Download Proteomic analysis of Escherichia coli with experimentally

Research in Microbiology 161 (2010) 268e275
www.elsevier.com/locate/resmic
Proteomic analysis of Escherichia coli with experimentally induced
resistance to piperacillin/tazobactam
Kênia Valéria dos Santos a, Cláudio Galuppo Diniz b, Luciano de Castro Veloso a, Hélida Monteiro
de Andrade c, Mario da Silva Giusta d, Simone da Fonseca Pires d, Agenor Valadares Santos d,
Ana Carolina Morais Apolônio a, Maria Auxiliadora Roque de Carvalho a, Luiz de Macêdo
Farias a,*
a
Departamento de Microbiologia, Instituto de Cieˆncias Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 30.161-970 Belo Horizonte MG,
Brazil
b
Departamento de Parasitologia, Microbiologia e Imunologia, Universidade Federal de Juiz de Fora, Bairro Martelos, Juiz de Fora CEP 36036-900 MG, Brazil
c
Departamento de Parasitologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte MG, Brazil
d
Departamento de Bioquı´mica e Imunologia, Instituto de Cieˆncias Biológicas, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Belo Horizonte
MG, Brazil
Received 12 January 2010; accepted 17 March 2010
Available online 8 April 2010
Abstract
The worldwide emergence of antibiotic-resistant bacteria poses a serious threat to human health. In addition to the difficulties in controlling
infectious diseases, the phenotype of resistance can generate metabolic changes which, in turn, can interfere with hostepathogen interactions.
The aim of the present study was to identify changes in the subproteome of a laboratory-derived piperacillin/tazobactam-resistant strain of
Escherichia coli (minimal inhibitory concentration [MIC] ¼ 128 mg/L) as compared with its susceptible wild-type strain E. coli ATCC 25922
(MIC ¼ 2 mg/L) using 2-D fluorescence difference gel electrophoresis (2D-DIGE) followed by matrix-assisted laser desorption/ionization timeof-flight/time-of-flight (MALDI-TOF/TOF MS). In the resistant strain, a total of 12 protein species were increased in abundance relative to the
wild-type strain, including those related to bacterial virulence, antibiotic resistance and DNA protection during stress. Fourteen proteins were
increased in abundance in the wild-type strain compared to the resistant strain, including those involved in glycolysis, protein biosynthesis,
pentose-phosphate shunt, amino acid transport, cell division and oxidative stress response. In conclusion, our data show overall changes in the
subproteome of the piperacillin/tazobactam-resistant strain, reporting for the first time the potential role of a multidrug efflux pump system in E.
coli resistance to piperacillin/tazobactam.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords: 2-D DIGE; Comparative proteome; Escherichia coli; Piperacillin/tazobactam; Resistance
1. Introduction
* Correspondence and reprints.
E-mail addresses: [email protected] (K. Valéria dos Santos),
[email protected] (C.G. Diniz), [email protected] (L. de Castro
Veloso), [email protected] (H. Monteiro de Andrade), mgiusta@
yahoo.com.br (M. da Silva Giusta), [email protected] (S. da Fonseca
Pires), [email protected] (A.V. Santos), [email protected]
(A.C. Morais Apolônio), [email protected] (M.A. Roque de Carvalho),
[email protected] (L. de Macêdo Farias).
0923-2508/$ - see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.resmic.2010.03.006
With almost seven decades of widespread use of antibiotics,
bacterial resistance is increasingly commonplace amongst
important human and animal pathogens. It is well recognized
that antimicrobial-resistant microorganisms can acquire
unexpected genetic information, thus expressing new physiologic and molecular characteristics that may interfere with the
management of infectious diseases (Diniz et al., 2000;
Linares-Rodriguez
and
Martinez-Menendez,
2005).
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
In addition, treatment with subinhibitory concentrations of
some antibiotics may also influence the virulence properties of
microorganisms and their relationships with host immune
defenses (Diniz et al., 2004; Lorian and Gemmel, 2005). The
elucidation of the molecular details of drug resistance is a very
active area of research that crosses many disciplinary boundaries. An understanding of the mechanism(s) by which drug
resistance develops leads to improvements in extending the
efficacy of current antimicrobials and in the management of
infectious diseases (Cash, 2000).
Currently, proteomics is a challenging field that has been
growing rapidly in the postgenomic era. Mass spectrometry
used in combination with various protein resolution methods
and bioinformatic tools have become routine methods for
proteomic research (Jungblut et al., 2008). Although not
widely used, proteomic methodologies contribute towards
determining antimicrobial resistance mechanism(s) and other
cell metabolic alterations through the capacity to analyze
overall changes in bacteria (Cash, 2000). The pattern of
protein species related to antimicrobial resistance has been
researched in a variety of microorganisms and with different
antimicrobial agents (Andrade et al., 2008; Bore et al., 2007;
Cash et al., 1999; Coldham and Woodward, 2004; Diniz
et al., 2004; Lis and Bobek, 2008; Mcatee et al., 2001;
Pieper et al., 2006; Xu et al., 2006; Yoo et al., 2007).
To date, there is no information in the literature concerning
the influence of the piperacillin/tazobactam resistance
phenotype on bacterial characteristics. Piperacillin is a potent,
broad-spectrum ureidopenicillin, and when combined with the
triazolymethyl penicillanic acid sulfone beta-lactamase
inhibitor tazobactam, results in a broader spectrum of activity
against beta-lactamase-producing Gram-negative, Gram-positive and anaerobic organisms. Piperacillin-tazobactam is
currently recommended for the treatment of intra-abdominal,
lower respiratory tract, skin and skin structure and gynecologic infections (Dos Santos et al., 2007). Bacterial resistance
to piperacillin/tazobactam has been related to: alterations in
drug binding to their cellular targets; alterations in cell
membrane permeation of Gram-negative bacteria and, more
frequently, high-level expression of extended spectrum betalactamases (ESBLs) (Marre et al., 1984; Pitout et al., 2008;
Rice et al., 2000).
Thus, in the present study, we examined the subproteome of
a laboratory-derived strain of Escherichia coli representing the
acquisition of piperacillin/tazobactam resistance. E. coli has
been frequently used as a model organism in structural and
functional studies aimed at understanding bacterial physiology
and gene expression. This is because E. coli not only has the
entire genome sequence available, but is also one of the most
important pathogenic bacteria, both for humans and animals.
2. Materials and methods
2.1. E. coli strains and antibiotics
E. coli ATCC 25922 was used as wild-type strain. Selection
of resistant bacteria was carried out by serially subculturing
269
the wild-type strain onto brain heart infusion (BHI) agar plates
(Difco, Spark, MD, USA) containing a linear gradient of
piperacillin/tazobactam (PTZ). BHI-antibiotic gradient plates
(90 by 90 mm) were prepared as previously described (Bryson
and Szybalski, 1952). Starting plates had maximal concentrations of 2 MIC for PTZ (1 mg ml). Overnight E. coli
culture was diluted in BHI to an optical density (OD) at
550 nm of 1.2, and 100 ml aliquots (109 CFU) were homogeneously spread onto BHI-PTZ gradient plates. Following
24 h of incubation at 37 C, the leading edge of growth was
sampled with a loop and subcultured into BHI without antibiotics. Overnight cultures were then diluted and plated as
described for the initial passage. Gradient plate antibiotic
concentrations were increased twofold once growth was
observed at approximately half of the plate distance (45 mm).
A piperacillin/tazobactam preparation (TazocinÒ) was
purchased commercially (Lederle Piperacillin) and the antibiotic solutions were freshly prepared according to the
instructions of the manufacturer. During the experiments, all
cultures were routinely incubated at 37 C in a bacteriological
chamber.
2.2. Antimicrobial susceptibility testing
MICs of pipercillin/tazobactam were determined by the
agar dilution method, according to the recommendations of the
Clinical and Laboratory Standards Institute (CLSI) (NCCLS,
2003). MICs were defined as the lowest concentration of
antimicrobial resulting in a marked change in the appearance
of growth compared to the control plate, described in the CLSI
protocols.
2.3. Measurement of growth rates
Overnight cultures of both strains (wild-type and the
derived resistant) grown in BHI broth were adjusted to 0.05
OD (550 nm) with fresh BHI broth to yield a starting inoculum
of approximately 106 cells ml1. Cultures were then incubated
at 37 C in the bacteriological chamber; then, every 20 min
until reaching 350 min of incubation, an aliquot was removed
to measure the OD.
2.4. Protein extraction
The protein extracts were obtained simultaneously for wildtype and the derived PTZ-resistant strain. Cell pellets were
obtained when cell growth reached the late exponential phase
(OD 0.8e1.0 at 550 nm), in BHI broth. The cells were washed
three times in 40 mM TriseHCl pH 7.5 by centrifugation at
5000 g for 10 min at 4 C. The pellet was then resuspended in
lysis buffer solution [7 M urea, 2 M thiourea, 4% cholamidopropyl dimethylammonio-1-propanesulfonate (CHAPS),
40 mM Dithiothreitol (DTT), 2% IPG Buffer (pH 3e10), 40 mM
Tris base and a protease inhibitor mix (GE Healthcare, Upsala,
Sweden)]. Crude cell-free extracts were obtained by cell
disruption in a French press (American Instrument Co., Silver
Spring, MD, USA) at 2000 psi followed by centrifugation at
270
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
12,000 g for 60 min at 4 C and protein content was measured (2D Quant Kit e GE Healthcare).
2.5. DIGE analysis
Samples were minimally labeled with cyanine-derived
fluors (CyDyes) according to the instructions of GE Healthcare. To determine quantitative differences between the
proteomes, the protein sample of the susceptible wild-type
strain was labeled with Cy3 and the protein sample of the
PTZ-resistant strain with Cy5. A pooled internal standard
representing equal amounts of both protein samples was
labeled with Cy2. For each labeling reaction, 160 mg protein
was incubated with 320 pmol CyDye for 30 min. To stop the
reaction, 1 ml 10 mM lysine was added and the mixture was
incubated for 10 min. All labeling incubations were carried out
on ice and protected from light. Experiments were accomplished with three biological replicates for both bacterial
strains.
2.6. Isoeletric focusing (IEF)
Following labeling, samples labeled with each CyDye were
pooled (480 mg total protein) and adjusted to a final volume of
340 ml with a rehydration solution (7 M urea, 2 M thiourea,
2% CHAPS, 40 mM DTT, 2% immobilized pH gradient (IPG)
buffer, pH 3e10, trace bromophenol blue). Samples were then
applied to IPG strips (18 cm, pH 3e10 NL; GE Healthcare)
for passive rehydration overnight at room temperature.
Rehydrated IPG strips were subjected to IEF for 65,000 Vh on
an Ettan IPGphor system (GE Helthcare). After IEF, each strip
was incubated for 15 min in 10 ml 50 mM TriseHCl buffer,
pH 8.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.002%
BPB, and 125 mM DTT, followed by a second incubation step
in the same buffer solution, excluding DTT, which was
replaced by 125 mM iodacetamide.
2.7. SDS-PAGE
IPG strips were transferred to a 12% polyacrylamide gel
within low-fluorescence glass plates (GE Helthcare) and run
on a Protean II system (Bio-Rad) connected to a Multitemp II
cooling bath (Amersham Biosciences). Electrophoresis was
performed in Tris/glycine/SDS buffer. Proteins were separated
at 200 V until the dye front reached the bottom of the gel.
2.8. Image analysis
Following electrophoresis, images were acquired and pixel
intensity was obtained using a Typhoon scanner (GE Healthcare), and differential in-gel analysis was performed using
ImageMaster 2D Platinum software (GE Healthcare). With this
software, the normalized spot volume ratios from Cy3 or Cy5
labeled spots were quantified relative to the Cy2-labeled
internal standard from the same gel. The Cy2-labeled standard
was then used to standardize and compare normalized volume
ratios of Cy3- and Cy5-labeled proteins between gels,
representing three independent experiments to generate statistical confidence for abundance changes by using Student’s t test
and analysis of variance. Those spots that had a p-value <0.05
were considered as having differential expression.
2.9. Spot handling
Spots with differential expression were manually excised,
and gel fragments were washed in 25 mM ammonium bicarbonate/50% acetonitrile until completely destained. After
drying, gel fragments were placed on ice in 50 ml of protease
solution (sequence grade-modified trypsin, Promega Biosciences, CA, at 20 ng/ml in 25 mM ammonium bicarbonate) for
30 min. Excess protease solution was then removed and
replaced by 20 ml 25 mM ammonium bicarbonate. Digestion
was performed at 37 C for 16 h. Peptide extraction was
performed twice for 15 min with 30 ml 50% acetonitrile/5%
formic acid. Trypsin (Promega) digests were then concentrated
in a SpeedVac (Savant, USA) to about 10 ml and desalted using
Zip-Tip (C18 resin; P10, Millipore Corporation, Bedford,
MA). The sample extract was mixed with matrix (5 mg/ml
recrystallized a-cyano-4-hydroxycinnamic acid) in a final
volume of 1 ml in proportions 1:1 and then spotted on the
target for MALDI-TOF-TOF (Autoflex III, Bruker Daltonics,
Billerica, USA) analysis.
2.10. Protein identification
MS and tandem MS analysis were performed using
a MALDI-TOF-TOF AutoFlex IIIÔ (Bruker Daltonics, Billerica, USA) instrument in positive/reflector mode controlled
by FlexControlÔ software. Instrument calibration was achieved by using peptide calibration standard II (Bruker Daltonics) as a reference and a-cyano-4-hydroxycinnamic acid
was used as matrix. Samples were spotted to MTP
AnchorChipÔ 600/384 (Bruker Daltonics) targets using
standard protocols for the dried droplet method. Trypsin and
keratin contamination peaks were excluded from the peak lists
used in the database searching. Each spectrum was produced
by accumulating data from 200 consecutive laser shots. The
results from the MS/MS were used to search the NCBInr
protein database using MASCOTÒ software. Search parameters are shown as follows: type of search, peptide mass
fingerprint; enzyme, trypsin; fixed modification, carbamidomethylation (Cys); variable modifications, oxidation (Met);
mass values, monoisotopic; peptide charge state, 1þ;
maximum missed cleavages, 1; and peptide mass tolerance of
0.05% Da (50 ppm). The statistical analyses of the sequences
were determined by the probability-based Mowse score
offered by MASCOTÒ software. A p-value of less than 0.05
was considered significant and used to generate the results.
3. Results and discussion
An E. coli strain resistant to piperacillin/tazobactam
(Ec-PTZ) was obtained from the wild-type strain (Ec-WT) after
10 serial passages on plates containing an antibiotic gradient,
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
according to the methods described. Incubation of E. coli with
piperacillin/tazobactam produced 256-fold MIC increments
(from 0.5 to 128 mg ml1). Resistance to piperacillin/tazobactam has been reported in E. coli, and it is often related to
high-level expression of ESBLs CTX-M, SHV-1 and TEM-1
(Marre et al., 1984; Pitout et al., 2008; Rice et al., 2000).
The development and selection of resistant bacteria are
affected by several factors, including the biological fitness
costs and the ability to compensate for such costs. Antibiotic
resistance often confers a metabolic cost because the resistance mutations typically occur in genes of target molecules
with essential functions in the cell. These cost-associated
mutations confer a reduction in bacterial fitness which can be
measured as relative growth rates within and outside the host
(Andersson, 2003), explaining the slight delay in the growth
rate of the PTZ-resistant strain compared to the wide-type
strain (Fig. 1). The ability of resistant bacteria to persist in
a population and in the community is dependent on several
factors, such as biological fitness. In this situation, antibiotic
resistance can be stabilized with fitness-restoring compensatory mutations and may allow fully resistant strains to compete
successfully with susceptible strains in an antibiotic-free
environment (Andersson, 2003).
To gain insight into the physiological changes conferring
resistance to the Ec-PTZ strain, we compared protein contents
between this mutant and the wild-type strain using the DIGE
technique. This allows for simultaneous separation by 2D
electrophoresis and quantitative detection of fluorescentllabeled proteins from different samples.
The analysis of DIGE gels revealed numerous protein
species abundance changes in the resistant strain in relation to
the susceptible wild-type strain. Approximately six hundred
spots were visualized, 30 of them were increased in abundance
in Ec-WT and 58 in Ec-PTZ. A total of 82 spots were excised
from the gel and 26 were successfully identified by MS,
representing 18 different proteins (Fig. 2, Table 1).
Several proteins were found in multiple spots on the gels.
OmpA (spots 139, 140, 144 and 182), glyceraldehyde-3-
Fig. 1. Growth patterns of wild-type E. coli strain (-) and derived PTZresistant strain ( ) in BHI. Growth was measured by determining absorbance
at 550 nm.
271
phosphate dehydrogenase A, GAPDH-A (spots 169, 170, 171
and 176) and superoxide dismutase Fe-SOD (spots 186, 187
and 188) migrated with different charges and masses during
electrophoresis. A different position in a 2-DE gel had to be
the result of a different chemical structure of the protein. Each
covalent chemical modification of a protein leads to a new
protein species. Thus, these spots may correspond to different
protein species with posttranslational modifications (Jungblut
et al., 2008).
Among the protein species that decreased in abundance in
the piperacillin-tazobactam-resistant strain are enzymes
involved in energy metabolism (6-phosphogluconate dehydrogenase [6PGD], transaldolase B [TALB], glyceraldehyde3-phosphate dehydrogenase A [GAPDH-A]) and in synthesis
and transport of proteins (elongation factor Ts [EFTS],
glutamine binding periplasmic protein [GLNH] and trigger
factor [TIG]) (Table 1). These metabolic changes may be
a consequence of the biological cost of antibiotic resistance,
although other forms of these proteins may exist which were
not resolved on these 2-DE gel. Moreover, these costs are
likely to be ameliorated by subsequent evolution, as discussed
by Andersson (2003).
Protein species involved in the oxidative stress response
also decreased in abundance in Ec-PTZ, such as three protein
species of iron-dependent superoxide dismutase (Fe-SOD) and
an alkyl-hydroperoxide reductase subunit C (AHPC) (Table
1). Oxidative stress is a major mutagen in aging colonies.
Several studies have demonstrated that deficiency in the
expression of SODs causes a variety of oxygen-dependent
phenotypic defects in E. coli, including a high rate of spontaneous mutagenesis (Farr et al., 1986; Imlay and Fridovich,
1992). Thus, the decreased abundance of SOD in Ec-PTZ
and the implications of this enzyme deficiency in bacterial
rate mutation may have some correlation with the increased
abundance of DNA protection during protein starvation
(DPS), also observed in this bacterial strain, although the
activity of these protein species has not been investigated in
this work.
The chaperone clpB was increased in abundance in the
resistant strain. This protein is part of a stress-induced multichaper one system in E. coli; it is involved in the recovery of
the cell from heat-induced damage, affecting processing of
protein aggregates. Thus, the increased abundance of clpB
suggests that the PTZ-resistant strain may also be more
resistant to heat damage than the wild-type strain.
In the resistant strain, there was decreased abundance of
protein species related to the oxidative stress response and
energy metabolism. Although enzyme assays and other physiological data are still needed to reach definitive conclusions
regarding overall changes in bacterial physiology, based on
these proteomic data, we can make some observations: the
consequences of decreased protection against oxidative stress
may have been compensated for by increased levels of DPS, as
previously discussed. Moreover, the likely loss of energy
generation caused by the decreased abundance of proteins
involved in the glycolytic pathway and pentose pathway may
be a consequence of the cost that resistance imposes on the
272
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
Fig. 2. DIGE analysis of differences in protein content between the wild-type strain (Ec-WT-Cy3) and the derived piperacillin/tazobactam-resistant strain (Ec-PTZCy5). (A) Fluorescent 2D gel images used in the presented DIGE analysis. IEF was performed with 480 mg of protein using 18 cm, 3e10NL pH range strips.
SDS-PAGE was performed on 12% polyacrylamide gels. Circles indicate protein species that are increased in abundance in the PTZ-resistant strain compared to
wild-type strain, while squares indicate protein species that are increased in abundance in the wild-type strain relative to PTZ-resistant strain. The numbers refer to
the spot identification used in the Table 1. (B) Three-dimensional images of intensities of protein spots derived from both strains from area indicated on (A) by
white rectangle. This gel is representative of three independent replicates.
fitness of bacteria. As compensation, enzymes related to energy
metabolism that were highlighted in the resistant
strain consisted of a formate acetyl transferase 1 (PFLB) and
a fumarate reductase iron-sulfur subunit (FRDB), both
functional in anaerobiosis. The first converts pyruvate (the final
product of glycolysis) to formic acid in the process of
fermentation. The second is responsible for interconversion
between fumarate and succinate in the citric acid cycle in
anaerobic respiration (Neijssel and Teixeira de Mattos, 1994).
An ATP-synthase (ATPB) also increased in abundance in the
resistant strain. However, whereas the activity of such enzymes
depends on products of the glycolytic pathway, these cells
remain disabled in energy efficiency and thus may have a lower
growth rate, as observed in this work on Ec-PTZ (Fig. 1)
Among the proteins that increased in abundance in the
resistant strain was the outer membrane protein TolC, which
belongs to the multidrug efflux pump system. TolC-increased
abundance is consistent with reports that this protein combines
with AcrAB, forming the major typical multidrug efflux pump
in E. coli (Sulavik et al., 2001). Xu et al. (2006) applied
proteomic methodology to characterize functional outer
membrane proteins of E. coli K-12 resistant to tetracycline and
ampicillin. Different outer membrane proteins due to antibiotic resistance were identified, including TolC, OmpC and
YhiU. Lin et al. (2008) showed, for the first time, the outer
membrane proteome of an E. coli strain resistant to nalidixic
acid. Again, the TolC protein together with other outer
membrane proteins was increased in abundance in the resistant
strain. Lok et al., (2008) also used proteomics to study strains
of E. coli with resistance to silver. Increased abundance of
TolC was observed in the resistant strain, corroborating the
results of Achard-Joris and Bourdineaud (2006), in which tolC
mutants were more sensitive to metals such as cadmium,
mercury and zinc. Thus, the increased abundace of TolC in the
Ec-PTZ strain suggests for the first time the importance of
a multidrug efflux pump system for resistance of this bacterium to piperacillin/tazobactam, since E. coli ATCC 25922
(wild-type strain) is a negative control for ESBL production.
OmpA was also increased in abundance (three protein species)
in the Ec-PTZ strain. OmpA is one of the most abundant and
well-studied proteins of the cell wall of E. coli. In addition to
its structural role, OmpA serves as a receptor of colicins and
several phages, and it is required in F-conjugation (Smith
et al., 2007). OmpA is also important for bacterial virulence,
since it has been associated with: i) invasion of brain microvascular endothelial cells by type 1 fimbrial modulation (Teng
et al., 2006); ii) invasion of the intestinal epithelium (Mohan
and Venkitanarayanan, 2007); iii) biofilm formation (Orme
et al., 2006); iv) serum resistance in a neonatal rat model by
interference with complement activation, inhibition of
cytokine induction and the ability to multiply within macrophages (Wooster et al., 2006).
Table 1
Quantitative abundance and MS analysis of protein species in an E. coli strain resistant to piperacillin/tazobactam.
Scorea
MWb
Protein
Peptide sequence
Gene
Biological process
Abundance
vol. ratio p < 0.05
Swiss-Protc
140
34/506
37292
Outer membrane protein A
ompA
Cell invasion, serum resistance,
conjugation, phage recognition
[ 33.16
OMPA_ECOLI
153
34/188
18684
DNA protection during DPS
LGYPITDDLDIYTR NHDTGVSPVFAGGVE
YAITPEIATR DGSVVVLGYTDRIGSDAYNQGLSER
AALIDCLAPDR
ANDVRKAIGEAKTALIDHLDTMAER
dps
[ 17.87
DPS_ECOHS
144
62/314
37292
Outer membrane protein A
DNA condensation, response
to stress
Cell invasion, serum resistance,
conjugation, phage recognition
[ 10.36
OMPA_ECOLI
134
35/43
56484
151
63/140
18648
Alkyl hydroperoxide
reductase subunit F
Outer membrane protein X
100
35/44
95697
Chaperone protein clpB
139
34/329
37292
Outer membrane protein A
147
62/135
27732
126
50/318
85588
Fumarate reductase iron-sulfur
subunit
Formate acetyltransferase 1
132
131
34/168
62/411
53708
50324
Outer membrane protein tolC
ATP-synthase subunit beta
136
35/61
52275
163
26/30
51449
171
51/173
35681
170
51/229
35681
185
50/54
20748
169
51/266
35681
177
187
50/183
51/90
30518
21253
Inosine-50 -monophosphate
dehydrogenase
6-phosphogluconate
dehydrogenase
Glyceraldehyde-3-phosphate
dehydrogenase A
Glyceraldehyde-3-phosphate
dehydrogenase A
Alkyl hydroperoxide reductase
subunit C
Glyceraldehyde-3-phosphate
dehydrogenase A
Elongation factor Ts
Superoxide dismutase [Fe]
161
62/259
47949
Trigger factor
175
51/63
35183
Transaldolase B
LGYPITDDLDIYTR NHDTGVSPVFAGGVEYA
ITPEIATR DGSVVVLGYTDRIGSDAYNQGLSER
AALIDCLAPDR
ARSIIVATGAKWR ATGAKWRNMNVPGEDQYR
LLPNTNWLEGAVER
YRYEEDNSPLGVIGSFTYTEK SVDVGTWIAGVGYR
AATQLEGKTMRLLR AMVRIDMSEFMEK
FGELDYAHMK
LGYPITDDLDIYTR NHDTGVSPVFAGGVEYAIT
PEIATR DGSVVVLGYTDRIGSDAYNQGLSER
AALIDCLAPDR
VEALANFPIER DFLIATLKPR
ompA
ahpF
Oxidative stress response
[ 3.8
AHPF_ECOLI
ompX
Cell invasion, serum resistance,
antimicrobial resistance
Stress response
[ 3.61
OMPX_ECOLI
[ 3.55
CLPB_ECOLI
clpB
ompA
Cell invasion, serum resistance,
conjugation, phage recognition
[ 3,16
OMPA_ECOLI
frdB
Electron transport
[ 2.68
FRDB_ECOLI
pflB
Fermentation
[ 2.15
PFLB_ECOLI
tolC
atpD
Antimicrobial resistance, transport
ATP synthesis
[ 2.10
[ 2.02
TOLC_ECOLI
ATPB_ECOHS
guaB
[ 1.77
IMDH_ECOLI
gnd
Purine biosynthesis,
GMP biosynthesis
Pentose-phosphate shunt
Y1.35
6PGD_ECOLI
WDEVGVDVVAEATGLFLTDETAR VPTPNVSVVDLTVR
gapA
Glycolysis
Y1.39
G3P1_ECO57
WDEVGVDVVAEATGLFLTDETAR VPTPNVSVVDLTVR
gapA
Glycolysis
Y1,45
G3P1_ECO57
NGEFIEITEKDTEGR LGVDVYAVSTDTHFTHK
ATFVVDPQGIIQAIEVTAEGIGR
WDEVGVDVVAEATGLFLTDETAR VPTPNVSVVDLTVR
ahpC
Oxidative stress response
Y1.78
AHPC_ECOLI
gapA
Glycolysis
Y1.91
G3P1_ECO57
tsf
sodB
Protein biosynthesis
Oxidative stress response
Y1.93
Y1.99
EFTS_ECOHS
SODF_ECOLI
tig
Cell division
Y2.02
TIG_SALCH
talB
Pentose-phosphate shunt
Y2.36
TALB_ECOL6
GDWQNEVNVR THNQGVFDVYTPDILR
SGVLTGLPDAYGR LAQFTSLQADLENGVNLEQTIR
TSTFLDVYIER
FNVGLVAITDVQNAR AQYDTVLANEVTAR
IVQVIGAVVDVEFPQDAVPR LVLEVQQQLGGGIVR
NIAIEHSGYSVFAGVGER DVLLFVDNIYR
YTLAGTEVSALLGR
SEGVLQRIRETRAK SYRGMGSLGAMSKGSSDR
HGHSEGVLQR PNACKDEQGR
QIADDYQQALR
VAALEGDVLGSYQHGAR EHNAEVTGFIR
SFELPALPYAK DALAPHISAETIEYHYGK
VAEAIAASFGSFADFK
AGEEFTIDVTFPEEYHAENLK ANDIDVPAALIDSEIDVLR
VVVGLLLGEVIR
LYQPQDATTNPSLILNAAQIPEYR LYNDAGISNDR
EHGYETVVMGASFRELAESEGAIER
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
Spot
(continued on next page)
273
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
SODF_ECOLI
G3P1_ECO57
Y4.25
Y8.74
gapA
sodB
Glycolysis
GLNH_ECOLI
Y3.22
glnH
Oxidative stress response
OMPA_ECOLI
Y2.74
ompA
Cell invasion, serum resistance,
conjugation, phage recognition
Amino-acid transport
SODF_ECOLI
Y2.48
OmpX, similarly to OmpA, is an integral membrane protein
that was also increased in abundance in Ec-PTZ strain. The E.
coli outer membrane protein OmpX belongs to a family of
highly conserved bacterial proteins that have been assigned
key functions in promoting bacterial adhesion and entry into
mammalian cells (Mecsas et al., 1995). Another function that
has been attributed to OmpX is resistance to antimicrobials.
Stoorvogel et al. (1987) demonstrated that overexpression of
this protein causes downregulation in expression of the major
pore proteins OmpF and OmpC, resulting in decreased
susceptibility to beta-lactams. Resistance to piperacillin/tazobactam has been associated with reduced cell wall permeability of TEM-1-producing strains of E. coli (Marre et al.,
1984) and to a decrease in the quantity of a minor 45 kDa
outer membrane protein in K. pneumonia (Rice et al., 2000).
Thus, we should consider the potential role of OmpX, together
with a multidrug efflux pump system, in E. coli resistance to
piperacillin/tazobactam.
The numerous changes in protein abundance observed in
the derived drug-resistant strain suggest that resistant microorganisms may develop molecular changes in an effort
towards adaptation to adverse environmental conditions
(Linares-Rodriguez and Martinez-Menendez, 2005; Justice
et al., 2008) with implications for many aspects of bacterial
metabolism, which may be reflected in their virulence
parameters. This is suggested by the increased abundance of
OmpA and OmpX in the Ec-PTZ strain. In fact, it is known
that some antimicrobial drugs can stimulate bacterial adhesion
and toxin production and can interfere with the phagocytic
process (Lorian and Gemmel, 2005; Ohlsen et al., 1998). It has
been shown that the phenotype of metronidazole resistance
derived from Bacteroides fragilis ATCC 25285 encompassed
a broad range of traits, including differences in gene/protein
expression (Diniz et al., 2004) and pathogenic properties
(Diniz et al., 2000).
However, since classical proteomic approaches alone
mainly provide information on the relative amounts of protein
species and only rarely information on the activity of these
protein species, it is necessary to complement these findings
by metabolomics and interaction studies to determine the true
functional level of the biological system under investigation in
this work.
In conclusion, our data reveal overall changes in the subproteome of the piperacillin/tazobactam-resistant strain, suggesting that the misuse of antimicrobial agents might interfere
with management of infectious diseases not only due to the
selection of resistance, but also by interfering with cell
physiology in its entirety. Furthermore, we report for the first
time the potential role of a multidrug efflux pump system in E.
coli resistance to piperacillin/tazobactam.
[ increase, Ydecrease.
a
Required/found.
b
Molecular weight.
c
Accession number.
Glyceraldehyde-3-phosphate
dehydrogenase A
35681
51/103
176
21253
51/90
188
Glutamine-binding
periplasmic protein
Superoxide dismutase [Fe]
27173
50/53
184
Outer membrane protein A
37178
50/66
182
Superoxide dismutase [Fe]
51/90
186
21253
SFELPALPYAK DALAPHISAETIEYHYGK VAEAIAA
SFGSFADFK
LGYPITDDLDIYTR NHDTGVSPVFAGGVEYAITPEIATR
DGSVVVLGYTDR
NVDLALAGITITDERAIDFSDGYYK
AVGDSLEAQQYGIAFPK
SFELPALPYAK DALAPHISAETIEYHYGK
VAEAIAASFGSFADFK
WDEVGVDVVAEATGLFLTDETAR
sodB
Oxidative stress response
Swiss-Protc
Protein
MWb
Scorea
Spot
Table 1 (continued )
Peptide sequence
Gene
Biological process
Abundance
vol. ratio p < 0.05
274
Acknowledgements
This study was supported by grants from the Conselho
Nacional de Desenvolvimento Cientı́fico e Tecnológico
(CNPq) and the Fundação de Amparo à Pesquisa do Estado de
Minas Gerais (FAPEMIG). We thank Dr. Simone Gonçalves
K. Vale´ria dos Santos et al. / Research in Microbiology 161 (2010) 268e275
dos Santos, Luzia Rosa Resende and José Sérgio Barros de
Souza for their support.
References
Achard-Joris, M., Bourdineaud, J.P., 2006. Heterologous expression of
bacterial and human multidrug resistance proteins protect Escherichia coli
against mercury and zinc contamination. Biometals 19, 695e704.
Andersson, D.I., 2003. Persistence of antibiotic resistant bacteria. Curr. Opin.
Microbiol. 6, 452e456.
Andrade, H.M., Murta, S.M., Chapeaurouge, A., Perales, J., Nirdé, P.,
Romanha, A.J., 2008. Proteomic analysis of Trypanosoma cruzi resistance
to benznidazole. J. Proteome Res. 7, 2357e2367.
Bore, E., Hébraud, M., Chafsey, I., Chambon, C., Skjaeret, C., Moen, B.,
Møretrø, T., Langsrud, Ø., Rudi, K., Langsrud, S., 2007. Adapted tolerance
to benzalkonium chloride in Escherichia coli K-12 studied by transcriptome and proteome analyses. Microbiology 153, 935e946.
Bryson, L., Szybalski, W., 1952. Microbial selection. Science 116, 45e51.
Cash, P., 2000. Proteomics in medical microbiology. Electrophoresis 21,
1187e1201.
Cash, P., Argo, E., Ford, L., Lawrie, L., McKenzie, H., 1999. A proteomic
analysis of erythromycin resistance in Streptococcus pneumoniae. Electrophoresis 20, 2259e2268.
Coldham, N.G., Woodward, M.J., 2004. Characterization of the Salmonella
typhimurium proteome by semi-automated two dimensional HPLC-mass
spectrometry: detection of proteins implicated in multiple antibiotic
resistance. J. Proteome Res. 3, 595e603.
Diniz, C.G., Cara, D.C., Nicoli, J.R., Farias, L.D., Carvalho, M.A.R., 2000.
Effect of metronidazole on the pathogenicity of resistant Bacteroides strains
in gnotobiotic mice. Antimicrob. Agents Chemother. 44, 2419e2423.
Diniz, C.G., Farias, L.M., Carvalho, M.A.R., Rocha, E.R., Smith, C.J., 2004.
Differential gene expression in a Bacteroides fragilis metronidazoleresistant mutant. J. Antimicrob. Chemother. 54, 100e108.
Dos Santos, K.V., Diniz, C.G., Coutinho, S.C., Apolônio, A.C.M., SousaGaia, L.G., Nicoli, J.R., Farias, L.M., Carvalho, M.A.R., 2007. In vitro
activity of piperacillin/tazobactam and ertapenem against Bacteroides
fragilis and Escherichia coli in pure and mixed cultures. J. Med. Microbiol. 56, 798e802.
Farr, S.B., D’Ari, R., Touati, D., 1986. Oxygen-dependent mutagenesis in
Escherichia coli lacking superoxide dismutase. Proc. Natl. Sci. USA 83,
8268e8272.
Imlay, J., Fridovich, I., 1992. Suppression of oxidative envelope damage by
pseudoreversion of a superoxide dismutase-deficient mutant of Escherichia
coli. J. Bacteriol. 174, 953e961.
Jungblut, P.R., Holzhütter, H.G., Apweiler, R., Schlüter, H., 2008. The
speciation of the proteome. Chem. Cent. J. 18, 2e16.
Justice, S.S., Hunstad, D.A., Cegelski, L., Hultgren, S.J., 2008. Morphological plasticity as a bacterial survival strategy. Nat. Rev. Microbiol. 6,
162e168.
Lin, X.M., Li, H., Wang, C., Peng, X.X., 2008. Proteomic analysis of nalidixic
acid resistance in Escherichia coli: identification and functional characterization of OM proteins. J. Proteome Res. 7, 2399e2405.
Linares-Rodriguez, J.F., Martinez-Menendez, J.L., 2005. Resistência a los
antimicrobianos y virulencia bacteriana. Enferm. Infecc. Microbiol. Clin.
23, 86e93.
Lis, M., Bobek, L.A., 2008. Proteomic and metabolic characterization of
a Candida albicans mutant resistant to the antimicrobial peptide MUC7
12-mer. FEMS Immunol. Med. Microbiol. 54, 80e91.
Lok, C.N., Ho, C.M., Chen, R., Tam, P.K., Chiu, J.F., Che, C.M., 2008. Proteomic identification of the Cus system as a major determinant of
constitutive Escherichia coli silver resistance of chromosomal origin. J.
Proteome Res. 7, 2351e2356.
Lorian, V., Gemmel, G.C., 2005. Effect of low antibiotic concentrations on
bacteria: effects on ultrastructure, virulence, and susceptibility to immunodefenses. In: Lorian, V. (Ed.), Antibiotics in Laboratory Medicine.
Lippincott Williams & Wilkins, Philadelphia, pp. 493e555.
275
Marre, R., Borner, K., Schulz, E., 1984. Different mechanisms of TEM-1 and
Oxa-1 mediated resistance to piperacillin in E. coli. Zentralbl. Bakteriol.
Mikrobiol. Hyg. 258, 287e295.
Mcatee, C.P., Hoffman, P.S., Berg, D.E., 2001. Identification of differentially
regulated proteins in metronidozole resistant Helicobacter pylori by proteome techniques. Proteomics 1, 516e521.
Mecsas, J., Welch, R., Erickson, J.W., Gross, C.A., 1995. Identification and
characterization of an outer membrane protein, OmpX, in Escherichia coli
that is homologous to a family of outer membrane proteins including Ail of
Yersinia enterocolitica. J. Bacteriol. 177, 799e804.
Mohan, N.M.K., Venkitanarayanan, K., 2007. Role of bacterial OmpA and
host cytoskeleton in the invasion of human intestinal epithelial cells by
Enterobacter sakazakii. Pediatr. Res. 62, 664e669.
NCCLS, 2003. Methods for Dilution Antimicrobial Susceptibility Tests for
Bacteria that Grow Aerobically. Approved Standard M7eA6. National
Committee for Clinical Laboratory Standards, Wayne, PA.
Neijssel, O.M., Teixeira de Mattos, M.J., 1994. The energetics of bacterial
growth: a reassessment. Mol. Microbiol. 13, 172e182.
Ohlsen, K., Ziebuhr, W., Koller, K.-P., Hell, W., Wichelhaus, T.A., Hacker, J.,
1998. Effects of subinhibitory concentrations of antibiotics on alpha-toxin
(hla) gene expression of methicillin-sensitive and methicillin-resistant
Staphylococcus aureus isolates. Antimicrob. Agents Chemother. 42,
2817e2823.
Orme, R., Douglas, C.W., Rimmer, S., Webb, M., 2006. Proteomic analysis of
Escherichia coli biofilms reveals the over expression of the outer
membrane protein OmpA. Proteomics 6, 4269e4277.
Pieper, R., Gatlin-Bunai, C.L., Mongodin, E.F., Parmar, P.P., Huang, S.T.,
Clark, D.J., Fleischmann, R.D., Gill, S.R., Peterson, S.N., 2006.
Comparative proteomic analysis of Staphylococcus aureus strains with
differences in resistance to the cell wall-targeting antibiotic vancomycin.
Proteomics 6, 4246e4258.
Pitout, J.D., Le, P., Church, D.L., Gregson, D.B., Laupland, K.B., 2008.
Antimicrobial susceptibility of well-characterised multiresistant CTX-Mproducing Escherichia coli: failure of automated systems to detect resistance to piperacillin/tazobactam. Int. J. Antimicrob. Agents 32, 333e338.
Rice, L.B., Carias, L.L., Hujer, A.M., Bonafede, M., Hutton, R., Hoyen, C.,
Bonomo, R.A., 2000. High-level expression of chromosomally encoded
SHV-1 beta-lactamase and an outer membrane protein change confer
resistance to ceftazidime and piperacillin-tazobactam in a clinical isolate
of Klebsiella pneumoniae. Antimicrob. Agents Chemother. 44, 362e367.
Smith, S.G., Mahon, V., Lambert, M.A., Fagan, R.P., 2007. A molecular Swiss
army knife: OmpA structure, function and expression. FEMS Microbiol.
Lett. 273, 1e11.
Stoorvogel, J., Van Bussel, M.J.A.W.M., Van De Klundert, J.A.M., 1987.
Cloning of a beta-lactam resistance determinant of Enterobacter cloacae
affecting outer membrane proteins of Enterobacteriaceae. FEMS Microbiol. Lett. 48, 277e281.
Sulavik, M.C., Houseweart, C., Cramer, C., Jiwani, N., Murgolo, N., Greene, J.
, Didomenico, B., Shaw, K.J., Miller, G.H., Hare, R., Shimer, G., 2001.
Antibiotic susceptibility profiles of Escherichia coli strains lacking
multidrug efflux pump genes. Antimicrob. Agents Chemother. 45,
1126e1136.
Teng, C.H., Xie, Y., Shin, S., Di Cello, F., Paul-Satyaseela, M., Cai, M.,
Kim, K.S., 2006. Effects of ompA deletion on expression of type 1
fimbriae in Escherichia coli K1 strain RS218 and on the association of E.
coli with human brain microvascular endothelial cells. Infect. Immun. 74,
5609e5616.
Wooster, D.G., Maruvada, R., Blom, A.M., Prasadarao, N.V., 2006. Logarithmic
phase Escherichia coli K1 efficiently avoids serum killing by promoting
C4bp-mediated C3b and C4b degradation. Immunology 117, 482e493.
Xu, C., Lin, X., Ren, H., Zhang, Y., Wang, S., Peng, X., 2006.
Analysis of outer membrane proteome of Escherichia coli related
to resistance to ampicillin and tetracycline. Proteomics 6,
462e473.
Yoo, J.S., Seong, W.K., Kim, T.S., Park, Y.K., Oh, H.B., Yoo, C.K., 2007.
Comparative proteome analysis of the outer membrane proteins of in vitroinduced multi-drug resistant Neisseria gonorrhoeae. Microbiol. Immunol.
51, 1171e1177.