Quantitative Proteomic Analysis of Cell Wall and Plasma Membrane Fractions from Multidrug-Resistant Acinetobacter baumannii Sung-Ho Yun,† Chi-Won Choi,† Sang-Oh Kwon,†,| Gun Wook Park,‡,| Kun Cho,‡ Kyung-Hoon Kwon,‡ Jin Young Kim,‡ Jong Shin Yoo,‡,| Je Chul Lee,§ Jong-Soon Choi,†,| Soohyun Kim,† and Seung Il Kim*,† Division of Life Science, Korea Basic Science Institute, Daejeon, 305-806, Korea, Mass Spectronmetry Research Center, Korea Basic Science Institute, Ochang, Cheongwon-Gun, Chungcheongbuk-Do, 363-883, Korea, Department of Microbiology, Kyungpook National University School of Medicine, Daegu 700-422, Korea, and Graduate School of Analytical Science and Technology, Chungnam National University, Daejeon 305-764, Korea Received June 22, 2010
Acinetobacter baumannii is a Gram-negative, nonmotile aerobic bacterium that has emerged as an important nosocomial pathogen. Multidrug-resistant (MDR) A. baumannii is difficult to treat with antibiotics, and treatment failure in infected patients is of great concern in clinical settings. To investigate proteome regulation in A. baumannii under antibiotic stress conditions, quantitative membrane proteomic analyses of a clinical MDR A. baumannii strain cultured in subminimal inhibitory concentrations of tetracycline and imipenem were performed using a combination of label-free (one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry) and label (isobaric tag for relative and absolute quantitation) approaches. In total, 484 proteins were identified, and 302 were classified as outer membrane, periplasmic, or plasma membrane proteins. The clinical A. baumannii strain DU202 responded specifically and induced different cell wall and membrane protein sets that provided resistance to the antibiotics. The induction of resistance-nodulation-cell division transporters and protein kinases, and the repression of outer membrane proteins were common responses in the presence of tetracycline and imipenem. Induction of a tetracycline resistant pump, ribosomal proteins, and ironuptake transporters appeared to be dependent on tetracycline conditions, whereas β-lactamase and penicillin-binding proteins appeared to be dependent on imipenem conditions. These results suggest that combined liquid chromatography-based proteomic approaches can be used to identify cell wall and membrane proteins involved in the antibiotic resistance of A. baumannii. Keywords: Acinetobacter baumannii • multidrug-resistant • membrane protein • 1-DE-LC-MS/MS • iTRAQ
Introduction Multidrug-resistant (MDR) Acinetobacter baumannii is an important nosocomial pathogen, particularly in intensive care units of the hospitals.1,2 This opportunistic pathogen can colonize patients and cause a variety of human infections, including pneumonia, urinary tract infections, septicemia, and meningitis. A. baumannii was susceptible to the majority of antibiotics during the 1970s, but it readily developed resistance to virtually all available antimicrobial agents over time and MDR A. baumannii became prevalent in many hospitals.3 Hospital outbreaks of MDR A. baumannii and its control in hospital environments are of great concern in clinical settings. * To whom correspondence should be addressed. Seung Il Kim, Ph.D., Division of Life Science, Korea Basic Science Institute, 52, Yeoeun-Dong, Yusung-Ku, Daejeon, 305-333, South Korea. E-mail:
[email protected]. Fax: 8242-865-3419. † Division of Life Science, Korea Basic Science Institute. | Chungnam National University. ‡ Mass Spectronmetry Research Center, Korea Basic Science Institute. § Kyungpook National University School of Medicine. 10.1021/pr101012s
2011 American Chemical Society
Despite numerous reports concerning the epidemic spread of MDR A. baumannii, little is known about the mechanisms by which these organisms develop MDR and epidemicity.4 Several antimicrobial-resistance mechanisms of A. baumannii have been identified, including β-lactam hydrolysis,5 aminoglycoside modification,6 modification of antibiotic targets,7 active efflux,8 and changes in outer membrane proteins (OMPs).9,10 Several OMPs of A. baumannii strains have been identified and characterized as porins for the influx of drugs.9-13 Cell wall and membrane proteomic analyses have become hot issues because these proteins are responsible for antimicrobial resistance and pathogenicity. However, few reports exist concerning the identification and quantitative analysis of cell wall and membrane proteomes.11,14-16 The comparison of inner and outer membrane proteins between MDR A. baumannii A6 and reference strain A. baumannii ATCC19606T revealed that antibiotic resistance or virulence was highly associated with the differential expression of inner membrane proteins and OMPs.14 Recently, whole-genome sequencing of A. baumannii not only provided information about pathogenic determinants Journal of Proteome Research 2011, 10, 459–469 459 Published on Web 11/05/2010
research articles and antibiotic resistance, but also enabled proteomic research to clarify pathogenic mechanisms at the protein level.17-21 A clinical isolate, A. baumannii DU202 from a Korean hospital, exhibited MDR to many antibiotics, including ampicillin, cephalosporins, kanamycin, tetracycline, imipenem, and chloramphenicol.22 Our previous study using two-dimensional electrophoresis-tandem mass spectrometry (2-DE-MS/MS) demonstrated that the membrane expression of OMPs was decreased in A. baumannii DU202 in response to tetracycline, whereas the secretion of OMPs and periplasmic proteins was increased.23 Additionally, the secretion of outer membrane vesicles (OMVs) was first discovered in this strain.24 OMVs containing primarily OMPs, β-lactamase, and several cytoplasmic proteins were suggested to be vehicles for the secretion of effector molecules. Recently, various antibiotics have been tried to treat MDR A. baumannii infections.25-27 Carbapenems, polymyxins, tetracycline derivatives such as tigecycline are included in antibiotics as active ingredients for treatment of MDR A. baumannii. In the present study, we performed comparative analyses of the cell wall and membrane proteomes of A. baumannii DU202 cultured in Luria-Bertani (LB) medium supplemented with subminimal inhibitory concentrations (MICs) of imipenem or tetracycline, to characterize proteome regulation under antibiotic stress conditions. We selected the two antibiotics because imipenem is still an active antibiotic of carbapenems for treatment of MDR A. baumannii and tetracycline derivatives (minocycline and tigecycline) are considered as potentially active antibiotics. Even though most clinical A. baumannii including clinical strain DU202 are resistant to tetracycline, efflux pumps are common in resistance to tetracycline and its derivatives in A. baumannii. We combined two methods, one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry (1DE-LC-MS/MS) and isobaric tag for relative and absolute quantitation (iTRAQ), for the quantitative analysis of cell wall and membrane proteomes. In contrast to previous studies, the comparative analysis of A. baumannii DU202 was performed underdifferentcultureconditions(withorwithoutantibiotics).14,16 Through this study, we will have a better understanding of resistant mechanisms of MDR A. baumannii to active antibiotics in a proteomic perspective and our results show that the proteomes of A. baumannii are differentially regulated in the presence of antibiotics, which may be responsible for the antibiotic resistance of this bacterium.
Materials and Methods Bacterial Cultivation and Preparation of Protein Samples. A. baumannii DU202 was cultured in LB broth until reaching an optical density of 0.7-0.8 at 600 nm (OD600). The diluted bacteria, at about 1.0 × 105 colony-forming units (cfu)/ mL, were inoculated under three different culture conditions: LB broth with no antibiotic (control), LB broth supplemented with 500 µg/mL tetracycline, and LB broth supplemented with 50 µg/mL imipenem. The bacteria were grown at 37 °C with shaking at 180 rpm until reaching an OD600 of 0.7-0.8 (living cells in culture media were estimated at 1.0 × 109 to 2.0 × 109 cfu/mL). These cells were used for sample preparation. Soluble (cytosol) fractions were prepared according to previously described methods.22 Preparation of membrane-associated (membrane and cell wall) fractions was performed according to modified methods described by Molloy et al.28 The cell pellet was washed three times with 50 mM Tris-HCl (pH 8.0) and 460
Journal of Proteome Research • Vol. 10, No. 2, 2011
Yun et al. collected by centrifugation at 115 000g for 20 min. The protein content was quantified by the BCA method.29 To maximize protein induction by antibiotics, A. baumannii DU202 cultured in LB supplemented with tetracycline (500 µg/mL) or imipenem (50 µg/mL) was subcultured more than 10 times under the same antibiotic conditions and then harvested for comparative proteome analysis according to the procedure described by Xu et al.30 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and In-Gel Digestion. Protein samples were fractionated by 12% SDS-PAGE (Mini-Protean; Bio-Rad Laboratories, Inc., Hercules, CA) according to a previously described method.22 The gels were stained with Coomassie brilliant blue (CBB) and each 15 µg of protein sample was loaded on the gel. The gel lanes containing the membraneassociated fractions were sliced into 15 fractions based on molecular weight (MW; Figure 1), and the gels slices were prepared for digestion with trypsin (Promega, Madison, WI). The gel slices were destained in a solution of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate,31 rinsed more than three times with distilled water, and rinsed with 100% acetonitrile (ACN) to stop the destaining reaction. After drying in a speed vacuum concentrator (Model Spin 31; Hanil Co., Ltd., Seoul, Korea), the slices were incubated with 10 mM dithiothreitol and 100 mM ammonium bicarbonate at 56 °C to reduce disulfide bonds in the sample proteins. For the alkylation of cysteines, 55 mM iodoacetamide was added. The slices were then washed with 2 or 3 vol of distilled water by vortexing and were dried completely in a speed vacuum concentrator. After suspending the gels in 30 µL of 50 mM ammonium bicarbonate, 7-8 µL of trypsin solution (0.1 µg/ µL) was added, and tryptic digestion was carried out at 37 °C for 12-16 h. The tryptic peptides were extracted using 50 mM ammonium bicarbonate, followed by 50% ACN containing 5% trifluoroacetic acid (TFA). For 1-DE-LC-MS/MS analysis, each peptide extract was lyophilized in a vacuum concentrator and then dissolved in 0.5% TFA. For multidimensional protein identification technology (MudPIT), the tryptic peptide extracts were pooled, lyophilized in a vacuum concentrator, and dissolved in 0.5% TFA solution for 2D-LC fractionation. LC-MS/ MS and MudPIT analysis procedure were summarized in Figure 2. LC-MS/MS Analyses. A 10-µL sample of tryptic peptides was concentrated on a MGU-30 C18 trapping column (LC Packings, Amsterdam, The Netherlands) and eluted directly onto a C18 reverse-phase (RP) column (10 cm ×5 µm i.d.; Proxeon Biosystems, Odense, Denmark) at a flow rate of 120 nL/min. Peptides were eluted using a gradient of 0-65% ACN for 70 min. All MS and MS/MS spectra were acquired using a Thermo Finnigan LTQ FT mass spectrometer (San Jose, CA). Each full MS scan (m/z range of 400-2000) was followed by three MS/ MS scans of the most abundant precursor ions in the MS spectrum, with the dynamic exclusion feature enabled. Protein identification and quantification were performed using Mascot version 2.2 software (Matrix Science, Inc., Boston, MA). The A. baumannii ATCC 17978 protein database Uniprot site (www. uniprot.org) was used for the analysis of the MS/MS data. Carbamidomethylation of cysteine, oxidation of methionine, and propionamidation of cysteine were considered variable protein modifications. For MudPIT, the pooled tryptic peptides were loaded by an autosampler (Thermo Finnigan) onto a microcapillary column (300 × 0.25 mm) packed with strong cation-exchange (SCX) material (polysulfethyl A, 5-mm par-
Proteomic Analysis of Cell Wall/Membrane of A. baumannii
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Figure 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of A. baumannii DU202 cultured in LB medium supplemented with imipenem or tetracycline. (A) Soluble (cytosol) (1) and membrane-associated (cell wall and membrane) fractions (2) of A. baumannii DU202 cultured in LB medium supplemented with imipenem. (B) Membrane fraction of A. baumannii DU202 subcultured >10 times in LB medium (1), LB medium supplemented with tetracycline (2), or LB medium supplemented with imipenem (3).
Figure 2. Analytical scheme for quantitative analysis of membraneassociated protein fraction from A. baumannii DU202.
ticles, 300 Å pore size (Poly LC, Columbia, MD)). The peptides were eluted from the SCX column with an ammonium acetate step gradient (0, 50, 250, and 500 mM) and trapped on MGU30 C18 trap columns (LC Packings). The subsequent steps of the MS/MS analyses were same as for the LC-MS/MS analyses. iTRAQ. For iTRAQ of membrane protein fractions, protein samples were separated by SDS-PAGE and digested with trypsin as described above. After in-gel digestion, the solutions containing the digested peptides were acidified by addition of TFA to a final concentration of 0.1% and loaded onto ZipTip micropipet tips (Millipore Corp., Billerica, MA). The ZipTip micropipet tips were washed with 50 µL of 0.1% TFA, and the tryptic peptides were eluted with 7 µL of 60% ACN/0.1% TFA and vacuum-dried. The dried peptides were dissolved in 20µL reaction solutions containing 6 µL of 500 mM triethylammonium bicarbonate (TEAB), 9 µL of ethanol, and 5 µL of
iTRAQ reagents (114, 115, 116, and 117; Applied Biosystems, Inc., Foster City, CA). Proteins from the bacteria cultured in LB were used as a control and were labeled with iTRAQ reagents 116 and 117. The proteins from the imipenem- or tetracycline-treated bacteria were labeled with iTRAQ reagents 114 and 115, respectively. After 1 h of incubation at room temperature, the samples were mixed in a 1:1:1:1 ratio (v/v/ v/v) and vacuum-dried. The iTRAQ-labeled peptide mixtures were separated on SCX resin (polysulfethyl A, 5-mm particles, 300 Å pore size (Poly LC, Columbia, MD)) packed in silica tubing (3 cm × 250 µm i.d.) and on a C18 RP column, consisting of C18 resin packed into silica tubing (10 cm × 75 µm i.d.). The peptides were eluted with a six-step ammonium acetate gradient (0, 25, 50, 125, 250, and 500 mM). At each step, a salt plug was loaded onto the SCX column for peptide elution. The peptide fractions were collected onto trap columns for concentration and desalting. The peptides were eluted with 0% ACN and 80% ACN containing 0.1% TFA as mobile phases A and B, respectively, according to the following gradient: 5% B for 5 min, 5-20% B over 25 min, 20-60% B over 50 min, and 60-100% B over 5 min. At 20-s intervals, fractions were collected directly onto six 576-well matrix-assisted laser desorption/ionization (MALDI) plates, and R-cyano-4-hydroxycinnamic acid (CHCA; 5 mg/mL in 70% ACN/0.1% TFA) was added to each well using a Probot MALDI spotting device (LC Packings). LC-MS/MS was performed using a MALDI-TOF/ TOF spectrometer (4700 Proteomics Analyzer; Applied Biosystems) integrated with a Famos autosampler, Switchos switching pump, and Probot MALDI spotting device (LC Packings), with SCX/RP columns. Mass spectra were obtained using a neodymium-doped yttrium-aluminum-garnet (Nd:YAG) laser (355 nm, 200 Hz). Scans were performed in the reflector-positive mode with an acceleration voltage of 25 kV and an m/z range Journal of Proteome Research • Vol. 10, No. 2, 2011 461
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Table 1. Identification of Total Proteins in A. baumannii DU202 Cultured in LB Medium Supplemented with Imipenem predicted localization (CELLO v 2.5)
membrane-associated fraction
soluble fraction
total
cytoplasm inner membrane periplasmic space outer membrane extracellular no. of identified proteins
91 28 67 51 11 248
471 17 65 32 12 597
487 35 95 63 19 699
of 800-4000. MS/MS spectra were acquired using the datadependent acquisition method with 10 precursor ions selected from a previous reflector MS scan with 1-kV collision energy. Protein identification and quantification were performed using Mascot version 2.2 software (Matrix Science), as described above for the LC-MS/MS analysis. The peptide and fragment mass tolerances were 0.2 and 0.3 Da (Da), respectively. The iTRAQ 4-plex method was used for quantification. Proteins with a p-value e 0.05 were considered for identification. The resultant proteins were calculated to have a false discovery ratio (FDR) of 1024 µg/mL) and imipenem (>128 µg/ mL).22 The DU202 strain was grown in LB medium supplemented with tetracycline (500 µg/mL) or imipenem (50 µg/mL). Total Protein Identification in A. baumannii DU202. Before comparative proteomic analysis was performed under antibiotic stress conditions, we tried to identify the total proteome of A. baumannii DU202 cultured in LB medium supplemented with imipenem. The soluble protein (cytosol) fraction was prepared by centrifugation after cell disruption, and the membrane-associated (cell wall and membrane) protein fraction was prepared by sodium carbonate precipitation. Proteins from the two fraction types were separated by SDS-PAGE (Figure 1A). The results of the 1-DE-LC/MS-MS analysis are summarized in Table 1. In total, 597 and 248 proteins were identified from the soluble and membrane-associated fractions, respectively. Of these, 146 proteins were simultaneously identified from both fractions; thus, the total number of identified proteins was 699, which represents about 18.4% of the total genome of A. baumannii. Among the identified proteins from the membrane-associated fraction, about 59% (146 proteins) were OMPs, inner membrane proteins, or periplasmic proteins based on the subcellular location prediction program Cello version 2.0. To reduce the complexity and focus on cell wall 462
Journal of Proteome Research • Vol. 10, No. 2, 2011
and membrane proteins, we selected the membrane-associated fraction for the quantitative analysis of cell wall and membrane proteins. Comparative Analysis of Membrane-Associated Protein Fraction of A. baumannii Using Label-Free Approaches. The membrane-associated fraction was prepared from A. baumannii cultured in LB alone, LB supplemented with tetracycline, and LB supplemented with imipenem. The proteins were separated by SDS-PAGE, and the gels were sliced into 15 pieces based on MW (Figure 1B). LC-MS/MS analysis was performed twice, and proteins detected from both analyses were assigned according to culture conditions. The identified proteins differed according to culture conditions. A comparative analysis of proteins identified by 1-DE-LC-MS/MS analysis showed that 225 proteins were induced in all three conditions. However, 111, 17, and 26 proteins were specifically induced in the presence of tetracycline, imipenem, and LB only, respectively. For the quantitative analysis, the concentration of each protein was calculated using the exponentially modified protein abundance index (emPAI),34 and the emPAI values were converted into mole-percent (mol %) values to calculate the protein content. The relative concentrations of proteins induced in the presence of tetracycline or imipenem were compared with the protein concentrations induced in the absence of antibiotic (as a control). As expected, the protein identification yield by MudPIT analysis was lower than that by 1-DE-LC-MS/MS analysis (Table 2), owing to the difference in the complexity of the tryptic peptides analyzed. For MudPIT, whole tryptic peptide mixtures were pooled and injected into ion-exchange columns. In contrast, for 1-DE-LC-MS/MS, whole proteins were fractionated by SDS-PAGE, each fraction was trypticdigested, and each tryptic peptide mixture was separately injected into a RP column for MS/MS analysis. The results of the 1-DE-LC-MS/MS and MudPIT analyses are summarized in Table 2; the complete data of the 1-DE-LC-MS/MS and MudPIT analysis results are listed in the Supplementary Table 1. Comparative Analysis of Membrane-Associated Protein Fraction of A. baumannii by iTRAQ. iTRAQ was used as a labeling method for the quantitative analysis of cell wall and membrane fractions. The 71 proteins identified as proteins commonly induced under the three different conditions were quantitatively compared (Figure 3). The quantification results of these 71 proteins are presented in Table 3 and also in Supplementary Table 1. A sample of mass spectra (putative OMP, A1S 0884) is shown in Supplementary Figure 1. Protein variations were more evident in the tetracycline growth condition. Cell wall or membrane proteins accounted for 49 (69%) of the 71 proteins that were analyzed (Table 2). Proteomic Characterization of Membrane-Associated Protein Fraction from A. baumannii DU202 Cultured in Tetracycline. 1. AdeABC and Putative Resistance-Nodulation-Cell Division (RND) Family Transporters. AdeA (A1S_1751, 1752) and AdeB (A1S_1750) were upregulated in the presence of tetracycline, which suggests that the AdeABC efflux pump works to resist a high concentration of tetracycline (Table 3). The AdeABC efflux pump was associated with resistance to tetracycline, β-lactam, fluoroquinolone, and tigecycline in a MDR A. baumannii strain.35 AdeK (A1S_2737), another identified upregulated protein, is an AdeC-like protein and a member of the second RND-type efflux pump (AdeIJK) family of clinical A. baumannii strains.36 This result suggests that AdeABC and AdeIJK cooperate to play a primary role in pumping tetracycline
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Proteomic Analysis of Cell Wall/Membrane of A. baumannii
Table 2. Summary of Proteomic Results for Membrane-Associated Protein Fraction of A. baumannii DU202 Cultured in LB Medium, LB Medium Supplemented with Tetracycline, or LB Medium Supplemented with Imipenema analysis method:
1DE-LC-MS/MS
samples:
predicted localization (CELLO version 2.5)
transmembrane prediction (TMHMM version 2.0)
cytoplasm inner membrane periplasmic space outer membrane extracellular 0 1 2 3 4 5 g6
no. of identified proteins
MudPIT-MS/MS
LB
TC
IM
LB
TC
IM
iTRAQ
total
74 84 58 61 22 178 42 16 10 5 7 41 299
124 116 74 68 20 240 58 17 19 7 10 51 402
81 86 53 50 18 165 42 14 12 6 6 43 288
40 43 40 39 18 109 30 9 7 1 1 23 180
73 69 45 43 14 142 39 12 10 5 8 28 244
40 41 33 28 13 90 26 10 6 3 4 16 155
13 14 15 20 9 48 8 5 3
152 129 96 77 30 300 65 20 22 9 11 57 484
1 6 71
a
Abbreviations: IM, imipenem; LB, Luria-Bertani; TC, tetracycline; 1DE-LC-MS/MS, one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry; iTRAQ, isobaric tag for relative and absolute quantitation; MudPIT-MS/MS, multidimensional protein identification technology-tandem mass spectrometry.
Figure 3. Differential protein identification by label and label-free proteomic methods.
out of A. baumannii DU202. In contrast to the Ade efflux system, the β-lactamases (A1S_1517 and A1S_2367) were strongly downregulated in the presence of tetracycline, compared with LB alone (Table 3). Additionally, 11 RND transporters (A1S_0255, 0535, 0537, 0538, 0908, 1242, 1243, 2618, 2619, 2620, and 2736) were upregulated (Table 3), suggesting the global expression of RND transporters was induced in A. baumannii DU202 to overcome high concentrations of tetracycline. Other upregulated transporters were outer membrane lipoproteins (A1S_2613 and 2611) and toluene tolerance efflux transporters (A1S_3101 and 3102). A. baumannii ATCC 17978, which was used as a proteome DB, has putative a TetA efflux pump, tetA(A) (A1S_3117). However, we could not detect tetracycline resistant transporters. PCR screening of other tetracycline resistant genes revealed that A. baumannii DU202 contains tetA(B) and tetA(G1) instead of tetA(A) (Supplementary Figure 2A). Therefore, we reanalyzed the proteomic mass data using tetracycline resistant genes obtained from the Uniprot database (http://uniprot.org) as a new database and found the TetA(B) was induced in only in the tetracycline condition (Supplementary Figure 2C). However, an estimated amount of TetA, which was based on emPAI, was very small (about 0.05 mol %). Another reason for the difficulty in detection of TetA may be due to hydrophobicity of MFS TetA. Normally, MFS TetA efflux pumps have 12-10 transmembrane (TM) domains, respectively. Therefore, more efficient method for detection of proteins with multitransmembrane domains should be applied. Likewise, the tetracycline resistant proteins were not detected
in the previous proteomic analysis of Pseudomonas aeruginosa and Escherichia coli.30,37 2. BfmS. BfmS (A1S_0749) was strongly induced in the presence of tetracycline and imipenem (Table 3). BfmS contains 549 amino acids and three domains (HATPase, HisKA, and HAMP). It is a putative sensory histidine kinase of the novel two-component regulatory system BfmSR.38 Although BfmR plays a role in the morphology and formation of biofilms through the Csu pili chaperone-usher assembly system, BfmS is not related to biofilm formation and its function is not known. 3. Ferric Enterobactin Receptor. Iron is important for maintaining basic metabolic functions in bacteria. Ferrous iron acquisition is essential for bacterial infection and intracellular survival, as the concentration of free iron in a host is strictly limited. Therefore, iron acquisition systems have developed in pathogenic bacteria. Two enterobactin receptors (A1S_0980 and 0981) were upregulated in A. baumannii DU202 at a high concentration of tetracycline (Table 3). Enterobactin receptors have been shown to be responsible for iron acquisition.39 Other upregulated iron transport proteins were putative ferrous iron transporter protein B (A1S_0243), ferric hydroxamate siderophore receptor (A1S_1667), and putative ferric siderophore receptor protein (A1S_3339). 4. Ribosomal Proteins and RNA Polymerase. Tetracycline inhibits bacterial growth by interfering with protein synthesis at the ribosomal level. Although the affected proteins are soluble proteins found primarily in the cytosol fraction, A. baumannii DU202 responded to tetracycline stress by upregulating RNA polymerase and ribosomal proteins in cell wall and membrane fractions (Table 3). Thus, these results indicated that protein synthesis assembly was upregulated in A. baumannii DU202 in response to a high tetracycline concentration in order to overcome protein synthesis inhibition. RNA polymerase and ribosomal proteins were not significantly induced in the presence of imipenem (Table 3). 5. ATP Synthase and Related Proteins. ATP synthase (A1S_0148 and 0153) was upregulated in the presence of tetracycline. Succinate dehyrogenase subunits (A1S_2713 and 2714), cytochrome oxidase subunits (A1S_2166 and 2167), and malate:quinone oxidoreductase were also increased in the presence of tetracycline. As these proteins are involved ATP Journal of Proteome Research • Vol. 10, No. 2, 2011 463
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Journal of Proteome Research • Vol. 10, No. 2, 2011
A1S_2367
A1S_1517
A1S_2613
A1S_2611
A1S_3102
A1S_3101
A1S_2736
A1S_2620
A1S_2619
A1S_2618
A1S_1243
A1S_1242
A1S_0908
A1S_0538
A1S_0537
A1S_0535
A1S_0255
A1S_1754 A1S_2735 A1S_2737
A1S_1752
beta-lactamase OXA-95 Beta-lactamase
toluene tolerance efflux transporter toluene tolerance efflux transporter transport protein of outer membrane lipoproteins transport protein of outer membrane lipoproteins
putative RND family drug transporter putative RND family drug transporter putative RND family drug transporter putative RND family drug transporter RND family multidrug resistance secretion protein putative ABC family drug transporter putative RND family drug transporter putative RND family drug transporter putative RND family drug transporter putative RND family drug transporter RND family drug transporter
AdeB AdeA membrane fusion protein AdeA membrane fusion protein sensor protein AdeI AdeK
A1S_1750 A1S_1751
A1S_0049
sensor protein (BfmS) protein tyrosine kinase
protein name
A1S_0749
gene accession no. MW (Da)/pI
60222/5.91 81391/6.84
112565/8.07 15714/5.46 18396/9.3 40739/6.03 43649/9.11 52770/9.02 50182/8.97 51161/8.32 8612/9.98 31381/8.57 40932/6.56
79375/8.55 44260/7.75 17998/5.08 27913/6.53 41285/8.42 114478/6.11
24099/5.02 27290/6.08 25083/6.06 27362/6.89
30579/8.43 43110/9.37
gene name
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
periplasmic space
periplasmic space
periplasmic space
cytoplasm
inner membrane
extracellular
inner membrane
outer membrane
extracellular
outer membrane
outer membrane
inner membrane
outer membrane
extracellular
extracellular
outer membrane
outer membrane
inner membrane outer membrane outer membrane
periplasmic space
inner membrane cytoplasm
inner membrane
cytoplasm
cellular locationb
signal peptided
Ade
Y Y Y
N
N N
N
0
0
0
0
5
1
11
1
0
0
1
3
1
0
0
0
0.406
0.041
Lactamase Y Y
0.029
0.031 N
N
0.062
0.350
Transporter Y N
0.159
0.050
0.046
0.045
0.212
0.046
0.018
0.038
-
0.022
-
0.010 0.426 1.561
0.068
0.033 0.028
0.438
0.025
LB
N
N
N
Y
N
N
N
N
N
Y
RND Transporter 0 Y
2 0 0
0
12 0
2
Protein Kinase 1 N
transmembranec
0.298
0.023
0.047
0.077
0.111
0.323
0.244
0.141
-
-
0.265
0.032
0.082
0.061
0.162
0.067
-
0.017 0.568 0.938
0.159
0.052 0.407
0.716
0.080
IM
0.058
-
0.228
0.150
0.116
1.112
0.230
0.148
0.589
0.289
0.640
0.046
0.042
0.128
0.288
0.126
0.039
0.014 0.258 5.055
0.169
0.062 0.249
0.401
0.120
TC
Mol% by 1DE-LC-MS/MSe
0.909
0.112
-
-
-
0.505
0.177
-
0.124
-
-
0.067
-
-
-
-
-
0.539 2.699
-
0.018 -
0.191
-
LB
0.934
-
0.200
-
-
0.487
0.194
-
-
-
-
0.037
-
-
-
-
-
0.494 2.009
-
0.028 -
0.378
0.053
IM
-
-
0.126
-
0.126
3.036
0.311
0.050
0.123
-
0.046
0.196
-
-
-
-
-
0.537 3.445
0.123
0.066 0.256
0.461
0.071
TC
Mol% by MudPIT-MS/MSe
Table 3. Representative Proteins of Membrane-Associated (Cell Wall and Membrane) Fractions Differentially Expressed at Sub-MICs of Tetracycline and Imipenema
0.603
-
-
-
-
-
1.41
-
-
-
-
-
-
-
-
-
-
1.36 0.814
-
-
-
-
IM/LB
0.249
-
-
-
-
-
1.314
-
-
-
-
-
-
-
-
-
-
1.586 1.421
-
-
-
-
TC/LB
ratio by iTRAQ
research articles Yun et al.
A1S_3339
A1S_1667
A1S_0981
A1S_0980
A1S_0243
A1S_3317
A1S_3297
A1S_0170
A1S_2849
A1S_0884
A1S_2840
A1S_2538
A1S_0292
A1S_3204
A1S_2435
A1S_3197
A1S_3196
gene accession no.
putative ferrous iron transport protein B ferric enterobactin receptor ferric enterobactin receptor putative ferric hydroxamate siderophore receptor putative ferric siderophore receptor protein
putative outer membrane protein putative glucose-sensitive porin (OprB-like) putative outer membrane copper receptor (OprC) putative outer membrane protein putative outer membrane protein
putative outer membrane protein W outer membrane protein CarO outer membrane protein omp38
putative penicillin-binding protein (PonA) putative penicillin-binding protein (PonA) D-ala-D-alacarboxypeptidase; penicillin-binding protein 5 (Precursor) septum formation penicillin-binding protein 3, peptidoglycan synthetase
protein name
Table 3. Continued
26273/4.92
-
76362/5.87 32119/4.77 27663/4.59
67472/6.57 57655/5.35 19417/8.73 80591/5.38
81223/5.75
-
-
-
-
-
-
-
-
46789/5.54
21199/5.56
-
-
67617/9.78
-
22472/9.3
41756/8.22
-
-
38964/8.33
-
38427/5.32
52322/9.54
-
omp38
MW (Da)/pI
gene name
outer membrane
outer membrane
Extracellular
outer membrane
inner membrane
outer membrane
outer membrane
outer membrane
outer membrane
extracellular
outer membrane
outer membrane
signal peptided LB
0.247
Y
Y
Y
Y
0
0
0
0
Y
Y
N
N
Fe Binding Protein 11 N
0
0
0
1
-
-
-
0.038
0.049
2.137
2.498
0.267
0.679
-
-
-
0.012
0.036
1.506
0.948
0.394
0.538
9.230
0.152
Putative Outer Membrane Protein 0 Y 14.254
Y
Y
2.674
1
0
1.344
0.312
0.485
Outer Membrane Protein 0 Y 1.256
0.197
0.139
0.244
N
Y
N
1.935
IM
0.183
1
0
0
Penicillin-Binding Protein 0 N 0.337
transmembranec
2.616
periplasmic space
periplasmic space
periplasmic space
periplasmic space
periplasmic space
cellular locationb
0.060
0.072
0.349
0.353
0.118
0.636
0.552
0.059
0.272
3.827
1.355
0.105
1.298
0.292
0.320
0.155
0.486
TC
Mol% by 1DE-LC-MS/MSe
-
-
-
-
-
0.406
1.976
0.071
1.189
16.293
3.326
0.193
0.627
0.101
0.376
0.085
0.060
LB
-
-
-
-
-
0.122
1.978
0.084
0.700
17.383
7.492
0.212
0.422
0.159
0.625
0.187
0.131
IM
0.025
-
0.290
0.386
0.101
-
1.099
0.055
0.610
4.496
2.092
0.132
0.256
0.190
0.436
0.085
0.085
TC
Mol% by MudPIT-MS/MSe
-
-
1.247
-
-
0.802
0.638
1.153
0.612
0.776
0.793
0.768
0.644
-
-
-
1.416
IM/LB
-
-
2.791
-
-
0.439
0.513
0.711
0.667
0.453
0.497
0.629
0.626
-
-
-
1.041
TC/LB
ratio by iTRAQ
Proteomic Analysis of Cell Wall/Membrane of A. baumannii
research articles
Journal of Proteome Research • Vol. 10, No. 2, 2011 465
466
Journal of Proteome Research • Vol. 10, No. 2, 2011
phospho-Nacetylmuramoylpentapeptide transferase
ATP synthase subunit A ATP synthase subunit R
succinate dehydrogenase flavoprotein subunit succinate dehydrogenase iron-sulfur subunit cytochrome o ubiquinol oxidase subunit II cytochrome o ubiquinol oxidase subunit I probable malate:quinone oxidoreductase
A1S_3200
A1S_0148
A1S_2713
A1S_0923
A1S_2167
A1S_2166
A1S_2714
A1S_0153
A1S_3079
A1S_3076
A1S_3075
A1S_3074
A1S_3069
A1S_3064
A1S_3062
A1S_3057
A1S_3055
A1S_2731
A1S_2323
A1S_0867
A1S_0597
DNA-directed RNA polymerase subunit beta 50S ribosomal protein L20 30S ribosomal protein S7 30S ribosomal protein S2 50S ribosomal protein L21 50S ribosomal protein L17 30S ribosomal protein S4 50S ribosomal protein L15 30S ribosomal protein S5 50S ribosomal protein L5 50S ribosomal protein L16 30S ribosomal protein S3 50S ribosomal protein L22 50S ribosomal protein L4
protein name
A1S_0288
gene accession no.
Table 3. Continued
26786/7.46
38851/6.53 74492/6.6
-
60397/9.07
66955/5.94
-
mqo-
55363/5.29
atpA
32421/5.89
12908/10.27
-
atpB
21539/9.74
11799/10.2
-
rplD
27904/10.36
15457/11
20008/9.71
17246/10.11
15472/10.95
23255/10.09
13986/10.95
11467/10.07
11221/4.3
17688/10.13
13429/11.63
154954/6.9
MW (Da)/pI
rpsC
rplP
rplE
rpsE
rplO
rpsD
rplQ
rplU
-
rpsG
rplT
rpoC
gene name
signal peptided LB
N
N
N
N
N
N
N
N
N
N
N
N
N
0.232
0.092
periplasmic space
inner membrane
inner membrane
cytoplasm
periplasmic space
cytoplasm
1
15
3
Y
N
Y
N
0.480
0.093
0.997
1.857
1.261
0.185
1.031
1.650
4.263
Krebs Cycle and Respiratory 0 N 2.342
0
0.621
N
0.406
0
0.254
0.180
0.121
0.155
0.442
0.708
0.118
0.523
ATP Synthesis or Proton Pumping inner membrane 5 N 0.159
0.171
0.093
0.169
0.208
0.750
0.074
0.176
0.609
0.118
0.313
0.727
0.168
0.968
0.444
0.142
0.490
0.165
Peptidoglycan Synthesis 3 N
0
0
0
0
0
0
0
0
0
0
0
0
0
0.161
IM
2.971
0.125
1.208
2.165
4.045
0.527
0.210
0.120
0.385
0.490
0.563
0.374
4.484
0.268
0.440
0.654
0.972
0.644
1.655
0.434
0.476
0.339
TC
Mol% by 1DE-LC-MS/MSe
0.029
inner membrane
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
cytoplasm
periplasmic space
cytoplasm
transmembranec
Ribosomal Proteins and RNA Polymerase 0 N 0.076
cellular locationb
0.220
0.073
0.494
0.801
1.290
0.216
0.149
0.309
0.087
0.972
0.903
2.331
0.159
0.237
-
-
-
-
0.144
0.519
0.684
0.388
0.317
0.264
0.653
-
0.528
-
-
0.500
-
0.594
0.089
0.658
1.380
2.912
0.183
0.318
-
0.251
2.076
0.738
0.263
4.306
0.345
0.263
0.411
1.001
0.404
1.588
0.870
1.193
1.083
0.055
TC
0.531
0.028
IM
0.303
-
0.230
0.220
0.321
0.018
LB
Mol% by MudPIT-MS/MSe
1.187
1.232
1.473
1.32
1.557
1.231
-
-
1.662
1.894
1.983
1.738
1.445
1.473
-
-
-
-
-
3.099
2.221
-
-
1.047
0.975
-
-
-
-
-
-
-
-
-
-
-
-
TC/LB
-
IM/LB
ratio by iTRAQ
research articles Yun et al.
outer membrane 81279/5.6 -
outer membrane 26712/4.61 -
extracellular 16751/6.82 -
cytoplasmic inner membrane cytoplasmic 38047/4.58 69620/5.76 15765/5.62 -
periplasmic space 19297/8.38 -
periplasmic space periplasmic space 15709/8.83 14351/9.04 -
extracellular outer membrane extracellular 12853/4.7 17981/9.42 20061/6.91 -
A1S_2829
A1S_3459
A1S_3785
A1S_0798 A1S_2681 A1S_2072
A1S_3143
A1S_1321 A1S_0894
putative lipoprotein putative lipoprotein peptidoglycan-associated lipoprotein putative hemolysin outer membrane lipoprotein superoxide dismutase [Cu/Zn] cell division protein zipA cell division protein putative universal stress protein family putative uncharacterized protein putative uncharacterized protein putative tonB-dependent receptor protein A1S_1009 A1S_2987 A1S_2595
a Abbreviations: Da, dalton; MW, molecular weight; N, no; RND, resistance, nodulation, and cell division; Y, yes. b Prediction of cellular location by CELLO version 2.5. c Prediction of the no. of transmembrane regions by TMHMM version 2.0. d Prediction of the presence of signal peptides by SignalP version 3.0. e Mol% was calculated according to emPAI values.
0.729 0.025 Y 0
0.053
0.006
0.016
0.037
-
0.722
Y 1
0.682
0.181
0.099
-
-
0.262 0.333 N 0
0.763
0.122
0.022
0.716
2.315 1.794 0.121 1.099 N N N 1 2 0
0.236 0.881 0.087
1.397 1.446
0.393 2.155 0.018
0.122 0.262 -
0.412 0.378 -
0.478 0.684 0.557 2.909 3.865 1.101 2.240 Y 0
7.046
Y Y 0 0
1.368 0.098
0.605 -
0.162 0.211
2.571 0.457
0.978 -
0.064 0.167
1.461 0.508 0.612 0.728 0.324 1.428 0.928 0.203 2.046 0.340 0.216 2.787 0.120 0.511 1.294 1.047 0.339 1.086 0.937 0.364 1.776 Y Y Y 0 1 0
TC/LB IM/LB TC IM LB TC IM LB
Other
MW (Da)/pI gene name protein name gene accession no.
Table 3. Continued
cellular locationb
transmembranec
signal peptided
Mol% by 1DE-LC-MS/MSe
Mol% by MudPIT-MS/MSe
ratio by iTRAQ
Proteomic Analysis of Cell Wall/Membrane of A. baumannii
research articles synthesis or proton gradient maintenance in cells, these results suggest that more energy is required to resist high tetracycline concentrations. 6. OMPs. Porins play key roles in outer membrane permeability. Several OMPs (OmpA, CarO, and OmpW) were reported to be depressed in the presence of tetracycline or imipenem.10,13,16,23 Our results demonstrate that these proteins as well as other OMPs (A1S_0884, 3297, and 3317) were downregulated in response to tetracycline (Table 3). Although the reason for the downregulation of OMPs under antibiotic conditions is not clear, the downregulation of OMPs can decrease the intake of antibiotics. In our previous study, secretion of OMPs was increased in proportion to the concentration of tetracycline in A. baumannii DU202.23 Recent data revealed that OMVs, whose primary components are OMPs (A1S_2840, 0884, 3297, and 2849) and β-lactamase (A1S_2367), are released from A. baumannii DU202.24 However, RND transporters, except for AdeK and a RND-type efflux pump (A1S_0009), were not detected in the OMVs.24 In the present study, 11 RND transporters were identified in the cell wall or membrane fraction (Table 3). These findings suggest that the components, especially OMPs, of OMVs are specifically selected and regulated. We are currently searching for a possible role of OMVs in regulating OMPs on the cell surface in response to antibiotics. Downregulated OMPs included the putative glucosesensitive porin (OprB-like; A1S_2849), putative outer membrane copper receptor (OprC; A1S_0170), and two uncharacterized proteins (A1S_3297 and 3317). Additionally, the levels of Cu/ Zn superoxide dismutase (A1S_3143), putative tonB-dependent receptor protein (A1S_2829), putative lipoprotein (A1S_1009), and putative hemolysin (A1S_1321) were significantly decreased, while the levels of cell division protein (A1S_2681) and putative membrane-associated Zn-dependent protease 1 (A1S_1970) were increased in the presence of tetracycline. Proteomic Characterization of Membrane-Associated Protein Fraction from A. baumannii DU202 Cultured in Imipenem. Imipenem is an important carbapenem, a member of the β-lactam family of antibiotics. It has been widely used for the treatment of MDR A. baumannii infection and acts by inhibiting cell wall synthesis.16 Relatively fewer proteins were induced in the presence of imipenem (n ) 288) than with tetracycline (n ) 402). The number of commonly induced proteins under the two conditions was 259. Although the expression patterns of major membrane proteins under the two culture conditions were generally similar, several differences existed (Table 3). Levels of antibiotic resistance-related transporters (Ade, RND, and other transporters) were increased with imipenem, but slightly less so than in the presence of tetracycline. In contrast to the marked decrease in β-lactamase (A1S_2367) in the presence of tetracycline, induction of β-lactamase was noticeable in the presence of imipenem (Table 3). These results suggest that β-lactamase and transporters work cooperatively to achieve resistance to imipenem in A. baumannii DU202. Penicillin-binding proteins (A1S_3196, 3197, and 2435) were specifically increased in the presence of imipenem. As all β-lactam antibiotics bind to penicillin-binding proteins to prevent cell wall synthesis, the increase in penicillinbinding proteins was a response of A. baumannii DU202 to imipenem. Expression of phospho-N-acetylmuramoyl-pentapeptide-transferase (A1S_3200), which participates in peptidoglycan biosynthesis, was also increased in the presence of imipenem. The induction of some OMPs (A1S_0292, 2538, 2840, 0884, and 3317) was decreased in the presence of imipenem. Journal of Proteome Research • Vol. 10, No. 2, 2011 467
research articles The iTRAQ results showed that average decrease of eight representative OMPs was 46.7% in the presence of tetracycline and 22.7% in the presence of imipenem (Table 3). Further investigation is required to clarify the differences in protein induction. One possible explanation is the difference in the concentrations of the antibiotics. Additionally, proteins induced in the presence of imipenem include the cell division protein zipA (A1S_0798), the putative universal stress protein family (A1S_2072), and protein tyrosine kinase (A1S_0049). Taken together, these results reveal the existence of common and specific antibiotic resistance mechanisms used in response to imipenem and tetracycline, respectively, in A. baumannii DU202.
Conclusions Quantitative analysis of the membrane-associated fraction of A. baumannii DU202 was performed to identify antibiotic resistance-related proteins, using a label method (iTRAQ) and label-free methods (LC-MS/MS and MudPIT). Among the 484 identified proteins in the membrane-associated fraction, 62.4% of proteins (302) were plasma membrane or cell wall proteins. A total of 302 membrane proteins were classified as outer membrane (77), inner membrane (129), and periplasmic proteins (96); this is the largest number of proteins identified by proteomic analysis of A. baumannii.14 Prediction of transmembrane domains by TMHMM revealed that proteins with 1-3 TM domains were most effectively enriched (Table 2). A comparison of the label-free methods (LC-MS/MS or MudPIT) and label method (iTRAQ) demonstrated that >80% of the proteins obtained using iTRAQ had induction or repression patterns similar to those of the proteins obtained using freelabel methods. Our approaches may be useful for the quantification of cell wall and membrane proteins and for the identification of membrane proteins undetectable by 2-DE. Carbapenems and tetracycline derivatives are active drugs to MDR A. baumannii isolates. However, resistance to carbapenems and tetracycline derivatives is recently increasing in A. baumannii. The reduced efficacy of tigecycline and carbapenem in A. baumannii is primarily mediated by an efflux-based mechanism, such as AdeABC,40 and production of carbapenemhydrolyzing β-lactamases, respectively. However, the role of cell wall and membrane proteins associated with resistance to both antibiotics has not been understood yet. In the present study, a clinical isolate A. baumannii DU202, which is resistant to imipenem, but susceptible to minocycline and tigecycline, was used. Common responses to tetracycline and imipenem were the induction of RND family transporters (e.g., AdeABC and AdeJIK), and a protein kinase (BfmS), as well as repression of OMPs (e.g., OmpA and OmpW). Induction of a tetracycline resistant pump, ribosomal proteins, and iron up-take transporters (e.g., ferric enterobactin receptor) were specific to tetracycline, whereas induction of β-lactamase and a peptidoglycan synthesis enzyme were specific to imipenem. This proteome regulation in the cell wall and membrane in MDR A. baumannii cultured in an antibiotic stress condition may be associated with resistance to active tetracycline derivatives, such as tigecycline, and other carbapenems. Our proteomic result may open new insight into a more comprehensive understating of resistant mechanisms of MDR A. baumannii to active anti-acinetobacter drugs. Abbreviations: 1-DE-LC-MS/MS, one-dimensional electrophoresis-liquid chromatography-tandem mass spectrometry; emPAI, exponentially modified protein abundance index; iTRAQ, 468
Journal of Proteome Research • Vol. 10, No. 2, 2011
Yun et al. isobaric tag for relative and absolute quantitation; MALDI TOF, matrix-assisted laser desportion/ionization time-of-flight; MDR, multidrug-resistance; MIC, subminimal inhibitory concentration; MS, mass spectrometry; MS/MS, tandem MS; MudPIT, multidimensional protein identification technology; OMP, outer membrane protein; OMV, outer membrane vesicle; RND, resistance, nodulation, and cell division; SCX, strong-cation exchange; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; TM, transmembrane domain.
Acknowledgment. This work was supported by a grant (K-MeP T30100) from the Korea Basic Science Institute. Supporting Information Available: Supplementary Table 1, total proteins of membrane-associated fraction expressed at inhibitory concentrations of tetracycline and imipenem. Supplementary Figure 1, an example of MS/MS spectra of iTRAQ labeled peptides from a putative outer membrane protein, A1S 0884. Supplementary Figure 2, screening of tetracycline resistance genes present in A. baumannii DU202 and proteomic verification. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Peleg, A. Y.; Seifert, H.; Paterson, D. L. Acinetobacter baumannii: emergence of a successful pathogen. Clin. Microbiol. Rev. 2008, 21 (3), 538–82. (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) Abbo, A.; Navon-Venezia, S.; Hammer-Muntz, O.; Krichali, T.; Siegman-Igra, Y.; Carmeli, Y. Multidrug-resistant Acinetobacter baumannii. Emerging Infect. Dis. 2005, 11 (1), 22–9. (4) Dijkshoorn, L.; Nemec, A.; Seifert, H. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol. 2007, 5 (12), 939–51. (5) Poirel, L.; Marque, S.; Heritier, C.; Segonds, C.; Chabanon, G.; Nordmann, P. OXA-58, a novel class D β-lactamase involved in resistance to carbapenems in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2005, 49 (1), 202–8. (6) Seward, R. J.; Lambert, T.; Towner, K. J. Molecular epidemiology of aminoglycoside resistance in Acinetobacter spp. J. Med. Microbiol. 1998, 47 (5), 455–62. (7) Vila, J.; Ruiz, J.; Goni, P.; Jimenez de Anta, T. Quinolone-resistance mutations in the topoisomerase IV parC gene of Acinetobacter baumannii. J. Antimicrob. Chemother. 1997, 39 (6), 757–62. (8) Ruzin, A.; Keeney, D.; Bradford, P. A. AdeABC multidrug efflux pump is associated with decreased susceptibility to tigecycline in Acinetobacter calcoaceticus-Acinetobacter baumannii complex. J. Antimicrob. Chemother. 2007, 59 (5), 1001–4. (9) del Mar Tomas, M.; Beceiro, A.; Perez, A.; Velasco, D.; Moure, R.; Villanueva, R.; Martinez-Beltran, J.; Bou, G. Cloning and functional analysis of the gene encoding the 33- to 36-kilodalton outer membrane protein associated with carbapenem resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2005, 49 (12), 5172–5. (10) Limansky, A. S.; Mussi, M. A.; Viale, A. M. Loss of a 29-kilodalton outer membrane protein in Acinetobacter baumannii is associated with imipenem resistance. J. Clin. Microbiol. 2002, 40 (12), 4776– 8. (11) Dupont, M.; Pages, J. M.; Lafitte, D.; Siroy, A.; Bollet, C. Identification of an OprD homologue in Acinetobacter baumannii. J. Proteome Res. 2005, 4 (6), 2386–90. (12) Magnet, S.; Courvalin, P.; Lambert, T. Resistance-nodulation-cell division-type efflux pump involved in aminoglycoside resistance in Acinetobacter baumannii strain BM4454. Antimicrob. Agents Chemother. 2001, 45 (12), 3375–80. (13) Mussi, M. A.; Limansky, A. S.; Viale, A. M. Acquisition of resistance to carbapenems in multidrug-resistant clinical strains of Acinetobacter baumannii: natural insertional inactivation of a gene encoding a member of a novel family of beta-barrel outer membrane proteins. Antimicrob. Agents Chemother. 2005, 49 (4), 1432–40.
research articles
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(27) Gordon, N. C.; Wareham, D. W. A review of clinical and microbiological outcomes following treatment of infections involving multidrug-resistant Acinetobacter baumannii with tigecycline. J. Antimicrob. Chemother. 2009, 63 (4), 775–80. (28) Molloy, M. P.; Herbert, B. R.; Slade, M. B.; Rabilloud, T.; Nouwens, A. S.; Williams, K. L.; Gooley, A. A. Proteomic analysis of the Escherichia coli outer membrane. Eur. J. Biochem. 2000, 267 (10), 2871–81. (29) Krieg, R. C.; Dong, Y.; Schwamborn, K.; Knuechel, R. Protein quantification and its tolerance for different interfering reagents using the BCA-method with regard to 2D SDS PAGE. J. Biochem. Biophys. Methods 2005, 65 (1), 13–9. (30) Xu, C.; Lin, X.; Ren, H.; Zhang, Y.; Wang, S.; Peng, X. Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline. Proteomics 2006, 6 (2), 462–73. (31) Kim, Y. H.; Cho, K.; Yun, S. H.; Kim, J. Y.; Kwon, K. H.; Yoo, J. S.; Kim, S. I. Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis. Proteomics 2006, 6 (4), 1301–18. (32) Yu, C. S.; Chen, Y. C.; Lu, C. H.; Hwang, J. K. Prediction of protein subcellular localization. Proteins 2006, 64 (3), 643–51. (33) Moller, S.; Croning, M. D.; Apweiler, R. Evaluation of methods for the prediction of membrane spanning regions. Bioinformatics 2001, 17 (7), 646–53. (34) Ishihama, Y.; Oda, Y.; Tabata, T.; Sato, T.; Nagasu, T.; Rappsilber, J.; Mann, M. Exponentially modified protein abundance index (emPAI) for estimation of absolute protein amount in proteomics by the number of sequenced peptides per protein. Mol. Cell. Proteomics 2005, 4 (9), 1265–72. (35) Nemec, A.; Maixnerova, M.; van der Reijden, T. J.; van den Broek, P. J.; Dijkshoorn, L. Relationship between the AdeABC efflux system gene content, netilmicin susceptibility and multidrug resistance in a genotypically diverse collection of Acinetobacter baumannii strains. J. Antimicrob. Chemother. 2007, 60 (3), 483–9. (36) Damier-Piolle, L.; Magnet, S.; Bremont, S.; Lambert, T.; Courvalin, P. AdeIJK, a resistance-nodulation-cell division pump effluxing multiple antibiotics in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2008, 52 (2), 557–62. (37) Peng, X.; Xu, C.; Ren, H.; Lin, X.; Wu, L.; Wang, S. Proteomic analysis of the sarcosine-insoluble outer membrane fraction of Pseudomonas aeruginosa responding to ampicilin, kanamycin, and tetracycline resistance. J. Proteome Res. 2005, 4 (6), 2257–65. (38) Tomaras, A. P.; Flagler, M. J.; Dorsey, C. W.; Gaddy, J. A.; Actis, L. A. Characterization of a two-component regulatory system from Acinetobacter baumannii that controls biofilm formation and cellular morphology. Microbiology 2008, 154 (Pt. 11), 3398–409. (39) Zeng, X.; Xu, F.; Lin, J. Molecular, antigenic, and functional characteristics of ferric enterobactin receptor CfrA in Campylobacter jejuni. Infect. Immun. 2009, 77 (12), 5437–48. (40) Peleg, A. Y.; Adams, J.; Paterson, D. L. Tigecycline efflux as a mechanism for nonsusceptibility in Acinetobacter baumannii. Antimicrob. Agents Chemother. 2007, 51 (6), 2065–9.
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Journal of Proteome Research • Vol. 10, No. 2, 2011 469
