074) is up-regulated in the
strain C.
In the CF-lung, P. aeruginosa is exposed to reactive oxygen
species (ROS) produced by neutrophils. Superoxide dis-
mutase (SOD) plays an important role in scavenging oxygen
radicals. Two isoforms of SOD discriminated by the metal ion
at the active site have been reported in P. aeruginosa PAO.
Strain C expressed both the manganese- (SodM) and the iron-
cofactored (SodB) form of SOD whereas PAO expressed SodB
only, although at higher levels than in strain C. The C-subunit
of alkyl hydroperoxide reductase (AhpCF), another protein
involved in the oxidative stress response against exogenous
ROS [44], was up-regulated by strain C.
A highly up-regulated protein spot identified as the
mature form of elastase was found in the periplasmic sub-
proteome of strain C. Retention of elastase in the peri-
plasmic space has been shown to have a biological role,
namely, to participate in the cleavage of the 16 kDa form of
nucleoside diphosphate kinase (Ndk) to the 12 kDa form [40,
45] which predominantly synthesizes GTP necessary for
protein synthesis and virulence factor production. Accumu-
lation of enzymatically active elastase in the periplasmic
space was shown to indirectly influence the energy metabo-
lism of P. aeruginosa [40]. Although the 12 kDa form of Ndk
was not identified, the lower quantities of the 16 kDa form of
Ndk from the whole-cell proteome of strain C compared to
PAO could serve as an indirect evidence for an enhanced
conversion of Ndk from the 16 kDa to the 12 kDa counter-
parts. As an alternative function, however not mutually
exclusive, elastase is retained in the periplasm to be secreted
via membrane vesicles [46].
Chitin-binding protein (CbpD) precursor is one of the
major secreted proteins by P. aeruginosa PAO [47]. CbpD was
identified as a 43 kDa protein spot in addition to a 30 kDa
fragment, which has been shown to be cleaved by elastase
[47]. Though there was only a minor difference in the level of
expression of the full-length CbpD by P. aeruginosa C and
PAO, the 30 kDa fragment was found in huge amounts in
the strain C periplasmic sub-proteome. Chitin-binding pro-
tein (CbpD), which is expressed by many clinical isolates of
P. aeruginosa, plays a significant role as an adhesin, mediat-
ing colonization of eukaryotic cells [47].
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
3720 D. D. Sriramulu et al. Proteomics 2005, 5, 3712–3721
A variety of proteins involved in proper folding of dis-
ulfide-bond-containing proteins have been found up-regu-
lated in strain C. DsbA, a periplasmic protein that functions
as a thiol:disulfide oxidoreductase, is required for catalyzing
the oxidative folding and assembly of many secreted factors
in P. aeruginosa [42, 48]. Up-regulation of DsbA by strain C
might be important for proper folding of virulence factors
such as elastase and protease IV, which have disulfide bonds.
DsbA was also shown to affect the twitching motility pheno-
type mediated by type IV pili, which contain an intrastrand
disulfide loop [49]. A probable peptidyl-prolyl cis-trans isom-
erase – FkbP type (PA3262) was also up-regulated in
strain C, which might participate in the maturation of peri-
plasmic and secreted factors. LolA, a lipoprotein-specific
chaperone [50] known to participate in the translocation of
lipoproteins from inner membrane to the outer membrane
was produced in significant amounts by strain C. Up-reg-
ulation of trigger factor, a ribosome-associated chaperone
involved in the folding of nascent secretory and non-secre-
tory polypeptide chains [51] might be necessary under
stressful growth conditions. From the above observation, it
can be speculated that up-regulation of chaperones involved
in protein-folding plays a significant role in the proper pro-
duction and localization of several virulence factors and
membrane proteins under stressful conditions such as in the
CF-lung.
Peptide methionine-sulfoxide reductase (MsrA) reduces
methionine sulfoxide produced as a consequence of oxidation
by ROS to methionine with subsequent restoration of the bio-
logical activity of proteins [52]. MsrA was also reported to be
important for the production and maintenance of cell surface
structures especially adhesins [53]. Up-regulation of MsrA by
strain C might have a protective role on proteins, which inter-
act closely with the CF-lung environment, where bacterial cells
face enhanced concentration of exogenous ROS.
4 Concluding remarks
In this study, we compared the protein expression pattern of
P. aeruginosa strains derived from different niches. CF iso-
lates of P. aeruginosa are known to exhibit multiple pheno-
typic changes depending on the extent of chronic coloniza-
tion in the CF lung. Analysis of proteins from different cel-
lular compartments and the milieu (secreted proteins)
under predefined culture conditions gave us an overview
about the general adaptation strategy of bacteria living in
the unusual niche of the CF lung (Fig. 5). This adaptation
strategy, which is maintained even under in vitro conditions,
suggests that P. aeruginosa strain C performs ‘proteome
minimalism’, by reducing the expression of all unnecessary
metabolic pathways with the probable involvement of global
regulatory gene(s). Differential regulation of proteins
involved in general metabolism, virulence and substrate
acquisition by the CF lung isolate strain C indicate that
bacteria can tailor themselves according to the nutrient sta-
tus of the microenvironment of the host. Moreover, by fol-
lowing a compartmental proteomic approach it is possible
to characterize and to localize proteins involved in novel and
unknown functions and to find effective antibacterial tar-
gets. Particularly, elaboration of proteins from the peri-
plasmic space provided better understanding of the proces-
sing of virulence factors by a well-adapted P. aeruginosa
CF lung isolate.
We thank Dirk Wehmhoener and Maja Baumgaertner for
their technical guidance. S.D.D was a member of the European
Graduate College ‘Pseudomonas: Pathogenicity and Biotechnol-
ogy’. This work was further supported by the Mukoviszidose e. V.
the Karolinska Institutet (Elitforskartjänst to U.R) and the
Anna-Greta and Holger Crafoords fund.
Figure 5. Adaptation strategy
shown by P. aeruginosa strain C
during the course of chronic
colonization in the CF lung.
2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.de
Proteomics 2005, 5, 3712–3721 Microbiology 3721
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