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Extracellular proteome of a highly invasive multidrugresistant clinical strain of Acinetobacter baumannii Jose A. Mendez, Nelson da Cruz Soares, Jesus Martin Mateos, Carmen Gayoso, Carlos Rumbo, Jesús Aranda, Maria M. Tomas, and German Bou J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 11 Sep 2012 Downloaded from http://pubs.acs.org on September 13, 2012

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Journal of Proteome Research

Extracellular proteome of a highly invasive multidrug-resistant clinical strain of Acinetobacter baumannii

Jose Antonio Mendez1#, Nelson C. Soares1#, Jesús Mateos2, Carmen Gayoso1, Carlos Rumbo1, Jesús Aranda1, Maria Tomas1, Germán Bou1* #

1

Both authors contributed equally to this work

Laboratório de Microbiología. Instituto de Investigación Biomédica de A Coruña (INIBIC). Servicio de Microbiología. Complejo Hospitalario Universitario A Coruña (CHUAC), As Xubias s/n; La Coruña. Spain. 2Unidad de Proteómica. INIBIC, As Xubias s/n; La Coruña. Spain.

*To whom correspondence should be addressed. Germán Bou. Phone: +34 981176087 Fax: +34 981176097 Email: [email protected]

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Abstract The study of the extracellular proteomes of pathogenic bacteria is essential for gaining new insights into the mechanisms of pathogenesis and for the identification of virulence factors. Through the use of different proteomic approaches, namely Nano-LC and 2DE combined with MALDI-TOF/TOF, we have characterized the extracellular proteome of a highly invasive, multidrug-resistant strain of A. baumannii (clone AbH12O-A2). This study focused on two main protein fractions of the extracellular proteome: proteins that are exported by outer membrane vesicles (OMVs) and freely soluble extracellular proteins (FSEPs) present in the culture medium of A. baumannii. Herein, a total 179 non-redundant proteins were identified in the OMV protein fraction and a total of 148 non-redundant proteins were identified in FSEP fraction. Of the OMV proteins, 39 were associated with pathogenesis and virulence, including proteins associated with attachment to host cells (e.g. CsuE, CsuB, CsuA/B) and specialized secretion systems for delivery of virulence factors (e.g. P. pilus assembly and FilF), whereas the FSEP fraction possesses extracellular enzymes with degradative activity, such as alkaline metalloprotease. Furthermore, among the FSEP we have detected at least 18 proteins with a known role in oxidative stress response (e.g. catalase, thioredoxin, oxidoreductase, superoxide dismutase). Further assays demonstrated that in the presence of FSEPs, bacterial cells withstand much higher concentrations of H2O2 showing higher survival rate (approximately 2.5 fold) against macrophages. In this study we have identified unprecedented number of novel extracellular proteins of A. baumannii and we provide insight into their potential role in relevant processes such as oxidative stress response and defense against macrophage attack.

Keywords. Proteomics. Acinetobacter baumannii. Secretome. Outer membrane vesicles. Pathogenesis. Extracellular proteome. Virulence. 2-DE. MALDI-TOF/TOF


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Introduction Acinetobacter baumannii is a human pathogen that is often found in health care settings and is associated with several infectious diseases, including pneumonia, as well as urinary and bloodstream infections. Additionally, A. baumannii has recently been associated with cases of community acquired infections (usually among alcoholics),1,2 war-related injuries and survivors of natural disasters (e.g. survivors of the Asian tsunami in 2004).3 A. baumannii has acquired resistance to a wide spectrum of antibiotics used in clinical practice, which often makes treatment extremely difficult and the eradication of this bacterium from the healthcare environment almost impossible. The increasing number of cases of enhanced virulence of the MDR (multi-drug-resistant) A. baumanii is also of great concern.4,5 In spite of this, and although the epidemiology and antibiotic resistance of A. baumannii have been extensively revised, there remains an alarmingly large gap in our knowledge with regards to the pathogenesis and nature of the A. baumannii virulence. It has been suggested that a battery of bacterial virulence factors are required for pathogenic infections caused by A. baumannii.6 Consistent with this belief, Smith et al,.7 presented evidence suggesting that a large portion of the A. baumannii genome harbors a number of genes associated with virulence. It has also been demonstrated that a major surface protein, outer membrane protein A (OmpA), acts as a virulence factor playing an important role in cell death through both mitochondrial and nuclear targeting.8,9 It has also been found that two genes encoding for a putative protein tyrosine kinase and a putative polysaccharide export outer membrane protein are required for capsule polymerization and assembly, and have a major role in the pathogenesis of A. baumannii infection.6 Recently, a study using Galleria mellonella as an in vivo model compared the virulence of five different strains of A. baumannii and found that clinical isolates display the ability to express a number of virulence factors, including exoproducts 3 ACS Paragon Plus Environment


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with hemolytic, phospholipase and protease activities, and siderophore-based iron uptake mechanisms.10 There is a general consensus that the study of secreted proteins is required for the identification of virulence factors11 and represents a realistic opportunity for the development of new vaccines.12,13 Within this context, study of the extracellular proteome of A. baumannii is essential for gaining new insights into the pathogenesis of the species. However, despite the recognized importance of extracellular proteins, very few studies have investigated this protein fraction in A. baumannii. A study in a clinical isolate of A. baumannii identified 132 proteins of outer membrane vesicles,14 and more recently it was demonstrated that potential virulence factors, including OmpA and tissue degrading enzymes, were associated with A. baumannii outer membrane vesicles.15 Here, to complement these early studies, we have undertaken a

comprehensive study of the extracellular proteome of a highly invasive

multidrug-resistant (including carbapenems) A. baumannii clone (named AbH12O-A2), which infected more than 300 patients in the 12 de Octubre Hospital (Madrid, Spain) , 18 of whom died as consequence of the infection.5,16 In many studies, the extracellular proteome is often referred to as the secretome. However Desvaux et al.,17 have pointed out that several terms related to secretion are used interchangeably in the relevant literature and suggested that the term “secretome” should be restricted to describing components of translocation. These authors also proposed that the subset of proteins localized in the extracellular milieu, whether they are actively secreted or not, should be most rigorously described using the terms ‘extracellular cellular proteome’ or ‘exoproteome’. In this context, we will focus on two main fractions of the extracellular proteome: proteins that are secreted via OMVs and FSEPs present in the culture medium (extracellular milieu) of A. baumannii. We propose the use of different proteomic approaches for the separate analysis of these two extracellular sub-proteomes of AbH12O-A2 in order to 4 ACS Paragon Plus Environment

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explore these possibilities. In the first study of its kind, we present data that allows a direct comparison between the two extracellular protein fractions and discuss the potential role of the identified extracellular proteins within the context of antioxidant activity, host interaction, bacterial protection, pathogenesis and virulence.



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Materials and Methods

Bacterial Strain and Growth Condition. Acinetobacter baumannii AbH12O-A25 was grown overnight in Mueller Hinton broth (Fluka, Madrid, Spain) at 37 ºC with constant shaking. Fresh nutrient media (500 mL) was inoculated with a 1:100 dilution of the overnight culture and grown at 37 ºC with vigorous shaking. Cells were harvested during the early stationary phase of growth (OD600nm=1.6-1.9).

Isolation and Purification of OMVs OMVs were isolated as described by Rumbo et al.,.18 Briefly, cells were pelleted by centrifugation (2800× g, 40 min at 22 ºC), and the supernatant was filtered through a 0.22 µm membrane (low protein binding Millex-GP polyethersulfone membrane (Millipore, Bedford, U.S.A.) to remove residual bacteria and was then subjected to ultracentrifugation (200 000× g, 90 min, 4 ºC, rotor 70.Ti, Beckman, Munich, Germany). The supernatant, representing the FSEP fraction, was saved for processing as described in the section below. The OMV pellet was washed twice with PBS (Phosphate-buffered saline), pH 7.0 and resuspended in PBS, pH 7.4. The suspension was again filtered through a 0.22 µm membrane (Millex-GP) and spread on agar plates to test for any bacterial growth. The resultant filtrate was processed for protein extraction. For gel-free Nano-LC-MALDI-TOF/TOF (ABSciex, Foster city, CA) analyses, the OMV filtrate was treated by a modification of the method suggested by Deng et al.,19. Briefly, trichloroacetic acid TCA was added to the resuspended OMVs to a final concentration of 10% and the mixture was left overnight at 4 ºC to allow the proteins to precipitate. The proteins were pelleted by spinning at 16 000× g for 30 min in 2 mL low binding protein Eppendorf Safe-Lock tubes (Eppendorf AG, Hamburg, Germany), washed twice with cold acetone and 6 ACS Paragon Plus Environment

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air-dried. The protein pellet was then solubilized in 6 M urea and 2 M thiourea in 25 mM ammonium bicarbonate (pH 8.0). For 2-DE and 1-DE (one-dimension gel electrophoresis) analysis, 100 µL of the OMV filtrate was treated with a 2-DE Cleanup Kit (GE Healthcare, Piscataway, U.S.A), following the manufacturer´s instructions. In all cases the protein concentration in the extracts was determined with a Bio-Rad protein assay kit (Bio-Rad, Munich, Germany), by a modified Bradford assay,20 as suggested by Ramagli et. al.,21.

Preparation of the Free Soluble Extracellular Protein Fraction After the OMVs were separated (see above), the remaining supernatant was used to prepare the FSEPs fraction. Two different methods were used to isolate the FSEPs present in the supernatant, namely: a) Protein precipitation using TCA. The supernatant were precipitated using a modification of the method suggest by Deng et al.,19 as described above for OMV proteins.19 b) Protein precipitation using ammonium sulphate. For the functional assays OMV-free supernatant was treated as described by Ibrahim et al.,22 i.e. 400 mL of supernatant was brought to saturation with solid ammonium sulphate and the suspension was stirred at 4 oC for 3 h, then the precipitate was collected by centrifugation at 5525 × g at 4 oC for 1 h and the pellet suspended at 4 oC in PBS overnight. Any insoluble material still present after the suspension was removed by centrifugation at 5525× g for 1 h at 4 oC; the supernatant was collected and subject to gel-filtration with PD-10 columns (GE Healthcare, Buckinghamshire, UK). The fraction collected was sterilized by filtration through a 0.22 µm membrane (Millex-GP), then concentrated by ultrafiltration through a 3-kDa cutoff membrane (Millipore).



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Electron microscopy For microscopy, the OMV suspension was treated essentially as described in Rumbo et al.,.18 Briefly, the OMV suspension was fixed with 2.5% cold glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.4) for 2 h at 4 ºC and post-fixed with 1% osmium tetroxide in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 h at 4 °C. The vesicles were then visualized and photographed with a JEOL JEM 1010 transmission electron microscope (80 kV).

1-DE and 2-DE Gel Electrophoresis For 1-DE, 40 µg of protein were loaded onto an 8% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel of size 8.2 x 8.2 cm and 0.75 mm thick. Gels were then stained with Coomassie blue G-250 and the revealed lanes were cut in a linear fashion from top to the bottom into 6 slices of approximate size 0.5 cm x 1.3 cm x 0.75 mm and subjected to in-gel digestion. For 2-DE analysis, 25-40 µg of protein were applied to 13 cm NL pH 3.0–10.0 IPG strips (GE Healthcare) (FSEPs) or to 13 cm pH 4–7 IPG strips (GE Healthcare) (OMVs) to achieve better resolution (separation and clarity) of the banding patterns in the OMV gel and treated as described by Soares et al.,23. Briefly, the proteins were solubilized in 8 M urea, 2% (w/v) CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1propanesulfonate), 40 mM dithiothreitol (DTT) and 0.5% (v/v) corresponding IPG buffer (GE Healthcare). IEF (Isoelectric Focusing) was carried out at 30 V for 12 h, followed by 250 V for 1 h, 500 V for 1.5 h, 1000 V for 1.5 h, a gradient to 8000 V for 1.5 h and maintenance at 8000 V for a further 4 h, all at 20 ºC. Reduction with DTT and alkylation with IAA were performed after IEF separation. SDS-PAGE was performed on 12% polyacrylamide gels. The analytical gels were stained with Sypro Ruby, using the staining protocol of Invitrogen Molecular Probes, and with silver stain, as described previously.23 The preparative gels, loaded with at least 400 µg of protein, were stained by colloidal Coomassie staining and used 8 ACS Paragon Plus Environment

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for picking protein spots. In order to confirm reproducibility, at least three in 1-DE and four in 2-DE biological replicates of the two fractions were processed in the analytical gels, but one biological sample for each method of proteins extractions (1-DE) or pool contain biological replicates (2-DE) of the two fractions were characterized. Image Acquisition and 2-DE Gel Analyses Fluorescence image acquisition of Sypro Ruby stained gels was performed with a FUJIFILM Luminescent Image Analyzer LAS-3000 (460 nm/605 nm Ex/Em) (FUJIFILM, Tyoko, Japan). The silver and Coomassie blue stained gels were scanned in an ImageScanner densitomer, version (v).3.3 (GE Healthcare,). Comparative analyses of 2-DE gels were as described by Soares et al.,23 All experiments were carried out in triplicate. The 2-DE gels were analyzed with Image Master Platinum 6.0 Software (GE Healthcare), according to the manufacturer’s instructions. Spot detection parameters were adjusted to: smooth 2; MinArea 34, Saliency 10.

In-Solution and in-Gel Protein Digestion In-solution tryptic digestions were carried out as described by Lopez-Ferrer et al.24 Briefly, the protein extract (100 µg) was solubilized in 6 M urea, and 2 M thiourea in 25 mM ammonium bicarbonate (pH 8) and DTT was added to a final concentration of 10 mM, and the mixture incubated for 1 h at 37 ºC. Iodoacetamide (IAA) was then added to a final concentration of 50 mM and the mixture incubated for approximately for 45 min at room temperature in darkness. The mixture was then diluted 4 fold to reduce the concentration of urea, 2 µg of trypsin (Roche Applied Science, Mannheim, Germany) added, and the mixture was incubated at room temperature overnight. Two biological samples of the two fractions were analyzed.



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For in-gel digestion, proteins were reduced with 10 mM DTT in 25 mM ammonium bicarbonate for 1 h at 56 ºC and then alkylated with 10 mg/mL IAA in 25 mM ammonium bicarbonate for 15 min in the dark. Digestion was performed overnight with 12.5 ng/L trypsin at room temperature. In all cases the resulting extracted peptide mixtures were desalted and concentrated with NuTips (Glygen, Columbia, MD).

MALDI Mass Spectrometry Analysis The 2-DE protein spots were treated as described by Soares et al.,.25 In summary, the samples were analyzed in a MALDI-TOF/TOF mass spectrometer 4800 Proteomics Analyzer (ABSciex), with 4000 Series ExplorerTM software (ABSciex). MALDI-TOF spectra were acquired in reflector positive ion mode, using 1000 laser shots per spectrum. Data Explorer version 4.2 (ABSciex) was used to analyze the spectra and to generate the peak picking list. All mass spectra were internally calibrated using autoproteolytic trypsin fragments and externally calibrated using a standard peptide mixture (Sigma-Aldrich, St. Louis, MO). TOF/TOF fragmentation spectra were acquired by selecting the 10 most abundant ions of each MALDI-TOF peptide mass map (excluding trypsin autolytic peptides and other background ions), and averaging 2000 laser shots per fragmentation spectrum. The parameters used to analyze the data were a signal to noise threshold of 20, minimum area of 100 and a resolution higher than 10000 with a mass accuracy of 20 ppm. For samples subjected to nanoflow liquid chromatography analysis (gel-free and 1-DE samples), the de-salted peptide mixture was separated by reverse phase chromatography in a nanoLC system (ABSciex) by loading onto a C18 silica-based column (New Objective, Woburn, MA) with an internal diameter of 300 Ả, via a trapping column. Peptides were eluted at a flow rate of 0.35 µL/min with 0.1% trifluoroacetic acid and a linear gradient of 1.9-38% acetonitrile applied over 2 h. Eluates were mixed with α-cyano matrix (4 mg/mL at a 10 ACS Paragon Plus Environment

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flow rate of 1.2 µL/min) and deposited on a MALDI plate using an automatic spotter (SunCollect, Sunchrome, Friedrichsdorf, Germany).The MS runs for each chromatogram were acquired and analyzed in a 4800 MALDI-TOF/TOF instrument (ABSciex), using a laser voltage of 3800 kV and 1500 shots/spectrum. Automated precursor selection with a mass range of 800-4000 m/z was carried out by a Job-wide interpretation method (up to 15 precursors/fraction, Signal to Noise lower threshold = 50) with a laser voltage of 4800 and 1500 shots/spectrum. The CID (Collision-induced dissociation) collision energy range was set at medium. A second Job-Wide precursor selection was made, excluding previously fragmented precursors and considering a Signal-to-Noise lower threshold of 30, to identify peptides originating from scarce proteins. Data from both Job-Wide acquisitions were used for data processing and subsequent protein identification.

Data Analysis For 2-DE database queries and protein identification, the monoisotopic peptide mass fingerprinting data obtained from MS and the amino acid sequence tag obtained from each peptide fragmentation in MS/MS analyses were used to search for protein candidates, using Mascot, version 1.9, from Matrix Science (www.matrixscience.com). Peak intensity was used to select up to 50 peaks per spot for peptide mass fingerprinting and 50 peaks per precursor for MS/MS identification. Tryptic autolytic fragment-, keratin-, and matrix-derived peaks were removed from the data set used for searching the database. The searches for peptide mass fingerprints and tandem MS spectra were performed in the NCBInr (National Center for Biotechnology Information non-redundant) database without any taxonomical restriction. Fixed and variable modifications were considered (Cys as S-carbamidomethyl and Met as oxidised methionine, respectively), allowing one trypsin missed cleavage. MS/MS ions searches were conducted with a mass tolerance of ±1.2 Da for the parent and 0.3-0.8 Da for 11 ACS Paragon Plus Environment


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fragments. Decoy search was done automatically by Mascot on randomized database of equal composition and size. Mascot scores for all protein identifications were higher than the accepted threshold for significance (at the p 1.3 (95.0%). The scoring model was defined by the Paragon. The False Discovery Rate was estimated by carrying out the search in parallel against a reverse nr database using PSPEP (Proteomics System Performance Evaluation Pipeline) on mode. Since the genome of this strain has not been sequenced, alignments of all proteins were performed to rule out duplications. The predicted subcellular locations of the proteins were obtained by PSORTb, version 2.0.4 (http://www.psort.org/psortb2/index.html).26 The presence of export signals for each protein found

was

predicted

by

PSORTb,

version

2.0.4,

LipoP

1.0

(www.cbs.dtu.dk/services/LipoP),27 TatP 1.0 (www.cbs.dtu.dk/services/TatP-1.0),28 Tatfind 1.4

(http://signalfind.org/tatfind.html)29

and

SecretomeP

2.0

(www.cbs.dtu.dk/services/SecretomeP)30. PsortB is designed to detect only LepB processed Sec (general secretory pathway) substrates, and does not predict lipoprotein location in inner membrane or outer membrane. The use of LipoP mainly predicts Sec signal peptides that are cleaved by LspA, but also predicts the location of peptides in inner membrane or the cytoplasm as well as LepB cleavage, but does not detect Tat substrates. The Tat (twin-arginine translocation) also involves a signal peptide. However, it contains two consecutive arginine residues and can be predicted with TatP 1.0, 12 ACS Paragon Plus Environment

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but does not detect lipoproteins that have Tat signal peptide, and moreover some very long signal peptides are not detected. Tatfind is used to predict Tat signal peptides, but does not require the presence of an adjacent LepB or LspA site, and does not have to be located at the protein N-terminus (although this may be an advantage when prediction of the start codon is wrong). The SecretomeP 2.0 server produces ab initio predictions of non-classical protein secretion i.e. not triggered by signal peptides. Antioxidant Activity Assay For further testing of the antioxidant capacity of the extracellular fractions, the OMVs were isolated as described above (see section: Isolation and Purification of OMVs), and the OMV pellet was then resuspended in 3 mL of disintegration buffer (7.8 g/L NaH2PO4, 7.1 g/L Na2HPO4, 0.247 g/L MgSO4 7.H2O) and sonicated on ice for 3 periods of 5 min. Debris was separated by centrifugation at 1500× g. The supernatant was centrifuged several times for 1 h at 4 oC at 12 000× g. For the FSEPs, the OMV-free supernatant was precipitated by sulfate ammonium precipitation (as described above). All samples (OMVs and FSEP) were subject to gel-filtration with PD-10 columns (GE Healthcare), in order to remove small molecules that could interfere with the measurement of protein antioxidant activity. The collected fraction was then concentrated by ultrafiltration, by the use of a 3 kDa cut-off membrane (Amicon). To measure the total antioxidant capacity (TAA) of the protein extracts, the Antioxidant Assay Kit (Sigma-Aldrich) was used according to the manufacturer’s instructions and as detailed in Soares et al.,25. Nine biological replicates were utilized.

Survival of A. baumannii in the presence of H2O2 Bacterial cells were collected at the exponential phase of growth (OD600nm = 0.5 - 0.6) by centrifugation at 3500× g for 10 min, and were washed twice with 0.9% NaCl and finally resuspended in the initial volume of 0.9% NaCl. After being treated as described above 13 ACS Paragon Plus Environment


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(including gel-filtration with PD-10 columns and ultrafiltration by the use of 3 kDa cut-off membrane), the suspension containing FSEPs or intact OMVs was added to the resuspended cells to a final concentration of 1.25, 5 and 20 µg/mL of total protein (2.5, 10 and 40 µg of total protein added per 1 x 106 Colony forming unit/mL (CFUs/mL)). H2O2 was then added (Sigma-aldrich, USA) to a final concentration of 2 mM H2O2 and the mixture was incubated for 10 min at 37 ºC. For control purposes, the same procedure was followed, although in this case 20 and 0 µg/mL of bovine serum albumin) (BSA) were used instead of FSEPs or intact OMVs. Bacterial cells incubated without H2O2, and with FSEPs (20 µg/mL of total protein) and bacterial cells incubated without H2O2, and with intact OMVs (20 µg/mL of total protein) were used as further controls. After incubation, cells were collected, diluted accordingly and plated onto MacConkey agar plates. After 24 h incubation at 37 oC, colonies were counted and CFUs were determined according to each dilution factor. The data correspond to at least five independent experiments. The survival of cells with and without the respective protein fraction (OMVs or FSEP) was measured by counting colonies or CFUs. The survival percentage was calculated from the CFUs, as described by Soares et al.,25. In vitro assay for A. baumannii survival within macrophages This assay was performed as described by Arandas et al.,31 Briefly, cells (5 × 105) from the mouse leukemic monocyte macrophage cell line, Raw 264.7, were allowed to adhere to the wells of a 24-well plate, and bacteria collected at the exponential phase of growth (OD600nm =0.6) were then added at a ratio of 10–20 bacteria per macrophage, together with either 10 µg/mL(65 µg of total protein added per 9 x 106 CFUs/mL) of FSEPs treated as described in the section above (Antioxidant Activity Assay), BSA (10 µg/mL), or 10 µg/mL of intact OMVs. Bacterial cells collected at the exponential phase of growth (OD600nm = 0.4 - 0.6) and added at a ratio of 10–20 bacteria per macrophage, were used as an additional control. The 14 ACS Paragon Plus Environment

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plates were centrifuged at 250× g for 5 min at room temperature to enhance and synchronize infection and then incubated for 1 h at 37 °C to permit phagocytosis. Free bacteria were subsequently removed by three washes with 0.9% NaCl. DMEM (Dulbecco's modified Eagle's medium) (BioWhittaker Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum and gentamicin (50 µg/mL) was added, and the cells were again incubated at 37 °C. Wells were sampled at 0 and 1 h by aspirating the medium, lysing the macrophages with 0.5 mL of 0.5% deoxycholate, rinsing each well with 1 mL of NaCl 0.9%, and plating 0.1 mL of the lysate onto MacConkey agar plates. Six wells were plated for each experiment at each time. The data correspond to three independent experiments. The survival percentage was calculated as the CFUs at 1 h divided by the CFUs at 0 h. Statistical analysis A Mann Whitney test was applied when appropriate, by use of the Statistical Package for Social Sciences version 18.0 (SPSS Inc, IL).



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Results Isolation of FSEPs and OMVs In order to characterize the extracellular proteome of A. baumannii, the sub-proteomes of both the FSEP and the OMV fractions were isolated for analysis (Figure 1). For this we have harvested the supernatants of cultures of the AbH12O-A2 isolate at early stationary phase to avoid cytoplasmic contamination.32 To isolate OMVs from the supernatant we have used an ultracentrifugation-based protocol,33 which has been successfully applied in our laboratory for the isolation of A. baumannii OMVs.18 In order to check whether the integrity of OMVs was compromised by this modified isolation method, resuspended OMV pellets were analyzed by electron microscopy. As illustrated (see supplementary Figure S1), the pellet was composed of intact OMVs. On the other side FSEPs suspensions were subject to filtration to remove any possible bacterial contamination followed by electron microscopy analysis. OMVs were not found in the analyzed suspensions (data not shown). These two fractions were therefore considered to represent the extracellular proteome of A. baumannii.

Characterization of the Acinetobacter baumannii extracellular proteome In order to obtain a more complete analysis of the extracellular proteome, the FSEP and OMV sub-proteomes were subjected to three different separation techniques, namely gel–free Nano-LC, 1-DE Nano-LC and 2-DE, all coupled with MALDI-TOF/TOF analysis. In the case of the 2-DE gels, proteins precipitated with ammonium sulfate were seen to produce a higher resolution and quality in gels than that observed with TCA precipitates and was therefore chosen as the preferred approach for all subsequent 2-DE separations (Figure S2). For the OMV fraction, a total of 179 non-redundant proteins were identified across all experiments. Out of those, 127 proteins were identified by 1-DE Nano-LC and 148 from gelfree Nano-LC. 20 proteins were identified by 2-DE (see Table 1 and supplementary material 16 ACS Paragon Plus Environment

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Table S1). In the FSEP fraction a total of 148 non-redundant FSEP were identified: 124 proteins from 1-DE Nano-LC, 44 from gel-free Nano-LC and 28 from 2-DE gels (see Table 1 and supplementary material Table S2). Our analysis indicated that 156 out 240 of the proteins here identified were predicted to be secreted by a variety of programs (see methods and supplementary Table S3). Most of the FSEP identified possessed export signals predicted by SecretomeP,30 followed by that from LipoP,27 PSORTb,26 TatP 27 and Tatfind 29. Only 6 proteins possessed export signals predicted by TatP 27 and no proteins with Tat signal peptides with an adjacent LspA cleavage site were identified (see supplementary material Table S3). A comparison between the sub-proteomes of OMVs and FSEPs revealed an overlap of 87 proteins (36.3%), whereas 92 proteins (38.3%) were exclusively present in OMVs and 61 proteins (25.4%) were specific to the FSEP fraction.

Functional classification of extracellular proteins According to their predicted biological functions, proteins present in OMVs were divided into 11 groups, whereas those of the FSEP fraction could be divided into 10. For both OMV and FSEP fractions, the largest groups consisted of proteins involved in pathogenesis and virulence (39 and 30, respectively), followed by a proteins of unknown function (30 and 22, respectively). However, there was also evidence for functional compartmentalization between two sub-proteomes. The OMV fraction contained many proteins associated with translation (29) and signaling (18) (Figure 2A and Table 1), whereas FSEPs contained those associated with transport and metabolism (19) and the response to oxidative stress (18) (Figure 2B and Table 1). Overall, the results clearly indicate that although the OMV and FSEP sub-proteomes share a substantial number of proteins (87), these fractions each have many unique proteins (92 in OMVs and 61 in the case of FSEPs), which display differing spectra of functionality. 17 ACS Paragon Plus Environment


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FSEPs provide additional antioxidant protection to bacterial cells Among the FSEPs there were several proteins with a known role in the response to oxidative stress (e.g. catalase (# 70), SOD (# 76), thioredoxin (# 66) and others, see Figure 2B and Table 1). This prompted us to verify whether the presence of this type of proteins could confer antioxidant activity. Using the total antioxidant activity assay described by, Huang et al,.34 it was revealed that the antioxidant capacity of the FSEP fraction (120 ± 26 mM per mg of protein extract) was 4.6 times higher than that verified in the OMV protein fraction (26,4 ± 20 mM per mg of protein extract). Previously, we demonstrated that A. baumannii bacterial cells at the exponential phase of growth have a low antioxidant capacity.25 Here, we wished to verify whether the antioxidant capacity of the FSEP fraction would confer resistance to bacterial cells against oxidative stress. To test this, A. baumannii bacterial cells at the exponential phase of growth were incubated with H2O2 (2 mM), together with 1.25, 5 and 20 µg/mL of FSEPs, and the survival rate was compared with that of bacterial cells incubated with H2O2 without added FSEPs. However, H2O2 is known to cause severe damage to proteins and the presence of exogenous proteins might influence the survival rate by providing alternative targets, thereby buffering this damage. Therefore, in order to enable the relation of any changes in the survival rate to the specific composition of FSEPs, an additional control was included, in which FSEPs was substituted with BSA (20 µg/mL) before incubating with H2O2 (Figure 3A). The assay revealed that with zero or low concentrations of FSEPs (1.25 µg/mL), the bacterial survival rate was close to 0% in response to challenge with H2O2. However, when cells were incubated with H2O2 together with 5 or 20 µg/mL of FSEPs, the bacterial survival rate increased to almost 100% (Figure 3A). No increase was observed when FSEPs were substituted with BSA (20 µg/mL) or OMVs (20 µg/mL) proteins (Figure 3B). 18 ACS Paragon Plus Environment

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FSEPs enhances the ability of bacterial cell to survive macrophage attack Macrophages are known to produce reactive oxygen species (ROS), which are essential for their microbiocidal attack35-39 Our observation that FSEPs can contribute to the extracellular antioxidant capacity prompted us to investigate whether FSEPs could confer protection against macrophage attack. For this purpose, bacterial cells at the exponential phase of growth were incubated in vitro with leukemic monocyte macrophages with or without 10 µg/mL FSEPs, BSA or OMVs proteins (Figure 4). After 1 h incubation with macrophages, the survival rate of the cells incubated together with FSEPs was 2.5 times higher than that of cells incubated without FSEPs or with BSA (Figure 4A). No increase was observed when FSEPs were substituted with 10 µg/mL of OMVs proteins (Figure 4B).



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Discussion Extracellular proteome of A. baumannii revealed the presence of unexpected proteins Bacterial extracellular proteins can be divided into two main groups: those that are exported via the outer membrane vesicles or those exported otherwise. In A baumannii there is little information concerning these two protein populations. Here we have employed different proteomic approaches to better characterize the extracellular proteome of A. baumannii. Results indicate that 1-DE coupled with Nano-LC-MALDI-TOF/TOF is a convenient method that can be used to identify a high number of the extracellular proteins, an observation made in other recent reports describing the OMV sub-proteome.14,

15

This approach can be

complemented and validated by gel-free based techniques (see supplementary Figure S2A and Table S1). We also demonstrate that techniques such as 2-DE with the addition of an improved protocol towards the extraction of OMVs proteins can be successfully applied to the analysis of extracellular sub-proteomes to provide complementary proteomic data (see supplementary Figure S3). The characterization of the A. baumannii extracellular proteome revealed that the majority of FSEP were predicted as secreted targets, whereas the OMV fraction was composed by membrane proteins together with others including cytoplasmatic proteins. However, both fractions contained some proteins predicted to be cytoplasmic (a total of 86 out of 240 proteins). The presence of proteins without a recognizable secretory signal might result from cytoplasmic contamination from broken cells, although such contamination is reported to be minor in in vitro cultures of bacteria at the early stationary phase of growth.32 In addition, the presence of these proteins in the extracellular milieu has been reported in many recent studies.12, 32, 33, 40, 41 It is plausible that A. baumannii utilizes a non-classical pathway for the secretion of non-canonical, extracellular proteins similar to that suggested for other bacterial species.

30,42,43

Additionally, a substantial overlap of proteins was observed between the two 20 ACS Paragon Plus Environment

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extracellular protein fractions. Portions of these overlapping proteins might result from the unspecific binding of some FSEPs to the OMVs. It is likely that this would occur in in vitro liquid cultures, where the two fractions could interact in the extracellular media over an extended period of time. This interaction would be severely reduced under in vivo conditions, where OMVs are secreted and internalized in the host cell after bacterial cell adhesion.44 Therefore, with a few exceptions, the discussion will focus on those proteins detected exclusively in either the FSEP or OMV fractions.

Extracellular proteome of A. baumannii revealed several proteins associated with pathogenesis and virulence Bacteria can export proteins via the outer membrane vesicles or via a number of specialized secretion systems. There is some information available concerning OMV formation and its proteome composition in A. baumnannii. However, the secretion systems and their protein substrates remain largely unknown. It was recently found that the genome of A. baumanniii ATCC 17978 7 contains a large putative alien island comprised of eight genes homologous to the Legionella/Coxiella Type IV virulence/ secretion apparatus, suggesting that this type of secretion occurs in A. baumannii. Here we have identified a P. pilus assembly protein, chaperone PapD (# 22), which forms part of a chaperon-usher secretion system, also denominated T7SS. Chaperone PapD mediates assembly of the pap fiber protein complex type 1 pili, in E. coli these pili are thought to be involved in bacterial adherence and invasion.45Accordingly, we also identified a filamentous type 1 putative assembly protein (CsuB (# 9) & FilF (# 14) ), similar to a proposed mechanism in E. coli 46 that may operate during A. baumannii infection. Moreover, blast (http://blast.ncbi.nlm.nih.gov/Blast.cgi)47, 48 analyses revealed a significant alignment between the hypothetical protein ACICU_01051 (# 15) and other putative tail fibers (data not shown). These structures have been associated with 21 ACS Paragon Plus Environment


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the T3SS invasion system, which appears to be used exclusively for secretion of virulence factors.11 Finally, proteins such as the large exoprotein (# 19) can be considered a hallmark of a two-partner secretion pathway, a specific secretion pathway for large protein virulence factors that are crucial for the virulence of several human pathogens.49, 50 Protein secretion systems are evidently associated with the translocation of virulence factors.

51

Here, by

employing proteomics strategies we present novel evidence indicating that secretion systems do indeed occur in A. baumannii. The results indicate that both sub-proteomes display several proteins associated with pathogenicity and virulence. In accordance with the model proposed by Jin et al.,15 in which A. baumannii OMVs are described as a major secretory vehicles for the delivery of virulence factors to host cells, characterization of the OMVs sub-proteome revealed additional proteins involved in cell adhesion, biofilm formation and motility (e.g. CsuE (# 49), CsuB (# 9), CsuA/B (# 45)), which may facilitate host cell attachment. Within the context of deliverance of virulence factors to host cell, we highlight the presence in OMVs as a hallmark of several specialized secretion systems, such as P. pilus assembly (# 22) and FilF (# 14), it is therefore possible that once OMV attachment occurs on host cells, these secretion systems would serve as a tool for delivering bacterial potential virulence factors to the host. Such systems would include the trypsin-like serine protease (# 26), metalloprotease (# 8), putative protease (# 39), lipases (# 36), phospholipase (# 20) and toxins such as bacteriolytic lipoprotein entericin B (# 30). Several virulence factors specific to the FSEP fraction were found, such as extracellular enzymes with degradative activity, including alkaline metalloprotease (protein (# 47) & (# 48)), secreted lipases (# 41), ZN-dependent oligopeptidase (# 31), and lactonohydrolase (# 42). Extracellular metalloproteoases are known to be virulence factors secreted from many bacterial species (reviewed in Wu et al., 52). For example, one of the three proteins comprising 22 ACS Paragon Plus Environment

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the anthrax toxin of Bacillus anthracis is a secreted, extracellular protease.53-55 The lipolytic activity of secreted lipases has been demonstrated to contribute to the persistence and virulence of C. albicans in human tissue.56 The success of a bacterial infection greatly depends on the ability of the bacteria to use external nutrients, e.g. proteins around bacteria must be degraded into amino acids and oligopeptides before they can be utilized for bacterial nutrition.52 Therefore, the proteolytic and lipolytic activities of extracellular proteins may play key roles in the establishment of A. baumannii infection. Considering the composition of OMVs and FSEP in terms of potential virulence factors, it is tempting to suggest that during infection, both sub-fractions of extracellular proteins may play rather complementary roles, which probably act synergistically. There were also a number of potential virulence factors present in both OMVs and FSEPs. These included OmpA (# 44), which as previously mentioned is thought to be involved in the induction of host cell death through both mitochondrial and nuclear targeting.

8, 57

More

recently, evidence suggested that OMVs act as a vehicle for the delivery of OmpA to host cells and induce cytotoxicity.15 However it is not clear how OmpA located in the vesicle membrane is translocated to the host cytosolic compartment.15 Furthermore OmpA was also found to be soluble in the medium supernatant. It should also be note that purified, soluble OmpA is capable of inducing cell host cytotoxicity.8, 57 It would therefore be interesting to carry out further research to determine whether membrane bound OmpA versus freely soluble OmpA present in the extracellular media are equal in rapidly and effectively inducing host cell death.



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Extracellular proteome of A. baumannii reveals several proteins associated with bacterial protection Here, a set of extracellular proteins associated with drug resistance including carbapenemhydrolyzing β-lactamase OXA-24 (# 58) as well as the constitutive AmpC β-lactamase (# 61) were identified. The A. baumannii clone AbH12O-A2 showed broad resistance to antimicrobials, being resistant to carbapenems and susceptible only to tigecycline and colistin.5 Additionally, the clone carries a pMMA2 plasmid that may be involved in mobilization of the bla

oxa-24

gene encoding carbapenemase OXA-24.16 We have identified

two proteins that are products of two ORFs located in the plasmid, namely carbapenemhydrolyzing β-lactamase OXA-24 (# 58) and a TonB-dependent receptor (# 2), suggesting that pMMA2 plasmid is being expressed with some gene products being exported to the extracellular medium. Several other proteins known to play a role in antidrug resistance, such as the putative class A β-lactamase (# 56), β-lactamase OXA 88 (# 59), were also identified (see Table 1). These results are therefore consistent with the multi-drug resistance profile of this strain. Furthermore, this dataset suggests that three different β−lactamase enzymes (# 58, #59, and #61) are released outside the bacterial cell, thus being able to hydrolyze and inactivate carbapenem antibiotics among other β−lactams. Studies concerning antibiotic resistance would benefit from attention to the so-far neglected, extracellular proteome and the molecular mechanisms underlying exportation of proteins such as cabapenemases. Characterization of the extracellular proteome revealed the existence of several proteins known to be associated with response to oxidative and/or reactive nitrogen intermediate (RNI)-induced stress. A large majority of these were exclusive to the FSEP fraction (e.g. oxidoreductase (# 64), thioredoxin (# 66), catalase (# 70), flavohemoprotein (# 75), SOD[Fe] (# 76)), whereas some were common to both extracellular protein fractions (e.g. alkyl hydroperoxide reductase (# 78) and Cu/Zn superoxide dismutase (# 82)). In a previous study, 24 ACS Paragon Plus Environment

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we have demonstrated that ROS and RNI accumulate along the growth curve. 25 In the present study, protein fractions were harvested during the early stationary phase. It is therefore possible that the growth phase accumulation of ROS/RNI may somehow be related to the presence of this type of protein in the extracellular medium. However, for most bacterial species, antioxidant proteins are considered to be intracellular and lack the sequence motifs required by specific secretion mechanisms. The presence of such proteins in the extracellular medium is therefore controversial, as it is not clear whether these proteins are released in the medium or are the result of contamination by cell lysis.42,58 A study in Mycobacterium tuberculosis demonstrated that superoxide dismutase SodA and catalase-peroxidase KatG are secreted by an unconventional export pathway involving SecA2.42 It is therefore possible that a similar secretion mechanism in A. baumannii is responsible for the secretion of some of the antioxidant proteins identified here. In a recent study, also in M. tuberculosis, it was demonstrated that the enzyme superoxide dismutase is abundantly exported in active form to the extracellular medium.59 Here we found that the FSEP extract has a high antioxidant capacity, suggesting that some of the ROSscavenging proteins identified are exported in their enzymatically active form25 to confer bacterial protection against oxidative stress. This protective feature of the FSEP fraction was confirmed by the observed higher survival rate of cells against exogenous H2O2 in the presence of FSEPs (Figure 3A). The concept that the resistance to oxidative stress begins outside the cell is of particular interest, especially considering that during infection, microbial pathogens are engulfed by phagocytic cells and then attacked by reactive oxygen and nitrogen species.35-38 The present results demonstrated that in the presence of FSEPs, bacterial cells show a higher survival rate against macrophages (see Figure 4A), suggesting that the antioxidant activity of ROS scavenger proteins such as catalase (# 70), SOD[Fe] (# 76), alkyl hydroperoxide reductase (# 78), and RNI scavengers such as flavohemoprotein (# 75), would 25 ACS Paragon Plus Environment


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neutralize the toxic effect of the oxygen metabolites generated by the macrophage. There is a precedent in other bacterial species for exported antioxidant proteins protecting against oxidative stress, and contributing to virulence in bacterial pathogens. 42, 60-62 For example, in M. tuberculosis, SOD and catalase Kat together with the respective secretion system, SecA2, are considered to be an important part of the virulence mechanism of this important human pathogen, acting to counter the bactericidal respiratory burst of macrophages. The results shown here allow us to conclude that A. baumannii FSEPs possess an efficient ROS/RNI scavenging mechanism that enhances bacterial survival against macrophage attack. Although other proteins, such as peptidyl cis-trans isomerase rotamatase (# 37), also known as PPIases, do not possess antioxidant activity, they may play a role during bacterial survival against macrophage attack. It has been suggested that PPIases can act as virulence factors by interacting with some proteins from the host cell membrane, thus helping render the host cell more susceptible to penetration via conformational changes through cis-trans isomerization of peptidyl-prolyl bonds.63,64 These types of proteins have been reported to affect the phagocytosis of Streptococcus pneumonia by macrophages.65 The large exoprotein (# 19) identified here belongs to the filamentous hemagglutinin/hemolysin (FHA) protein family. In Bordetella pertussis these types of proteins are considered to be important virulence factors and have been suggested to function as primary adhesives that facilitate bacterial binding to ciliated respiratory cells and macrophages during Bordetella infection.66 It is thought that FHA may play a role in allowing Bordetella to resist clearance by phagocytic cells, by inhibiting the microbiocidal activity of these cells.66 In summary, it is therefore likely that different kinds of FSEPs contribute in different ways to bacterial defense against macrophage attack.


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Concluding remarks This study provides a comprehensive overview of the extracellular proteome present in an in vitro liquid culture of A. baumannii and provides insight into the potential role of extracellular proteins in vivo. The OMV sub-proteome indicates that during host interactions, OMVs will probably act mainly as vehicles for the transport and deliverance of virulence factors to target cells thereby playing an important role in the pathogenesis of A. baumannii. On the other hand, the presence of proteins such those involved in oxidative stress responses among the FSEPs in the extracellular medium indicates that protection against ROS/RNI begins outside the cell and that this can be determinant in the protection against macrophage actions. Several extracellular proteins identified here provide insight into their potential role in important process such pathogenesis, infection and drug resistance. This study therefore draws attention to the importance for the inclusion of the extracellular compartment in future studies of the pathogenicity and multi-drug resistance of A. baumannii.



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Synopsis This study enables a comprehensive overview of the extracellular proteome of a multidrug resistance Acinetobacter baumannii isolate. We focused on two main protein sub-fractions: those secreted via outer membrane vesicles (OMVs) and those that exist freely soluble in the cell culture medium (FSEPs). Here we provide evidence at the proteome level which, indicates that OMVs function as a vehicle for delivering virulence factors to target cells. Proteomics and functional assays indicated that FSEPs play an important role in bacteria defense response to oxidative stress and macrophage attack.


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Acknowledgments JAM is supported by Río Hortega research contract (Instituto de Salud Carlos III). NCS acknowledges received funding from Xunta de Galicia post-doctoral program Angeles Alvariño Dez 2009. CR and JA were supported by post-doctoral (Sara Borrell) and doctoral grants, respectively, from the Instituto de Salud Carlos III. M. Tomás was financially supported by the Miguel Servet Programme (C.H.U.A Coruña and ISCIII). This work was funded by the Spanish Network for the Research in Infectious Diseases-REIPI-(Instituto de Salud Carlos III, RD06/0008/0025), FIS PI081613, PI10/00056, PS09/00687, PS07/90, PS07/51, and 08CSA064916PR from Xunta de Galicia. We thank Dr. Phil Jackson from ITQB/UNL for the useful comments on the manuscript. We acknowledge helpful advices from Dr. Cristina Ruiz-Romero and Dr. Patricia Fuentes (ProteoRed) and Ireneu Soares Costa for helping us with the synopsis cartoon.

List of abbreviation

OMVs

Outer membrane vesicles

FSEPs

Free soluble extracellular proteins

Nano-LC-MALDI-TOF/TOF Nanoliquid-chromatography coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer. 2-DE

Two-dimension gel electrophoresis

1-DE

One-dimension gel electrophoresis

MALDI-TOF/TOF

Matrix-assisted laser desorption/ionization

SOD

Superoxide dismutase

MDR

Multi-drug-resistant

OmpA

Outer membrane protein A

OD

Optical density

PBS

Phosphate-buffered saline 29 ACS Paragon Plus Environment


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TCA

Trichloroacetic acid

SDS-PAGE

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

CHAPS

(3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate)

DTT

Dithiothreitol

IEF

Isoelectric Focusing

IAA

Iodoacetamide

CID

Collision-induced dissociation

NCBInr

National Center for Biotechnology Information non-redundant

PSPEP

Proteomics System Performance Evaluation Pipeline

Sec

General secretory pathway

Tat

Twin-arginine translocation

TAA

Total antioxidant capacity

CFUs

Colony forming units

BSA

Bovine serum albumin

DMEM

Dulbecco's modified Eagle's medium

ROS

Reactive oxygen species

ORF

Open reading frames

RNI

Reactive nitrogen intermediate

FHA

Filamentous hemagglutinin/hemolysin


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FIGURE LEGENDS Figure 1. Flowchart indicating the steps taken for sample preparation of extracellular proteins. The pellet containing outer membrane vesicle pellet was then separated from the supernatant containing freely soluble, extracellular proteins. The protein were then extracted from the respective fractions and characterised by Nano-LC-MALDI-TOF/TOF or MALDITOF/TOF.

Figure 2.

Functional classification of the proteins identified in the A. baumannii

extracellular sub-proteomes. (A) Proteins from Outer Membrane Vesicle subproteome and (B) Freely Soluble Extracellular Proteins. In brackets is indicated the total numbers of proteins within the respective group.

Figure 3. Survival of A. baumannii cells collected at the exponential phase of in vitro growth after incubation with 2 mM H2O2 and different concentrations of total proteins of outer membrane vesicle (OMVs) or freely soluble extracellular proteins (FSEPs). (A) shows the effect cell survival rate after incubation with H2O2 and different concentrations of FSEPs : (1) 20 µg/mL of FSEPs, (2) 5 µg/mL of FSEPs, (3) 1.25 µg/mL of FSEPs,(4) control of 20 µg/mL of BSA (5), mock control, no FSEPs were added and (6) 20 µg/mL of FSEPs without H2O2. Data are the mean results of at least five independent experiments and correspond to the percentage of bacterial survival. Error bars indicate the standard deviations for replicate samples. *p < 0.05 vs. (4) control by Mann–Whitney U-test. (B) shows the effect cell survival rate after incubation with H2O2 and different concentrations of total proteins of OMVs (1) 20 µg/mL of total proteins of intact OMVs, (2) 5 µg/mL of total proteins of intact OMVs, (3) 1.25 µg/mL of total proteins of intact OMVs, (4) control of 20 µg/mL of BSA (5), mock control, no intact OMVs were added and (6) 20 µg/mL of total 40 ACS Paragon Plus Environment

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proteins of intact OMVs without H2O2. Data are the mean results of at least five independent experiments and correspond to the percentage of bacterial survival. Error bars indicate the standard deviations for replicate samples. *p < 0.05 vs. (4) control by Mann–Whitney U-test.

Figure 4. Survival of A. baumannii cells collected at the exponential phase of in vitro growth after incubation with different concentrations of FESPs and macrophages. (A) from left to right: (1) 500,000 Mouse leukemic monocyte macrophage cell line Raw 264.7 were incubated with 8.8 x 106 CFUs/mL of A. baumannii, (2,3) As for (1), but with A. baumannii substituted with 10 µg/mL of BSA and 10 µg/mL of FSEPs, respectively. Data are the mean values from 23 independent experiments. The information shows the percentage of intracellular bacterial survival at 1 h after incubation. *p < 0.001 compared with controls (Mann–Whitney U-test). (B) from left to right: (1) 500.000 Mouse leukemic monocyte macrophage cell line Raw 264.7 were incubated with 8.8 x 106 CFUs/mL of A. baumannii, (2) the same experiment but with 10 µg/mL of BSA and (3) the same experiment as (1) but with 10 µg/mL of intact OMVs protein. Data are the mean values from 23 independent experiments. The information shows the percentage of intracellular bacterial survival at 1 h after incubation. *p

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