Global Comparison of the Membrane Subproteomes between a

Global Comparison of the Membrane Subproteomes between a Multidrug-Resistant Acinetobacter baumannii Strain and a Reference Strain Axel Siroy,† Pascal Cosette,† Damien Seyer,† Christelle Lemaıˆtre-Guillier,‡ David Vallenet,§ Alain Van Dorsselaer,| Sophie Boyer-Mariotte,⊥ Thierry Jouenne,† and Emmanuelle De´ *,† IBBR Group, Laboratory “Polyme`res, Biopolyme`res, Membranes”, UMR 6522 CNRS, University of Rouen, “Laboratoire de Spectrome´trie de Masse BioOrganique”, UMR 7509 ECPM, University of Strasbourg, “Groupe de Recherche sur les Antimicrobiens et les Microorganismes”, GRAM, EA 2656, University of Rouen, and Centre Hospitalier Universitaire Charles Nicolle, France Received July 26, 2006

Acinetobacter baumannii causes severe infections in compromised patients. We combined SDS-PAGE, two-dimensional gel electrophoresis and mass spectrometry (LC-MS/MS and MALDI-TOF) to separate and characterize the proteins of the cell envelope of this bacterium. In total, 135 proteins (inner and outer membrane proteins) were identified. In this analysis, we described the expression by this bacterium of RND-type efflux systems and some potential virulence factors. We then compared the membrane subproteome of a clinical multidrug-resistant (MDR) isolate with that of a reference strain. We found that the MDR strain expressed lower levels of the penicillin-binding-protein 1b, produced a CarO protein having different primary and quaternary structures to that of the reference strain, and expressed OmpW isoforms. We also showed that the clinical strain has a high ability to form biofilms consistent with the accumulation of some outer membrane proteins (OMPs) such as NlpE or CsuD that have already been described as involved in bacterial adhesion. These features may partly explain the MDR emergence of the clinical isolate. Keywords: A. baumannii • proteome • membrane • multidrug resistance

Introduction Acinetobacter baumannii (previously known as Acinetobacter calcoaceticus var. anitratus) is a nonfermentative Gram-negative coccobacillus. It is part of the normal flora of human skin, and the gastrointestinal and upper respiratory tracts, and is also found in soil and water.1 However, this bacterium can develop a high resistance to many different antibiotics,2,3 including carbapenems,3,4 and can survive for considerably long periods in hospital environments.2,3 As a result, nosocomial outbreaks of multidrugresistant (MDR) A. baumannii have become increasingly common in hospitals worldwide, especially in intensive care5-7 and burns units.8,9 The most common infections due to this organism are bacteremia, meningitis, and respiratory and urinary tract infections (see the reviews by Bergogne-Berezin and Towner,2 Van Looveren et al.,3 and the references therein). * To whom correspondence should be addressed. E-mail: [email protected]. † UMR 6522 CNRS, Proteomic Platform, IFRMP23, University of Rouen. ‡ Proteomic Platform, IBMC. § Ge´noscope & UMR 8030, Centre National de Se´quenc¸ age. | UMR 7509 ECPM, Laboratoire de Spectrome´trie de Masse BioOrganique. ⊥ Groupe de Recherche sur les Antimicrobiens et les Microorganismes (EA 2656), IFRMP 23), University of Rouen, and Centre Hospitalier Universitaire Charles Nicolle. 10.1021/pr060372s CCC: $33.50

 2006 American Chemical Society

Some resistance mechanisms have already been described in A. baumannii. These include denaturing and/or modifying enzymes (e.g., β-lactamases,10 aminoglycosides-modifying enzymes3) and the possible modification of antibiotic targets (e.g., PBPs for β-lactams11 and both DNA gyrase and topoisomerase IV for quinolones12-15). Resistance to hydrophilic antibiotics, such as carbapenems, is often caused by alterations in membrane permeability due to a decreased influx and/or an increased efflux. The permeability barrier seems active in A. baumannii.11,16,17 Some active efflux pumps, such as the three-component AdeABC pump18,19 or a MATE-type pump called AbeM,20 have been described as responsible for the efflux of different classes of antibiotics or toxic compounds. The genes encoding TetA and TetB, the tetracycline and minocycline-specific MFS-type pumps, have also been described.21 Although porins play a key role in the outer membrane (OM) permeability barrier of Gram-negative bacteria, especially for antibiotic influx,22-24 there are little available data on these proteins in Acinetobacter species. Of those proteins, unidentified 45.5 and 47 kDa proteins were shown to have channel-forming function;25 the 37/43 kDa porin belonging to the OmpA-like protein family has been functionally characterized;26 and a protein called CarO, which is involved in carbapenem resistance, was shown to have ionophore properties.27,28 The underexpression of other outer Journal of Proteome Research 2006, 5, 3385-3398

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research articles membrane proteins (OMPs) may also be associated with the emergence of the resistance phenotype.29-31 In this study, we compared the membrane subproteomes of a clinical MDR A. baumannii strain isolated during an outbreak of nosocomial infections in a Rouen hospital with those of a reference (susceptible) strain. However, few data are available about the proteome of the cell envelope of Acinetobacter species.17,32 Therefore, we first characterized the protein constituents of the inner (IM) and outer (OM) membranes of the susceptible standard strain ATCC 196062,33-38 using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry (LC-MS/ MS). These approaches led us to identify 135 membrane proteins and 23 periplasmic proteins in A. baumannii. We then compared the proteomic maps of the reference and MDR strains using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MALDI-TOF). We showed that the MDR phenotype exhibited significant differences in the membrane protein patterns. In particular, some inner membrane proteins (IMPs) and OMPs involved in antibiotic resistance, virulence, or biofilm formation were differentially expressed in the clinical strain.

Experimental Procedures Bacterial Strain, Inoculum Preparation, and MICs Determination. A. baumannii ATCC19606 type strain was purchased from the CIP (CIP 70.34, Pasteur Institute Collection, Paris, France) and used as a reference strain.2,33-38 Clinical MDR A6 strain was isolated in 2000 from two patients hospitalized in the ICU of Charles Nicolle Hospital (Rouen, France). Bacteria from stock cell suspensions in Mueller-Hinton broth (Difco, Beckton Dickinson, Franklin Lakes, NJ) provided with 50% (v/ v) glycerol were first cultured at 37 °C in shaken flasks containing Mueller-Hinton broth. The growth kinetics were monitored in the same medium from the optical density (OD) at 546 nm (initial inoculum: 107 cfu/mL). After 18 h of incubation, the culture medium was centrifuged (3500g for 15 min) and the resulting pellet resuspended in sterile distilled water. This cell suspension was used as an inoculum for the subsequent culture. MICs were determined on Mueller-Hinton agar plates using the Etest method (AB Biodisks, Solna, Sweden) with a final inoculum of 104 cfu/mL. Results were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (formerly NCCLS). The presence of metalloβ-lactamase was assessed using the Etest MBL strip. Quantification of Crystal Violet-Stained Attached Cells. Attached cells were quantified using the protocol described by O’Toole and Kolter.39 Cells were grown for 48 h in MuellerHinton broth at 37 °C without agitation in microtiter dishes. Unattached cells were removed by rinsing the microdishes thoroughly with water, and attached cells were subsequently stained by incubation with 1% crystal violet (CV) for 20 min. The CV was then solubilized by adding 200 µL of ethanol and the OD measured at 570 nm (Genesys 2PC spectrophotometer, Spectronic Instruments, Inc., Rochester, NY). Membrane Protein Extraction. Membranes proteins were extracted as previously described.40 Briefly, 2 L of MuellerHinton broth was inoculated to an initial population of 107 cfu, and bacterial strains were then grown at 37 °C with medium shaking. All the following procedures were carried out at 4 °C or on ice. Bacteria at the late-exponential phase (5 × 108 cfu) were harvested by centrifugation (8000g for 20 min, JLA 10.500, Beckman-Coulter, Fullerton, CA). Inner and outer membranes 3386

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were separated using a sucrose gradient method and then stored at -80 °C. Briefly, harvested cells were washed with HEPES buffer (30 mM, pH 8), and the pellet was resuspended in 10 mL of a 20% sucrose solution (in HEPES buffer). A mixed solution of 0.5 mL of DNAse and RNAse at 1 mg/mL was added to the suspension, which was then sonicated. Peptidoglycan digestion was achieved by incubation with 1 mL of lysozyme at 1 mg/mL for 1 h at 37 °C with gently shaking. Unbroken cells were removed by a low-speed centrifugation (5000g, Sigma 2K15). The supernatant was mixed with 14 mL of HEPES buffer and settled on discontinued sucrose gradient made with 1 mL of a 70% sucrose and 6 mL of a 15% sucrose in HEPES buffer. After centrifugation (1 h, 39 000 rpm, SW-41, Beckman-Coulter), the bottom 2 mL in the tubes was harvested, mixed, and then settled on another gradient made with 70% (1 mL), 64% (3 mL), 58% (3 mL), and 52% (3 mL) solutions (in HEPES buffer). After the second centrifugation (18 h, 39 000 rpm, SW-41, BeckmanCoulter), the different rings appeared between each sucrose phase and were collected. They were then concentrated by ultracentrifugation (43 000 rpm, 70 lTi, Beckman-Coulter), resuspended in a minimum of deionized water, and conserved at -80 °C. The concentration of the samples was determined using the Bio-Rad protein assay (Bio-Rad, Hercules, CA). SDS-PAGE. SDS-PAGE was carried out under the following conditions: membrane extracts were diluted with deionized water to a final concentration of 1 mg/mL and mixed in equal parts with a reducing agent containing sample buffer (2% SDS w/v, 62.5 mM Tris-HCl, pH 6.8, 10% glycerol v/v, 5% β-mercaptoethanol, and trace of bromophenol blue). Samples were either heated (5 min, 95 °C in a water bath) or left untouched before being centrifuged (8000 rpm, 3 min, room temperature). The resulting supernatants were then loaded onto a polyacrylamide gel (7% stacking, 14% separating gel, 16 cm length, Hoefer SE 600, San Francisco, CA). Staining was by 0.1% (w/v) Coomassie brilliant blue G 250 solution. Each protein band was then cut into 2 mm slices and loaded onto a 96-well plate. Two-Dimensional Gel Electrophoresis. Membrane extracts containing 100 µg of proteins were solubilized in an isoelectric focusing (IEF) buffer of 5 M urea, 2 M thiourea, 0.5% (w/v) amidosulfobetaine-14 (ASB14), 2 mM tributyl phosphine, 10 mM dithiothreitol, 2% (v/v) carrier ampholytes (pH 3.5-10; Sigma), and 0.025 ‰ (w/v) Coomassie blue G 250 (Sigma)41 (final volume, 400 µL). The first-dimension gel separation was carried out with Immobiline Dry Strips NL (18 cm, pH 3-10NL, Amersham Pharmacia Biotech, Uppsala, Sweden). IEF was carried out using an IEF cell apparatus (Bio-Rad) as follows: 50V for 12 h, 250 V for 15 min, 3 h of a linear increase to 10 kV, and 10 kV (1 mA constant) for 10 h for a total of 105.6 kVh. The second dimension was obtained by SDS-PAGE using an 11% (w/v) polyacrylamide gel (width, 16 cm; length, 20 cm; thickness, 0.75 mm). After migration, proteins were visualized by colloidal Coomassie blue staining.41 Gel Analysis. All experiments were carried out in triplicate, and the corresponding gels were carried out in duplicate. Gels were scanned using the GS-800 Imaging densitometer (BioRad). 2-DE gels were analyzed using PDQuest software (version 7.0, Bio-Rad, Hercules, CA). For each protein sample (IMPs and OMPs), the 12 experimental gels were matched together to form a master gel so that the same spot in different gels had the same number. Both conditions (i.e., ATCC and A6 strains) were compared by constructing two different Replicate Groups, including all the gels in each condition. In each group, an

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Membrane Proteomes of A. baumannii

average quantity was determined for each spot and compared to the same one in the other group. Data Processing. Protein spots from A6 and ATCC strains were considered to be significantly quantitatively different if they fulfilled the following criteria: (i) overexpression in A6 X h - SEX g 10

and

(X h - SEX)/(Y h + SEY) > 2

(ii) underexpression in A6 Y h - SEY g 10

and

(X h + SEX)/(Y h - SEY) < 0.5

where X h and Y h are average spot volumes (n ) 6) in the A6 and ATCC strain, respectively. SEX and SEY are the standard errors on the means X h and Y h , respectively. The spot detection threshold, 10, and the 2-fold ratio for significant spot alteration have been arbitrarily chosen.41 Trypsin Digestion and Mass Spectrometry. Excised slices or plugs were rinsed and reduction/alkylation was carried out using the Massprep (Micromass, Altrincham, U.K.) Robot as follows: each piece of gel was washed with 100 µL of 25 mM ammonium carbonate and dehydrated with 100 µL of acetonitrile (ACN). This was repeated twice. Reduction was achieved by incubation for 1 h with 10 mM dithiothreitol (DTT) at room temperature. Alkylation was achieved by incubation with 25 mM iodoacetamide for 45 min at room temperature in the dark. Finally, gel spots were washed three times for 5 min, again alternating between 25 mM ammonium carbonate and ACN. Gel pieces were completely dried before tryptic digestion and rehydrated by trypsin addition. The volume of added trypsin was estimated visually according to the size of the piece of gel (about 3 vol of trypsin freshly diluted 12.5 ng/µL in 25 mM ammonium carbonate). Digestion was carried out overnight at 37 °C. Gel pieces were then centrifuged, and 60 µL of 35% H2O/60% ACN/5% formic acid (HCOOH) (v/v/v) was added to the peptide extracts. The mixture was sonicated for 30 min, the supernatant recovered, and the process repeated once with 100% ACN. Supernatants were pooled, transferred into a clean 96-well plate, and the peptide extraction volume was reduced to 10 µL by evaporation before analysis. For gel slices recovered from SDS-PAGE gels, tryptic digests were analyzed by nanoscale capillary liquid chromatographytandem mass spectrometry (nanoLC-MS/MS) using a CapLC capillary LC system (Micromass, Altrincham, U.K.) coupled with a hybrid quadrupole orthogonal acceleration time-of-flight tandem mass spectrometer (Q-TOF II, Micromass, Altrincham, U.K.). The sample (6.4 µL) was loaded and concentrated onto a C18 PepMap precolumn (LC Packings, Amsterdam, The Netherlands) at a flow rate of 30 µL/min and flushed for 3 min with 0.1% ACN before gradient elution of the peptides onto the separating column. The peptides were then separated on a reverse-phase capillary column (Pepmap C18, 75 µm i.d., 15 cm length) with a 200 nL/min flow generated by the CapLC delivering a flow rate of 4.5 µL/min split directly after the precolumn. The gradient profile was a linear gradient from 95% A (H2O/0.1% HCOOH v/v) to 60% B (ACN/0.1% HCOOH v/v) over 35 min, followed by a linear gradient to 95% B in 1 min. Mass data acquisitions were piloted by MassLynx software (Micromass). Automatic switching between MS and MS/MS modes was used when MS/MS was required, and the internal parameters of Q-TOF II were as follows: electrospray capillary voltage was 3.5 kV, cone voltage was 40 V, and source temperature was 120

°C. The MS survey scan was m/z 300-1500 with a scan time of 1s and an interscan time of 0.1s. When the intensity of a peak rose above a threshold of 10 counts, tandem mass spectra were acquired. Normalized collision energies for peptide fragmentation were set using the charge-state recognition files for +1, +2, and +3 peptides ions. The scan range for MS/MS acquisition was from m/z of 50 to 1500 with a scan time of 1 s and an interscan time of 0.1 s. Fragmentation was achieved with argon as the collision gas and with a collision energy profile optimized for various ranges of ion precursor mass. Mass data collected during a nanoLC-MS/MS analysis were processed and converted into a .PKL file for submission to the Global server 1.1 and Mascot search engines. Protein spots from 2-DE gels were identified by mass finger printing. MALDI-TOF analyses were carried out using the PrOTOF 2000 spectrometer (Perkin Elmer Sciex, Boston, MA). The peptide fingerprints were matched against computersimulated digests using the Mascot software (Matrix Sci., London, U.K.) using a custom database of all A. baumannii proteins available in the NCBI nonredundant protein databank. This database (called AbNCBI and used in Tables 2-4) also contains all the sequences of proteins determined in A. baumannii ATCC15839 (US Patent 656295; 4126 entries), available in the NCBI patented sequences databank (http:// www.ncbi.nlm.nih.gov). Computational Analysis. Peptide sequences were analyzed with the BioEdit v5.0.9 software from Tom Hall using the Gapped BLAST algorithm42 against A. baumannii ATCC 15839 or the Genoscope Acinetobacter baylyi ADP1 (http:// www.genoscope.cns.fr/agc/mage) protein databases.43 Protein function was predicted using InterProScan44 accessible at http://www.ebi.ac.uk. For hypothetical proteins, the protein location compartment in the bacterial cell was predicted using the PSORTb45 program accessible at http://www.psort.org.

Results and Discussion The comparison of OM and IM subproteomes between a susceptible and an MDR A. baumannii strains using 2-DE may allow the resistant phenotype of the MDR clinical strain to be explained. However, it is well-known that analyzing membrane proteins using 2-DE is complex due to difficulties in extracting and solubilizing these inherently hydrophobic proteins.46 Thus, a prerequisite of the study was to determine the membrane protein patterns (i.e., from the inner and the outer membranes) of the ATCC 19606 standard strain using SDS-PAGE (to enhance membrane protein solubilization) coupled with LC-MS/MS analysis. Membrane Proteomes of the ATCC 19606 Strain. Proteomics requires the different steps to be reproducible. Figure 1A shows a representative image of the SDS-PAGE patterns of the IM and OM fractions and shows how reproducible the protein purification and separation are. We selected 56 and 46 bands for trypsic in-gel digestion and LC-MS/MS analysis from IMP and OMP samples, respectively. From these 102 bands, we recovered 107 IMPs, 23 periplasmic proteins, and 28 OMPs (see Table 1). The overall enrichment of proteins having a predicted membrane location was 12% and 48% for the OM and IM samples, respectively. Of the detected proteins, 10% were located in the periplasm compartment, and cytoplasmic contamination (69 proteins) was 30% (Figure 1B). These enrichment levels are similar to those reported in a study of Pseudomonas aeruginosa,47 thus, validating our extraction Journal of Proteome Research • Vol. 5, No. 12, 2006 3387

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envelope of A. baumannii ATCC19606. In A. baumannii, the RND-type efflux pumps AdeABC and AdeDE are involved in the resistance to diverse classes of antibiotics,18,52 and for β-lactams, the overexpression of AdeABC increases the resistance to imipenem.49 However, the role of AdeXY has not yet been described. We also describe, for the first time, the expression of the AdeT protein in the outer membrane. Several major facilitator superfamily (MFS) pumps have also been described in the efflux of quinolones and tetracycline,53-55 although we were unable to detect any in our samples.

Figure 1. SDS-PAGE analysis of the membrane fractions of A. baumannii ATCC 19606. (A) The reproducibility of the extraction method was tested on OM extracts (lanes 1-3) and IM extracts (lanes 4-6). (B) Distribution of the proteins identified on SDSPAGE gels, according to their cellular compartment.

protocol. However, we observed a significant contamination of the OM sample by IMPs (38%), which may be explained by the presence of complexes of both IMPs and OMPs. However, there was very little (3%) OMP contamination in the IM sample. The comparison of these data with those for A. baylyi ADP143 shows that the number of detected OMPs and IMPs was, respectively, 40 and 54% of the potential IM and OM proteomes. However, it is most unlikely that most open reading frames would be expressed under any specific growth conditions, so that the 135 detected membrane proteins are far more than 50% of the predicted membrane subproteomes. Functional Classification of Identified Proteins. We classified the 230 unique proteins identified from the ATCC 19606 according to their function (Table 1, for a complete list and further details, see the Table S1 in the Supporting Information). Most polypeptides had a high sequence identity with proteins of Pseudomonas species (data not shown), the genomes of which are most similar to that of Acinetobacter.43 The highest ratio of identifications (44%) was for proteins involved in metabolism. Thus, we identified membrane proteins belonging to the energetic metabolism, such as the ATP synthase complex, proteins involved in the respiratory chain, and enzymatic complexes, such as NADH or succinate dehydrogenase. However, most of these metabolism-involved proteins were mainly cytoplasmic contaminants. Consistent with data published on the A. baylyi ADP1 genome,43 this classification also revealed that one of the most abundant categories of membrane proteins concerned the transport function (20%). More precisely, in A. baylyi ADP1, 38% of the ABC transport systems are involved in amino acid transport, whereas this bacterium seems deficient in sugar transporters as only one ABC system is dedicated to this function.43 In the present study, we identified different proteins belonging to ABC systems (at least six ABC systems) and a homologue of the OprB protein of P. aeruginosa (AbNCBI number 5173), which may be involved in carbohydrate transport.48 The molecular basis of A. baumannii resistance to β-lactams include different mechanisms: (i) an increased expression of efflux pumps,18,49 (ii) an alteration of OM permeability by reducing porin expression,28-31 (iii) the presence of β-lactamases and modifications to their specificities,50,51 and (iv) modifications to the penicillin-binding-proteins (PBP) pattern.11 For resistance-nodulation-cell division (RND)-type efflux pumps, we found the AdeABC and AdeXY pumps in the 3388


Several OM proteins having undetermined channel-forming functions are suspected to be porins and may play a role in antibiotic resistance.17,25,26 We identified some of these proteins, such as the Omp33/36 protein,17,29,30 the OprD-like protein,31 and the porin CarO27,28,40 (see Table 1). We identified only one β-lactamase, the chromosomal AmpC cephalosporinase.56 We found no chromosomally encoded oxacillinase, OXA-69, which was thought to occur in A. baumannii.50 However, the periplasmic location of these enzymes may explain their absence. From the inner membrane fractions, we were able to identify two PBPs. We found a homologue of the PBP1b or PonB protein of P. aeruginosa or Escherichia coli which may be the target of cefsulodin, cephaloridine, and ceftriaxone antibiotics in these bacteria and may have some affinity with imipenem.57-60 We also observed the PBP6 or DacC protein, which may be, as demonstrated in E. coli, the target of cefoxitin, cefepime, and, to a lesser extent, of imipenem.58,60 Joly-Guillou61 reported that the severity of Acinetobacter infections depends upon the site of infection, patient susceptibility to infections, and the potential virulence of the colonizing strain. The circumstances that allow Acinetobacter to assume a pathogenic role are not well-understood. This organism is considered to be a low-grade pathogen, and thus, infections by Acinetobacter may involve numerous factors. These include virulence determinants such as the expression of siderophores, lipolytic enzymes, or capsular exopolysaccharide.38,61 We were able to identify proteins that may be involved in bacterial virulence, such as the Mip protein,62 the FepA-like siderophore receptor,36 and a homologue of the VacJ protein of Shigella flexneri that seems to be involved in the biosynthesis of the cell wall and is necessary for Shigella infection.63 The Mip protein (macrophage infectivity potentiator) has already been identified in Acinetobacter lwofii RAG-1.62 This Acinetobacter strain is known for its capacity to emulsify extracellular lipids due to a complex (named Emulsan) that is a synergistic association between a biopolymer (polysaccharide + fatty acid) and a lipase, LipA.62 The mip gene is found in the wee operon that controls the production of the biopolymer and seems to be controlled by the same promoter as the wzABC genes responsible for export of the polymer.62,64 Although the precise function of this OMP in pathogenicity is still unknown, its expression seems necessary for macrophage infection by different species, such as Legionella pneumophila, Trypanosoma cruzi, Chlamydia trachomatis, and Neisseria gonorrhoae.65 Surprisingly, we found no BauA siderophore receptor that was previously described in A. baumannii ATCC19606,36 but we identified one homologue of a FepA-like protein.43 Both receptors have only 19% identity (Table 1, see Virulence section). Finally, 20% of the detected proteins were unknown and/or hypothetical. The abundance of these proteins is not surprising,

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Table 1. Selection of Identified Proteins from the Enriched Membrane Samples of A. baumannii ATCC 19606 theorical function

band

gene

AbNCBI

coverage (%)

oprD-like ompE oprB ompA carO ompW

5419 5331 5173 5219 6069 6316 8157

24 24 14 44 8 21 30

adeA adeB adeX adeY adeT adeC

7383 7238 4199 4276 5978 4198

34 14 23 16 17 30

IM IM IM IM IM IM IM IM IM IM periplasm

(2) mscL (OM) mscS (OM) (OM)

(IM)

5316 4694 6016 6762 6082 7102 6967 8054 5433 4252 6341

60 22 24 24 21 10 34 31 21 26 24

periplasm

(IM)

4404

17

periplasm

(IM)

6567

43

periplasm

(IM)

6762

26

General secretion pathway protein G Preprotein translocase Signal peptide peptidase Preprotein translocase-ATPase

IM IM IM IM-bound

xcpT secD sppA (OM) secA

7928 5153 7147 5244

59 26 45 35

OMP85-associated lipoprotein Chaperone - PPIase Chaperone Thiol:disulfide interchange protein Putative OMP85 OMP85-associated lipoprotein

IM IM periplasm periplasm OM OM

(OM) (2) (IM) (2)

yfgL surA skp dsbA omp85 nlpB

7939 7836 7281 5631 7498 5074

28 18 36 22 40 15

lolD

7153

38

lolA

4215

20

exbB exbD tolQ

8205 7935 6313

15 28 11

compartmenta

MW

pI

description

45538 48295 43989 36177 30235 24422 19182

5.8 4.8 5.2 5.1 4.5 4.6 5.1

Basic amino acids porin Fatty acids porin Glucose porin Unspecific OmpA-like porin (HMP) Omp33/36 Unspecific porin Hydrophobic compounds porin

OM OM OM OM OM OM OM

24/21 4 21 2 27 15

43434 112652 43806 114551 34214 50547

7.8 7.6 9.1 5.9 9.4 8.7

RND-type efflux pump RND-type efflux pump RND-type efflux pump RND-type efflux pump RND-type efflux pump OM Channel (RND pump)

IM IM IM IM OM OM

(2)

46/50 11 8 31 34 42 35/41 47 1 28 27

15782 54685 61529 29318 30347 28469 23211 20048 58926 31059 34918

6.3 9.2 5.2 9.5 5.2 9.3 6.1 6.7 4.8 5.5 9.4

28

34504

34

29213

36

29318

Mechanosensitive channel Putative mechanosensible channel Putative ABC superfamily transport protein ABC transporter-binding component Putative ABC-transporter Putative transporter exhibiting a RDD domain Potassium transporting ATPase C chain pH adaptation-potassium efflux system Putative transporter Stomatin related protein Sulfate transport, periplasmic binding component (ABC) 9.4 Phosphate transport, periplasmic binding component (ABC) 5.7 Amino acids transport, periplasmic binding component (ABC) 9.5 Glutamate/aspartate transport, periplasmic binding component (ABC) 6.1 8.9 6.6 5.2 8.1 8.5 9.4 9.5 5.2 6.3

Porins 16 15 17 23 27 31 43 Active Efflux

Transport

Type II Secretion Pathway 51 21017 1 66755 30 40288 1 102510 OMP Assembly 23 38186 System 9/13 65100 53 16498 43/41 20736 1 91207 37 22107 Lipoprotein Releasing System 37 25098

6.1 Lipoprotein releasing system, ATP binding component 40 22378 9.6 Lipoprotein releasing system, chaperone TonB-Coupled Transmembrane Proton Pathway 47 22041 9.4 TonB associated protein 55 14525 4.4 TonB associated protein 43 25369 5.6 TonB associated protein Proteases 12/9 65843 5.8 ATP-dependent metalloendopeptidase 29 25793 9.7 Peptidoglycan-binding peptideMetalloendopeptidase domain 25/19 39105 5.4 Serine protease 16 46464 9.2 Serine protease LPS Transport and Assembly 1 68469 7.8 Putative MsbA-like ABC transporte 4 88813 5.0 Organic solvant tolerance protein -LPS assembly and transport Fatty Acids Biosynthesis and Transport 9/3 97304 6.6 Glycerol-3-phosphate acyltransferase 53 17792 9.7 Putative sterol carrier protein Cell Wall/Capsule/Structure 14 50435 5.0 Capsule biosynthesis 19 43753 8.8 Cell envelope integrity 41 17519 5.8 Peptidoglycan associated lipoprotein

(2)

IM periplasm

(IM)

IM IM IM

kdpC kup terC

IM IM

(2) ftsH (OM) lysM/M23

5495 4603

32 29

periplasm periplasm

(2) algW (OM) mucD

4487 6315

47 19

6700 7660

24 24

IM OM

ostA

IM unknown

(2) (IM)

gpat

6286 5028

25 44

IM periplasm OM

(OM) K30 (OM) tolB oprL

5732 6322 6310

20 20 76

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Table 1 (Continued) theorical function

band

MW

pI

description

Peptidoglycan Biosynthesis 9 88231 6.6 Transglycosylase/Transpeptidase (PBP1b) 27/23 39747 7.2 D-ala-D-ala-carboxypeptidase (PBP6) 14 70368 7.8 Soluble Transglycosylase-Lytic muraminidase Resistance to Antimicrobial Molecules 42 29566 9.0 Bacitracine resistance protein 24 40731 9.2 ss-lactamase Virulence 5 106322 8.4 ImcF related-cell surface reorganization protein 34 29436 5.6 NLPA-type lipoprotein 32 31574 8.4 NLPA-type lipoprotein 33/31 23602 6.4 Putative macrophage infectivity potentiator 45 15758 4.7 Copper resistance lipoprotein NlpE 24 31049 4.9 Peptidoglycan associated lipoproteinassociated with virulence in intracellular pathogens 5 80552 5.6 TonB-dependent siderophore receptor 53 15868 10.0 Putative surface allergen Competence 19 39491 5.5 Putative competence protein Motility/Adherence 47 16060 4.6 Usher pili assembly system 31 27100 9.3 Usher pili assembly system 8 89402 6.0 Usher pili assembly system 25 33548 5.3 Usher pili assembly system 9 69659 7.2 Pilus assembly protein Adaptation 45 20999 9.8 Toluen tolerance protein - ABC transporter 34/44 30862 5.6 Heat shock protein-metalloendopeptidase 50 16761 6.8 Like-SM ribonucleoprotein 45 22148 9.2 Cu/Zn superoxide dismutase 53 20881 9.5 Organic solvant tolerance protein-OstA domain 41 24114 5.0 Toluen tolerance protein 34 25220 5.9 Putative lipoprotein involved in folding pathway Metabolism: Respiratory Chain 48 16367 9.2 NADH dehydrogenase I chain A 37 25551 6.2 NADH dehydrogenase I chain B 15/10 68580 5.6 NADH dehydrogenase I chain C,D 51 18928 5.3 NADH dehydrogenase I chain E 20/15 48621 5.8 NADH dehydrogenase I chain F 2/3 97988 5.6 NADH dehydrogenase I chain G 47 20431 6.0 NADH dehydrogenase I chain I 1 67879 8.1 NADH dehydrogenase I chain L 18 71092 7.0 Cytochrome o ubiquinol oxidase subunit I 40/23 37276 6.4 Cytochrome o ubiquinol oxidase subunit II 1 55010 6.9 Cytochrome D ubiquinol oxidase subunit I 3 41571 8.5 Cytochrome D ubiquinol oxidase subunit II 17/11 56977 8.6 Malate: quinone oxidoreductase 18 61484 6.5 Ubiquinone biosynthesis protein 14/10 69327 5.8 Electron transfert flavoprotein-ubiquinone oxidoreductase 55 16493 5.9 Riboflavin synthase ss chain 26 31510 4.9 Electron-transfer flavoprotein R-subunit Krebs Cycle 12/10 61556 8.6 Putative Acetyl-coA synthase-E2 component 19 51527 5.9 Dihydrolipoamide DH-E3 component 23 41529 4.9 Succinyl-CoA synthase beta chain 16/6 66996 5.9 Succinate dehydrogenase-flavoprotein subunit 52/34 26803 7.5 Succinate dehydrogenase-iron-sulfur (catalytic) subunit 53 14497 9.8 Succinate dehydrogenase-cytochrome b556 subunit 3 79505 5.4 Malate synthase G Glycolysis 25 41648 7.0 L-lactate dehydrogenase 1 83557 7.2 Quinoprotein alcohol dehydrogenase 3390


compartmenta

gene

AbNCBI

coverage (%)

IM IM periplasm

(2) (IM)

ponB dacC slt

4373 4349 4294

25 48 11

IM periplasm

(IM)

bacA ampC

5314 4460

20 39

6926

30

mip nlpE vacJ

7028 5549 5613 5711 8044

42 39 43 49 33

fepA bet v1

7650 6929

18 31

OM

comL

4661

30

OM OM OM OM OM

csuA/B csuC csuD csuE filF

7055 6996 7017 7078 5990

33 23 14 18 10

IM periplasm periplasm periplasm periplasm OM

OM unknown

(IM) (IM) (2) (IM)

(IM)

IM IM IM periplasm periplasm

OM(40) ttg2D (2) htpX hfq (OM) sod_Cu (IM) ostA-like

6074 5485 6964 6050 6718

33 11 36 38 30

periplasm OM

(IM)

6144 5866

62 15

4653 4707 4651 4783 4742 4656 4727 4785 7056 7040 7214 7556 5727 7944 7970

27 65 29 40 22 28 44 5 11 22 38 9 29 31 25

5019 7164

20 36 14 24 37 16

IM IM IM IM IM IM IM IM IM IM IM IM IM IM IM IM periplasm

ttg2C yraP

(2) (2) (2)

(2)

cyoB cyoA bac cydB

(2) (2)

(OM)

ubiB Etf-QO

IM IM IM IM

(2) (2)

5460 4191 4194 4256

IM

(2)

4178

23

4231

26

7603

18

4914 5208

54 15

IM IM IM IM

(OM)

research articles

Membrane Proteomes of A. baumannii Table 1 (Continued) theorical function

band

MW

ATP Synthesis 19/13 55397 7/13 50274 45 19508 22 32096 56/46 13405 Pyrimidine Biosynthesis 25 33821 Nucleosides-Related 37 23707 52 15462 53 11729 Amino Acids-Related 37 31254 4 138285

pI

compartmenta

description

AbNCBI

coverage (%)

41 19 20 39 20

5.3 5.0 4.6 9.1 5.9

ATP synthase R chain ATP synthase ss chain ATP synthase δ chain ATP synthase γ chain ATP synthase F0 - subunit B

IM IM IM IM IM

(2)

4880 4822 4912 4894 4908

5.6

Dihydroorotate dehydrogenase

IM

(IM)

4529

27

5.2 5.5 4.4

Phosphoriboisomerase A Nucleoside diphosphate kinase Thioredoxin

IM IM periplasm

rpiA ndk

7542 7960 7886

37 34 23

8.0 5.7

Phosphatidylserine decarboxylase Bifunctional protein, proline dehydrogenase-P5C dehydrogenase D-amino acid DH-small subunit Aspartyl/Asparaginyl ss-hydroxylase family

IM IM

psd putA

4997 6746

25 38

4875 8015

18 45

IM

7941

38

IM

6153

68

IM

5775

63

IM

6923

26

IM IM

5652 8108

42 46

IM IM

8037 6126

60 16

IM

7913

10

IM IM IM periplasm OM

5009 7325 7510 6608 5562

18 40 28 24 46

6870

39

6167 5757 6190

70 23 24

7151 5248 7768 6336 7449 7803 7488 5590 5200 6580 5061 4172 4641 5113 6739 6635

12 44 18 34 43 38 38 20 43 42 22 35 36 45 15 53

16 47613 6.0 34 35917 9.5 Proteins of Unknown Function 37 22584 4.4 Protein of unknown function-TPR-like domain 48 21736 6.8 LemA-like hypothetical protein Metabolism: Proteins of Unknown Function 47 21286 9.1 Putative peptidoglycan-associated OMP-OmpA/ motB domain 54 13944 8.4 Conserved hypothetical protein of unknown function-DUF333 32 23167 4.5 FHA domain protein 53 14285 9.9 Protein of unknown function-Rhodanese-like domain 49 18917 9.6 Conserved hypothetical protein 28 33810 5.1 Conserved hypothetical protein of unknown function-DUF477 47 17916 9.4 Conserved hypothetical integral membrane protein-YGGT family 38 26145 5.3 Short chain dehydrogenase 39 27018 9.5 Short chain dehydrogenase 18 48368 8.8 Short chain dehydrogenase 46 18885 9.0 Indole/base-induced protein 46 10001 8.0 Putative peptidoglycan associated OMP-OmpA/ MotB domain 11/19 48984 5.7 Putative peptidoglycan associated protein-OmpA/ MotB domain 46 25596 4.5 OMP of unknown function 22 39245 5.9 Putative OMP of unknown function-DUF306 43 21354 5.4 Conserved hypothetical protein of unknown function-DUF477 Hypothetical, Putative 38 31813 8.8 Putative membrane protein 44 21586 9.9 Putative membrane protein 44 25468 9.1 Putative membrane protein 55 11323 6.8 Putative membrane protein 54 13869 4.8 Putative membrane protein 49 13600 9.5 Putative membrane protein 32 29199 5.7 Putative membrane protein 40 24925 4.9 Hypothetical protein 46 22094 9.6 Hypothetical protein 49 19074 9.6 Putative OMP 55 13670 9.5 Putative OMP 29 26326 5.0 Putative OMP 13 50161 5.9 Putative OMP 44 22717 8.9 Putative OMP 49 15140 8.7 Putative OMP 30 8860 4.4 Putative OMP a

gene

IM IM

OM OM OM unknown

IM IM IM IM IM IM IM IM IM periplasm periplasm OM OM OM OM OM

(2) (2)

(IM)

(OM)

(IM)

yceL

(2) omp25 (IM)

(OM)

(IM) (IM) (IM) (IM) (IM)

Locations determined by the PSORTb program available at http://www.psort.org. In parentheses: compartments where proteins were identified.

as about 55% of the open reading frames of A. baylyi ADP1 encode unknown proteins.43

MICs Determination. As we have demonstrated here, the standard ATCC 19606 strain had a susceptible phenotype, Journal of Proteome Research • Vol. 5, No. 12, 2006 3391

research articles

Siroy et al.

Figure 2. 2-DE gels of OMPs and IMPs of A. baumannii ATCC19606 and A6 strains. Experimental 2-DE gels of OM extracts of ATCC 19606 (A) and A6 (B) strains were obtained from 100 µg of proteins. The gels were stained by Colloidal Coomassie blue. The same conditions were used for IM extracts of ATCC19606 (C) and A6 (D) strains. Table 2. Antibiotic Susceptibilities of A. baumannii Strainsa MIC in µg/mL strains

Imi

Caz

Cep

TzP

Atm

Tm

An

Cip

ATCC A6

1 >256

8 >256

16 32

0.25 >256

8 64

8 16

32 >256

1 >32

a Imi, Imipenem; Caz, Ceftazidime; Cep, Cefepime; TzP, Piperacilline + Tazobactam; Atm, Aztreonam; Tm, Tobramycin; An, Amikacin; Cip, Ciprofloxacin.

according to the tested antibiotics, whereas the A6 strain was resistant to these drugs (see Table 2). Although the A6 strain had a longer generation time (84 min) than the reference strain (61 min), this could not explain its MDR phenotype. Indeed, this strain was highly resistant to all β-lactams tested (including imipenem). PCR assays using specific primers revealed the presence of an OXA-40-type β-lactamase66 (S. Boyer-Mariotte, personal communication). The expression of this enzyme may contribute, when associated with an alteration in membrane permeability such as the overexpression of some efflux pumps, to the high level of resistance to carbapenems observed in the MDR strain.49 Although SDS-PAGE produced a high solubilization of the membrane proteins, this technique does not easily allow the quantitative comparison of different experimental membrane 3392


proteomes. Consequently, we used two-dimensional gel electrophoresis to compare the membrane subproteomes of the MDR and reference strains. Comparison of Standard and MDR Strains’ Membrane Subproteomes. We separated 172 (IM) and 112 (OM) spots (Figure 2) fulfilling the criteria described in Experimental Procedures, which were considered for quantitative and qualitative analysis. These good results are partly due to the incorporation of the zwitterionic detergent ASB-14 in the IEF buffer before running the gels. This detergent improves the solubility of membrane proteins over that of IEF buffer containing CHAPS or SB 3-10.67 However, only 67 IMPs and 75 OMPs were identified. The position of each identified spot on the 2-DE gels is shown in Figures 3 (OMPs) and 4 (IMPs). We observed significant differences between the subproteomes of the two strains. About 36% and 56% of spots detected for IMP and OMP patterns, respectively, were different (see Tables 3 and 4). This is not surprising given the antibiograms of the two strains. However, although the 1D-PAGE technique showed that the standard strain expressed the RND pumps AdeABC and AdeXY, we did not observed this using the 2-DE approach. This was due to the poor resolution at alkaline pH for the 2-DE gels.68 Indeed, A. baumannii transporters have basic isoelectric points (Table 1) and thus could not be recovered.


research articles

Figure 3. Comparison of OMPs expression in A. baumannii ATCC 19606 and A6 strains. Identified proteins are numbered from 1 to 75 on a 2-DE gel of OMPs from the A6 strain. 4; Proteins overexpressed (at least 2-fold) by the A6 strain; 2, proteins overexpressed (at least 5-fold) by the A6 strain; 3, proteins underexpressed (at least 2-fold) by the A6 strain; 1; proteins underexpressed (at least 5-fold) by the A6 strain.

Figure 4. Comparison of IMPs expression in A. baumannii ATCC 19606 and A6 strains. Identified proteins are numbered from 1 to 67 on a 2-DE gel of IMPs from the A6 strain. 4; Proteins overexpressed (at least 2-fold) by the A6 strain; 2, proteins overexpressed (at least 5-fold) by the A6 strain; 3, proteins underexpressed (at least 2-fold) by the A6 strain; 1; proteins underexpressed (at least 5-fold) by the A6 strain.

We identified the principal OM porins which may be involved in β-lactams resistance and particularly in imipenem resistance. These were the CarO, Omp33/36, and OprD-like proteins (see Table 3).17,28,30,31 For the Omp33/36 and OprDlike proteins, we observed no significant difference in their expression between the two strains. In contrast, the CarO protein had different migration behavior. Two groups of spots (39 and 22) were present in the standard strain OMP pattern but were absent in the MDR strain pattern (Table 3 and Figure 3). This is typical of oligomeric OM proteins, which migrate as rungs of a ladder due to their association with lipopolysaccharide (LPS) molecules.69 This suggests the existence of CarO dimers and trimers in association with different amounts of bound LPS.69 This difference in CarO behavior between the susceptible and MDR strains may reflect differences in the interaction between CarO and LPS due to modifications in the protein and/or LPS structure. Indeed, in E. coli, the migration and multiple banding patterns of OmpF protein trimers depended on the chemotype of the associated LPS.70 The mass spectra fingerprints of the CarO recovered from the two strains (see Figure S1 in the Supporting Information) showed that the primary structure of the protein was probably modified and suggests that the CarO porin diffusion pathway may be modified. Mass spectra analyses of other proteins likely involved in the antibiotic pathway (e.g., Omp33/36 and OprDlike proteins) did not suggest such primary structure modifications (not shown). Interestingly, we found four isoforms of OmpW (see spots 62, 63, 66, and 67 in Figure 3 and Table 3), the expression of which was strain-dependent. Mass spectra analysis did not allow us to characterize post-translational modifications. OmpW,

which migrated in SDS-PAGE with an apparent mass of 22 kDa (see Table 1), belongs to the OM protein family of small monomeric β-barrels such as OmpA of E. coli or NspA of Neisseria meningiditis.71,72 Its crystal structure determined in E. coli shows an eight-stranded β-barrel.71,72 The hydrophobic channel formed within this barrel suggests that OmpW may be involved in transporting small hydrophobic molecules across the bacterial outer membrane. OmpW is also a homologue of the OprG protein in the Pseudomonaceae family.73 OprG expression is highly dependent on growth conditions, including high growth temperatures and Mg2+ deficiency, and also on certain modifications to the lipopolysaccharide composition.73 In particular, this OMP has been suggested to be involved in low-affinity iron uptake due to the direct relationship between its expression and the iron concentration in the medium.74 Differences in the expression of OprG occur in P. aeruginosa strains exhibiting different antibiotic resistance.75 For the virulence factors, we found that VacJ63 and Mip62 proteins identified in the 1D-PAGE analysis had the same expression levels in both strains. However, the FepA-like protein had a higher expression level in the MDR strain, which also expressed two other siderophore receptors (spots 20, 27, and 19 in Figure 3). This subproteome analysis allowed us to show another aspect of the membrane modifications that took place in the MDR strain. This strain accumulated significant levels of the lipoprotein NlpE (Figure 4 and Table 4). Otto and Silhavy76 showed that this OMP may sense and generate an adhesionspecific signal to a two-component system in E. coli: the CpxRA pathway. This system belongs to a large family of homologous two-component systems that enable bacteria to respond to Journal of Proteome Research • Vol. 5, No. 12, 2006 3393

research articles

Siroy et al.

Table 3. Comparison of the Expression Level of Identified Outer Membrane Proteins in A. baumannii ATCC 196606 and A6a theorical function

spot

Mr

pI

protein

AbNCBI

ATCC

Transport, secretion

3 29 5 2 60 36 45 48 70 46 59 53 39 22 56 43 20 63 66 67 62 10 23 25 28 6 52 37 69 44 14 15 16 57 47 58 12 26 19 18 61 68 50 54 64 51 30 49 40 33 38 8 34 31 35 55 24 71 32 1 73 13 65 72 9 11 17 41 21 75 7 74 4 27

102510 61529 91207 96834 24422 48295 43814 36177 36177 45538 25596 30235 24422 24422 24422 36177 75312 19182 19182 19182 19182 88813 69446 71760 57166 95142 40487 50435 17519 39491 89402 89402 74709 29436 31049 23602 75424 75702 80552 80261 23238 22083 39245 25409 21736 33161 48984 43058 41461 50274 55397 80205 48621 66996 51527 31510 61112 15827 52575 151851 22653 71776 22919 14963 71677 44361 67659 48621 31853 19556 101862 13681 105242

5.2 5.2 5.2 4.9 4.6 4.8 6.3 5.1 5.1 5.8 4.5 4.5 4.6 4.6 4.6 5.1 5.7 5.1 5.1 5.1 5.1 5.0 4.7 4.9 4.9 5.2 7.5 5.0 5.8 5.5 6.0 6.0 5.0 5.6 4.9 6.4 5.1 5.4 5.6 5.5 4.4 5.8 5.9 4.6 6.8 5.9 5.7 5.9 4.9 5.0 5.3 5.6 5.8 5.9 5.9 4.9 5.0 5.6 5.9 5.2 9.4 5.4 5.6 5.7 4.7 5.0 5.1 5.1 9.5 6.3 5.4 6.2 5.5

SecA ABC transporter OMP85 OMP85-like CarO OmpE DcaP OmpA OmpA OprD Omp25 Omp33-36 CarO CarO CarO OmpA OprC OmpW OmpW OmpW OmpW OstA Hsp70 Hsp90 GroEL Hsp100 DnaJ K30 OprL ComL CsuD CsuD Putative TonB receptor NLPA lipoprotein VacJ lipoprotein Macrophage infectivity potentiator TonB-dependent receptor TonB-dependent siderophore receptor FepA-siderophore receptor Ferric enterobactin receptor Conserved hypothetical protein Conserved hypothetical protein Conserved hypothetical protein Hypothetical protein LemA-like hypothetical protein Hypothetical proteinsTPR pattern Peptidoglycan-associated lipoprotein Hypothetical proteinsTPR pattern Hypothetical OMP ATP synthase F1 ss ATP synthase F1 R Catalase NADH DH F1 Succinate DH Dihydrolipoamide DH Electron transfert flavoprotein R subunit 30S S1 50S L9 IMP DH RNA polymerase β chain Putative transcription regulator UvrAC-excinuclease ABC Superoxide dismutase Ribosomal protein 30S S6 Sigma70 Elongation factor Elongation factor NADH DH F1 Putative short chain DH Putative transferase Pyruvate decarboxylase E1 YfiA Ribonucleoside diP reductase R subunit No match

5244 6016 7498 7024 6316 5331 4261 5219 5219 5419 6167 6069 6316 6316 6316 5219 4834 8157 8157 8157 8157 7660 4932 8058 5508 6730 4984 5732 6310 4661 7017 7017 5144 7028 8044 5613 5347 5537 7650 5942 5002 7220 5757 5054 6153 6391 6870 4472 8185 4822 4880 6830 4742 4256 4191 7164 7620 7084 6297 8132 7381 4365 4346 7101 4238 4649 5348 4742 4992 4383 6037 5059 4668

) ) ) ) ) ) ) ) ) ) ) ) ++ ++

Porins

Adaptation

Capsule Cell wall Competence Motility Virulence

Hypothetical

Metabolism

Metabolism

a

Spot numbers refer to Figure 3. +, Proteins overexpressed (at least 2-fold); ++, proteins overexpressed (at least 5-fold).

3394


A6

) ) ) ) ) ) ) ) ) ) ) ) ++ + +

++ ++ ) ) ) ) ) ) ) ) ) ++ ) ) ) ) ++

) ) ) ) ) ++ ++ + ) ) ) ) ) ) ) ) ) ) ) ) ) ++

++ + ) ) ) ) ) ) ) ) ) ++ ) ) ) ) ++ + + ) ) ) ) )

++ ) ) ) ) ) ) ) ) ) ) ) ) ) + + ++ + + + + + + +

++

research articles


Table 4. Comparison of the Expression Level of Identified Inner Membrane Proteins in A. baumannii ATCC 196606 and A6a theorical function

Transport, Secretion

Adaptation

Virulence Hypothetical

Metabolism

a

spot

Mr

pI

protein

AbNCBI

ATCC

48 42 1 56 58 39 34 37 52 35 21

33638 30347 102510 25369 25098 30347 36177 25596 24422 30235 62546

6.4 5.2 5.2 5.6 6.1 5.2 5.1 4.5 4.6 4.5 6.2

4444 6082 5244 6313 7153 6082 5219 6167 6316 6069 6937

) ) ) ) ) ) ) ) ) ++

4 16 2 64 66 50 49 41 53 54

69446 57166 95142 15775 15940 31429 23602 29436 15758 22584

4.7 4.9 5.2 5.6 6.1 6 6.4 5.6 4.7 4.4

4932 5508 6730 7343 7302 6464 5613 7028 5711 7941

) ) ) ) ) ) ) )

48984 21736 50161 32332 26326 47833 48984 48984 55397 50274 13405 69327 48621 97988 68580 48621 22919 18089 66996 26803 37276 34556 39747 41648 78796 97988 89673 15827 51527 42514 50274 63560 88231 44960 128024 27014 91084 14962 66996 39747

5.7 6.8 5.9 5.1 5.0 9.1 5.7 5.7 5.3 5.0 5.9 5.8 5.8 5.6 5.6 5.8 5.6 5.6 5.9 7.5 6.4 6.5 7.2 7 5.1 5.6 5.9 5.7 5.9 5.2 5 8 6.6 7.3 6.5 5.2 5.5 5.7 5.9 7.2

BauB, iron transport ABC transporter Preprotein translocase SecA TolQsprotein uptake LolD-Lipoprotein releasing system ABC transporter-ATP binding OmpA Omp25 CarO Omp33-36 Paraquat inducible proteinputative ABC transporter DnaK - Hsp70 GroELsCpn60 Hsp100 UspA-like UspA-like UspA-like Macrophage infectivity potentiator NLPA Lipoprotein NlpE-Cu resistance Conserved hypothetical proteinsTPR-like domain Peptidoglycan-associated lipoprotein LemA-like hypothetical protein Putative OMP Probable OMP Probable OMP Hypothetical protein Peptidoglycan-associated lipoprotein Peptidoglycan-associated lipoprotein ATP synthase R chain ATP synthase ss chain ATP synthase FO-subunit B Electron-transfer flavoprotein NADH DH I chain F NADH DH I chain G NADH DH I chain C,D NADH DH I chain F Superoxide dismutase 50S L10 Succinate DH- Flavoprotein subunit Succinate DH- catalytic subunit Cytochrome o ubiquinol oxidase subunit II UbiE-Ubiquinone biosynthesis PBP6 L-lactate DH Elongation Factor G NADH DH I chain G Molybdopterin oxidoreductase 50S L9 Dihydrolipoamide DH-E3 subunit SucB, dihydrolipoamide succinyltransferase ATP synthase β chain BetA, choline DH-betaine synthesis PBP1b D-amino acid DH small subunit Pyruvate-ferredoxin oxidoreductase Short chain DH Putative acyl-CoA DH 30S S6 Succinate DH- Flavoprotein subunit PBP6 No match

55 59 25 33 36 28 18 17, 29 23 22, 32 60 19 24 6 12, 13 26 57 61 14 51 43 44 47 30 3 7 10 62 27 45 5 15 11 31 67 40 8, 9 63 20 46 38

6870 6153 4641 8169 4172 8089 6870 6870 4880 4822 4908 7970 4742 4656 4651 4742 4346 8144 4256 4178 7040 8005 4349 4914 4649 4656 7160 7084 4191 4239 4822 5679 4373 7566 6457 5632 4516 7101 4256 4349

A6

) ) ) ) ) ) ) ) ) ++

) ) ) ) ) ) + + ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ++ ++ + + + + + +

) ) ) ) ) ) ) ) ++ ) ) ) ) ) ) ++ ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) ) )

++ ++ ++ ++ + + + ++

Spot numbers refer to Figure 4. +; Proteins overexpressed (at least 2-fold); ++, proteins overexpressed (at least 5-fold).

various environmental parameters and are involved in surface sensing and biofilm formation (see the review by Lejeune77 and the references therein). We found two CsuD protein isoforms that had a higher expression level in the A6 MDR strain (Figure

4 and Table 4). This polypeptide, like its homologue in P. aeruginosa (PA4652), contains conserved domains that have been described in fimbrial usher proteins involved in the biogenesis of Gram-negative bacterial pili.78 It is well-known Journal of Proteome Research • Vol. 5, No. 12, 2006 3395

research articles that pili are required in the early stages leading to biofilm formation.79,80 As NlpE and CsuD have a higher expression level in the MDR strain, this strain may have a greater ability than the reference strain to adhere. Therefore, we compared the ability of both strains to initiate biofilm formation on an abiotic surface using the protocol previously described by O’Toole and Kolter.39 As expected, we found that strain A6 (OD570 ) 0.663 ( 0.100, p ) 0.95) had a greater ability to form biofilm than the ATCC strain (OD570 ) 0.318 ( 0.020, p ) 0.95).

Concluding Remarks Although the bacterium A. baumannii is the Acinetobacter species having the greatest importance in human medicine, there have been very few studies devoted to identify the molecular basis of its multiresistance. This present paper describes a first approach to characterize resistance mechanisms by proteomics. We focused our study on the inner and outer membranes of the bacterium, as these are highly involved in resistance in Gram-negative organisms and have described the membrane proteins of A. baumannii. This analysis revealed the expression of some RND-type efflux systems and highlighted some potential virulence factors in this bacterium. The comparison of the membrane proteomes of an MDR clinical isolate with that of a standard susceptible strain revealed modifications in certain proteins of the MDR strain cell envelope, such as the absence of a PBP (PBP1b protein), structural modifications to the CarO porin, and the presence of different isoforms of the hydrophobic channel OmpW. We also showed that this MDR strain had a high ability to form biofilms, consistent with the accumulation of OMPs already described as involved in bacterial adhesion. This enhanced biofilm-forming capability associated with the differential expression of certain proteins that could be involved in antibiotic resistance mechanisms may explain the emergence of multidrug resistance in the clinical strain. Mutagenesis approaches are currently in progress to confirm these results.

Acknowledgment. This work was supported by the Haute-Normandie region with a grant attributed to A.S. We thank G. Molle and J. M. Page`s for helpful discussions, and L. Coquet for technical support. Supporting Information Available: The peptide sequences of proteins identified from SDS-PAGE gels are shown in Table S1. CarO oligomers and the corresponding mass spectra in the reference and MDR strains are shown in Figure S1. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Wong, T. H.; Tan, B. H.; Ling, M. L.; Song, C. Multi-resistant Acinetobacter baumannii on a burns unit-clinical risk factors and prognosis. Burns 2002, 28 (4), 349-57. (2) Bergogne-Berezin, E.; Towner, K. J. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol. Rev. 1996, 9 (2), 148-65. (3) Van Looveren, M.; Goossens, H. Antimicrobial resistance of Acinetobacter spp. in Europe. Clin. Microbiol. Infect. 2004, 10 (8), 684-704. (4) Corbella, X.; Montero, A.; Pujol, M.; Dominguez, M. A.; Ayats, J.; Argerich, M. J.; Garrigosa, F.; Ariza, J.; Gudiol, F. Emergence and rapid spread of carbapenem resistance during a large and sustained hospital outbreak of multiresistant Acinetobacter baumannii. J. Clin. Microbiol. 2000, 38 (11), 4086-95.

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