Immuno-Reactive Molecules Identified from the ... - ACS Publications

Immuno-Reactive Molecules Identified from the Secreted Proteome of Aspergillus fumigatus Bharat Singh,†,‡ Michael Oellerich,§ Ram Kumar,† Manish Kumar,† Dharam P. Bhadoria,| Utz Reichard,⊥ Vijay K. Gupta,‡ Gainda L. Sharma,*,† and Abdul R. Asif*,§ Division of Diagnostics and Biochemistry, Institute of Genomics and Integrative Biology, University Campus, Mall Road, Delhi-110007, India, Department of Clinical Chemistry, University Medical Center Goettingen, Robert-Koch-Str.40, D-37075 Goettingen, Germany, Department of Medicine, Maulana Azad Medical College, Bahadur Shah Zafar Marg, New Delhi-110002, India, Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Medical Center Goettingen, Kreuzburgring 57, D-37075 Goettingen, Germany, and Department of Biochemistry, Kurukshetra University, Kurukshetra-136119, India Received June 16, 2010

The secreted proteomes of a three week old culture of an Indian (190/96) and a German (DAYA) Aspergillus fumigatus isolate were investigated for reactivity with IgG and/or IgE antibodies derived from pooled allergic broncho-pulmonary aspergillosis (ABPA) patients’ sera. Two dimensional Western blotting followed by mass spectrometric analysis of the reactive protein spots revealed 35 proteins from the two A. fumigatus strains. There were seven known A. fumigatus allergens among them (Asp f1-4, Asp f9, Asp f10, and Asp f13/15), whereas three proteins displaying significant sequence similarity to known fungal allergens have been assigned as predicted allergens (Dipeptidyl-peptidase-V precursor, Nuclear transport factor 2, and Malate dehydrogenase, NAD-dependent). Eight IgG and IgE reactive proteins were common in both strains; however, 12 proteins specifically reacted in 190/96 and 15 in DAYA. Further testing with sera of 5 individual ABPA patients demonstrated that 12 out of 20 immunoreactive proteins of 190/96 strain of A. fumigatus had consistent reactivity with IgE. Seven of these proteins reacted with IgG also. The 25 of 35 identified proteins are novel with respect to immunoreactivity with ABPA patients’ sera and could form a panel of molecules to improve the currently existing less-sensitive diagnostic methods. Through expressing recombinantly, these proteins may also serve as a tool in desensibilization strategies. Keywords: Aspergillus fumigatus • proteome • secretory antigen • allergens • diagnostic tool • allergic bronchopulmonary aspergillosis

Introduction Fungal disorders have emerged as a major threat to public health in the past few decades, and besides Candida, the species of Aspergillus are the most common pathogens involved in such infections.1,2 Aspergillus fumigatus are present worldwide and are known to cause four distinct clinically recognizable forms of hypersensitivity respiratory disorders, that is, allergic bronchopulmonary aspergillosis (ABPA), allergic As* To whom correspondence should be addressed. For Immunology part: Prof. Gainda L. Sharma, Division of Diagnostics and Biochemistry, Institute of Genomics and Integrative Biology, Mall Road, University Campus, Delhi110007, India. Tel.: +91-11-27667439, Fax: +91-11-27667471, E-mail: [email protected]. For Proteomics part: Dr. Abdul R. Asif, Department of Clinical Chemistry, University Medical Center Goettingen RobertKoch-Str.40, D-37075 Goettingen, Germany. Tel.: +49-551-3922945, Fax: +49551-3912505, E-mail: [email protected]. † Institute of Genomics and Integrative Biology. ‡ Kurukshetra University. § Department of Clinical Chemistry, University Medical Center Goettingen. | Maulana Azad Medical College. ⊥ Department of Medical Microbiology and National Reference Center for Systemic Mycoses, University Medical Center Goettingen. 10.1021/pr100604x

 2010 American Chemical Society

pergillus sinusitis, IgE-mediated asthma, and hypersensitivity pneumonitis.3 ABPA is primarily an immunologically mediated lung disease, which is very frequently associated with asthma. A. fumigatus releases large number of highly antigenic proteins and cell wall polysaccharides during the course of infection.4-6 They include enzymes, toxins, cell wall and growth related molecules, which facilitate A. fumigatus in colonization.7-9 These, often multifunctional molecules may be important for various cellular processes of A. fumigatus and ABPA is caused by an exaggerated hypersensitivity reaction to these antigenic molecules. However, further invasion of A. fumigatus in the deeper body parts leads to more fatal invasive aspergillosis.10 Strategies to detect and diagnose ABPA use recognition of specific immunity toward Aspergillus antigens, especially using crude or partially purified antigens of A. fumigatus. There is a lack of antigenic standardization between laboratories because of the use of local antigen preparations.11 Additionally, these antigen preparations have limited scope due to their low sensitivity and lack of specificity, which make them inept to detect the infection.12 Cross reactivity due to sharing of Journal of Proteome Research 2010, 9, 5517–5529 5517 Published on Web 09/09/2010

research articles common antigenic epitopes with other fungal allergens makes it difficult to use all A. fumigatus allergens for diagnosis.13 Use of these crude antigenic preparations of A. fumigatus for ABPA diagnosis is presently offered by renowned biotech companies like Siemens, Germany. On other hand, ELISA and radioimmunoassay based on the crude extracts with addition of recombinant allergens (rAsp f1, rAsp f2, rAsp f3, rAsp f4 and rAsp f6) of A. fumigatus have been used commercially for diagnosis of allergic aspergillosis (Immunoap, Phadia, Uppsala, Sweden).3,14,15 These investigations however, are limited to the IgE binding assay with nonspecific crude preparation and/or few number of recombinant molecules.16 Apart from diagnostic significance only rAsp f3 has been shown to give protective immunity in a mice model against aspergillosis.17 However, to identify potential vaccine candidates, few more molecules are needed to be screened which are IgG reactive with ABPA patients’ sera. Investigators have been using the cDNA library screening approach to improve available diagnostic tools, but picking up the right protein target using cDNA screening has proven to be difficult.18,19 Recent advances in mass spectrometric based proteomic techniques could potentially help to overcome these limitations and has already encouraged scientists to undertake such investigations.20 Initial reports showing the profile of IgE reactive proteins from A. fumigatus have emerged recently.21 So far, the elevated levels of total serum IgE, Aspergillus-specific IgE and IgG antibodies in patients’ sera are considered to be the major immuno-diagnostic criteria for allergic form of aspergillosis. There has been a lack of thorough studies using A. fumigatus antigens and patients’ sera to investigate the molecules reacting with the IgE and IgG fractions of ABPA patients’ sera. The present work uses the secreted fraction of molecules of threeweek cultures from two geographically distinct strains, 190/96 and DAYA, of A. fumigatus. The purpose of present study was to identify proteins that may improve the existing diagnostic tools enhancing sensitivity and specificity along with verification of previously known allergens and immunogenic molecules from A. fumigatus.

Materials and Methods A. fumigatus Strains. Two geographically distinct strains of A. fumigatus were used for the present study. The strain 190/ 96 isolated from an ABPA patient and characterized at Vallabhbhai Patel Chest Institute, Delhi, India was a kind gift to us. It was further typed and authenticated at Indian Type Culture Collection, Indian Agriculture Research Institute, New Delhi, India and coded as ITCC 6604. A second strain D141 was derived from Germany registered in the US (NRRL 6585; U.S. Department of Agriculture Peoria, IL). D141 coded as DAYA, was isolated from an aspergilloma that had developed in a 45 years old man with tuberculosis. Preparation of A. fumigatus Secretory Proteins. Both strains of A. fumigatus were cultured in 3 L conical flasks containing 500 mL L-asparagine medium.22 The flasks were inoculated with A. fumigatus conidia and incubated at 37 °C without shaking using incubator, C25KC (New Brunswick Scientific, Edison, USA) for three weeks to obtain a stationary culture. After separating the mycelia by filtering through two layers of filter paper, the culture filtrate was subjected to protein precipitation using 80% saturation with ammonium sulfate. Precipitated proteins were pelleted from culture filtrate by centrifugation at 18000 × g with a fixed angle rotor centrifuge (5810R, Eppendorf, Hamburg, Germany) for 30 min at 4 °C. Protein 5518

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Singh et al. pellets were dissolved in phosphate buffered saline (PBS) and dialyzed extensively against deionized H2O. The dialysate was further concentrated by using the dehydrating agent Polyethylene Glycol (PEG-15000). The concentrated extracts were then dialyzed against deionized H2O. The protein concentration in the sample was estimated by the method described by Bradford.23 Collection of ABPA Patient’s Sera and Determination of Total Serum IgE, Specific IgE and IgG. Blood samples (5.0 mL) were drawn from clinically diagnosed patients with ABPA after obtaining clearance from duly constituted Ethics Committee of the Institute. Basic criteria of Rosenberg et al (1977)24 subsequently revised by Nicolas et al (2001)25 and Greenberger et al (2002)26 were followed for diagnosis of ABPA. Apparently healthy individuals having no symptoms of any respiratory or other diseases were taken as the source of control samples. Informed written consent was obtained from both patients and control subjects before drawing the blood. Sera were analyzed for determination of level of Aspergillus specific antibodies, IgE and IgG, against secretory antigens of A. fumigatus and total serum IgE antibodies before pooling them for use in immunoblotting. Total serum IgE was determined by a sandwich ELISA using a commercially available kit (Bethyl Laboratories, Inc., Montgomery, TX). The monoclonal antihuman-IgE antibodies were used to coat 96 well polystyrene ELISA plates (Nunc maxisorp, Roskilde, Denmark) overnight at 4 °C. The blocking and subsequent steps were performed according to the protocol provided by the manufacturer. Plates were developed using 2, 2′-azino bis (3-ethylbenzthiazoline-6-sulfonic acid) and absorbance was recorded at 405 nm using an ELISA reader Spectra Max 384 plus (Molecular Devices, Sunnyvale, CA). Standard curves were generated and used to determine the IgE levels in the serum samples. The lower detection limit of the kit was 1.5 International Units per mL (IU/mL). All the serum samples were analyzed in duplicate and repeated at least twice. The mean of all measurements was taken for further statistical analysis and expressed in IU/mL. Specific IgE antibodies in sera of ABPA patients against Aspergillus were determined by an indirect ELISA according to the basic method outlined by Sepulveda.27 For this purpose, polystyrene microtiter plates (Nunc maxisorp, Roskilde, Denmark) were coated with 100 µL of secretory antigen (10 µg/ mL) diluted in 0.1 M carbonate buffer (pH 9.6). After coating overnight at 4 °C, the plates were washed three times with PBS, pH 7.4. After thorough washing, free sites were blocked by 1% BSA prepared in PBS at 37 °C for 2 h. After that, wells were thoroughly washed and 100 µL of serum diluted 1:10 with PBS and 0.05% BSA was added to each well followed by overnight incubation at 4 °C. Again after repeated washings, 100 µL of alkaline phosphatase labeled antihuman IgE (ε-chain specific, produced in goat, Sigma, USA Cat No. A3525) antibodies diluted 1:1000 with 0.05% BSA in PBS were added to each well and incubated at 37 °C for 4 h. Subsequently, 100 µL of pnitrophenyl phosphate (0.01%) in glycine buffer pH 10.4 (Sigma, St. Louis, MO) was added to the wells and incubated at 37 °C. The reaction was terminated after 45 min using 50 µL of 5 M sodium hydroxide solution. The absorbance was recorded at 410 nm by Spectra Max 384 plus (Molecular Devices, Sunnyvale, CA). Aspergillus specific IgG antibodies in sera of ABPA patients were determined by an indirect ELISA according to a method described by Sharma and Sarma.28 Polystyrene microtiter plates

A. fumigatus Vaccine Candidates and Diagnostic Tools

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(Nunc maxisorp, Roskilde, Denmark) were coated as described above for IgE detection. After thorough washing and blocking, the wells were incubated with 100 µL of serum diluted (1:100) in PBS for 1 h at 37 °C. After repeated washings, 100 µL of protein-A peroxidase diluted (1:1000) in PBS obtained from M/s Banglore Genei (Stock 5 mg/mL) was added to each well. The plates were incubated at 37 °C for 1 h and then wells were incubated with 100 µL of the substrate (0.01% orthophenyl diamine in 0.1 M citrate buffer, pH 4.0 containing 0.01% H2O2) at room temperature in the dark. The reaction was stopped after 10 min using 50 µL of 4 N sulphuric acid. ELISA absorbance was recorded at 492 nm by using a Spectra Max 384 plus (Molecular Devices, Sunnyvale, CA).

Matching the Immuno-Reactive Spots. The reactive protein spots obtained after immuno-blotting with ABPA patients’ sera were matched manually as well as with the help of Delta 2-DE v 4.0 (DECODON, Germany) to the corresponding silver stained 2DE gel. The Western blots were matched first with their own Ponceau stain images and then were manually compared with the subsequent silver stained gel using the landmarks inserted on the membrane during the 2DE procedure. To confirm if the correct protein spots had been excised from the corresponding gels, we repeated selected immuno-blots using PVDF membrane. We excised reactive spots directly from the PVDF membrane and gel from which incomplete protein transfer had been achieved. For this purpose 2DE gels were reproduced as described above and after reducing the time for transblotting and immuno-staining, antibodies were removed by 30 min washing with stripping buffer (62.5 mM Tris pH 6.8, 2% SDS, 100 mM 2-β mercaptoethanol) followed by five washes each for 5 min with PBS buffer. The reactive spot area was excised using the landmarks left during the procedure with the developed films and membrane spots were digested directly from the PVDF membrane. Additionally, gels from incomplete (reduced time) transblot experiments were stained with silver as described above and corresponding spots were excised after matching the gel with the Ponceau stain image of the PVDF membrane and with its own chemiluminescence exposed X-ray film. The PVDF membrane pieces were subjected to enzymatic digestion and Q-TOF analysis as described below. Enzymatic Digestion of Proteins and LC-MS/MS Analysis. The matched spots were excised and prepared for in-gel digestion by dehydration in a vacuum centrifuge (UNIVAPO 150H, UniEquip, Martinsried, Germany). The slices were rehydrated for digestion with 40 µL trypsin (10 ng/µL in 100 mM ammonium bicarbonate, pH 7.4; Promega, Mannheim, Germany) for 45 min on ice. The excess amount of trypsin solution was replaced by the same volume of 100 mM ammonium bicarbonate without trypsin. After overnight incubation at 37 °C, peptides were extracted and completely dried in a vacuum centrifuge. Tryptic digestion of proteins from PVDF membrane pieces was achieved as described previously.30 Membrane pieces were excised (2 mm2), and briefly washed with 50 µL of 100 mM ammonium bicarbonate buffer (pH 7.4) and then digested in 30 µL trypsin (10 ng/µL in 100 mM ammonium bicarbonate, pH 7.4; Promega, Mannheim, Germany) overnight at 37 °C. Peptides were extracted by repeated cycles of sonication in 0.1% TFA/50% ACN solution. The peptides were reconstituted in an aqueous solution of 0.1% (v/v) formic acid. For LC-MS/MS analysis, 1 µL of the reconstituted peptide samples was introduced into two consecutive C18-reversed phase chromatography columns (C18 PepMap, 300 µm × 5 mm, 5 µm particle size and C18 PepMap nanoanalytical column, 75 µm × 15 cm, 3 µm particle size, LC Packings, Germering, Germany) using an auto CapLC sampler (Waters, Eschborn, Germany). The chromatographically separated peptides were analyzed in a Q-TOF Ultima Global mass spectrometer (Micromass, Manchester, U.K.) equipped with a nanoflow ESI Z-spray source in the positive ion mode. The multiple charged peptide parent ions were automatically marked and selected in the quadrupole, fragmented in the hexapole collision cell and their fragment patterns analyzed by TOF. The data were acquired with the MassLynx (v 4.0) software on a Windows NT PC and further processed using ProteinLynx Global Server (PLGS, v 2.2, Micromass, Manchester, U.K.) as PKL (peaklist) under the fol-

Two-Dimensional Gel Electrophoresis (2DE) and 2DE Western Blotting. The concentrated and dialyzed secretory protein extracts were cleaned-up using 2D clean up kit (Amersham Biosciences) as recommended by the supplier. The protein pellet was dissolved in rehydration buffer (7 M Urea, 2 M Thiourea, 20 mM dithiothreitol, 0.5% Bioampholytes). Protein samples (40 µg)/7-cm immobilized pH gradient (IPG) strip with a linear pH range 3-10 and 4-7 (ReadyStrip, BioRad, Munich, Germany) were passively rehydrated. Isoelectric focusing was performed in a Protean IEF Cell (Bio-Rad, Munich, Germany). After equilibration each strip was loaded onto a vertical 12.5% polyacrylamide gel for SDS-PAGE. The gels were silver stained using a method described by Blum et al (1987)29 and scanned (CanoScan 8400F, Canon, Germany). The proteins of parallel gels were transferred onto nitrocellulose membranes (Amersham Biosciences) in a Mini Protean transfer unit (BioRad, Hercules, CA) overnight at 50 V at 4 °C. The transfer of proteins onto the membrane was visualized by reversible Ponceau staining followed by scanning. The blocking was carried out by treating the membranes with 2% BSA solution prepared in PBS containing 0.05% Tween-20 (PBST) with continuous shaking for 1.5 h at room temperature. Pooled immune sera from ABPA patients were used as the primary antibody, diluted (1:100) in PBST and kept shaking at room temperature for 3 h. The antihuman IgE (ε-chain specific, produced in goat, Cat No. A9667) and IgG (γ-chain specific, F(ab′)2, produced in goat, Cat No. A2290) antibodies conjugated with horseradish peroxidase (HRP) procured from SigmaAldrich, Germany, were diluted in PBST (1:3000) and used as the detector antibody separately for each blot for 1.5 h at room temperature. Excess secondary antibody was washed vigorously with PBST three times, each for 10 min. Finally, the membrane was washed once with PBS for 5 min before developing. The signal was detected by a chemiluminescence assay using ECL system (Thermo Scientific, Rockford, IL). The autoradiographs were developed using Kodak imaging films in accordance with the protocol provided by the supplier. For the reactivity of individual ABPA patients’ sera with secreted fraction of A. fumigatus strain (190/96), proteins were separated on narrow pH range IPG strips (pH 4-7). The separated proteins from gel were electro-transferred on to nitrocellulose membrane. The 2D IgE and IgG blots were developed separately with sera of 5 individual patients (S1-S5) which were selected randomly from the positive pool. The immunoblots were probed with detection antibodies (antihuman IgE and IgG conjugated with horseradish peroxidase) as described above. The blots showing IgE and IgG specific reactivity with pooled and individual patient’s sera are provided in Figures 4 and 5, respectively.

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Figure 1. 2DE 12.5% acrylamide gel separation of secreted proteins of A. fumigatus strains 190/96 (A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D) isolated from three weeks old culture filtrate by salting out method using 80% ammonium sulfate saturation. Forty microgram of protein was loaded onto linear pH gradient strips and separated in first dimension on the basis of their isoelectric points followed by separation in second dimension according to their molecular weight. After silver staining, protein spots were marked based on their reactivity with immune sera in Western blots. The marked spots were excised, in-gel digested, and subjected to liquid chromatography coupled with tandem mass spectrometry.

lowing settings; Electrospray, centrioid 80% with minimum peak width 4 channel, noise reduction 10%, Savitzky-Golay, MSMS, medium deisotoping with 3% threshold, no noise reduction and no smoothing. The peaklists were searched using the online MASCOT (http://www.matrixscience.com) algorithm against the Swiss-Prot 55.5 (389 046 sequences; 139 778 124 residues) and NCBInr protein database. The data were retrieved against the whole database with search parameters set as follows: enzyme, trypsin; allowance of up to one missed cleavage peptide; mass tolerance (0.5 Da and MS/MS tolerance (0.5 Da; modifications of cysteine carboamidomethylation and methionine oxidation when appropriate with auto hits allowed only significant hits to be reported. The proteins were identified on the basis of two or more peptides whose ions scores exceeded the threshold, P < 0.05, which indicated the 95% confidence level for these matched peptides. The proteins were accepted as identified if the threshold was exceeded and the protein spot possessed the correct molecular mass and pI value for the A. fumigatus isoform in the corresponding gels. All the experiments were repeated at least thrice, protein spots 5520


were digested from more than one gel and analyzed with mass spectrometry to ensure accurate identification. Functional Annotation and Prediction of Antigenic Index. List of known and predicted allergens31 of A. fumigatus was acquired from the Aspergillus database (www.aspergillus. org.uk) and functional annotation to all allergens as well as identified proteins was given by matching their accession numbers, obtained amino acid sequences at protein databases uniprot (www.uniprot.org) and kognitor32 (www.ncbi.nlm.nih. gov/COG/grace/kognitor). All acquired amino acid sequences of identified immuno-reactive proteins were analyzed by an in-silico sequence analysis tool DNAstar (DNAstar, windows 32 protean 5.07.1989-2003, DNAstar Inc. Madison, WI) for prediction of antigenic index of each protein. Prediction of Signal Peptide and Cellular Localization. Acquired amino acid sequences of all proteins were analyzed for presence of signal peptide by an in-silico Signal Peptide Prediction Tool SIG-Pred (http://bmbpcu36.leeds.ac.uk/prot_ analysis/Signal.html). Another web based in-silico sequence analysis tool “Subcellular Localization Prediction Tool, version

A. fumigatus Vaccine Candidates and Diagnostic Tools Table 1. Details of the ABPA Patients and Control Subjects Included in the Study

Number Age, years (range) Gender (M/F) Total IgE (IU/mL) (M ( SE) Specific IgE against secreted antigen (Abs. at 410 nm) (M ( SE) Specific IgG against secreted antigen (Abs. at 492 nm) (M ( SE)

control individuals

ABPA patients

12 25-65 8/4 328.8 ( 48 0.033 ( 0.006

13 28-60 5/8 1035.5 ( 170.8 0.42 ( 0.073

0.0424 ( 0.01

0.973 ( 0.115

3.0′′, PSORTb (http://www.psort.org/psortb/) was used to analyze the amino acid sequences of identified proteins for prediction of their cellular localization.

Results and Discussion The secreted fraction of two geographically different strains (190/96 and DAYA) of A. fumigatus were used to identify new immunogenic molecules reacting with pooled ABPA patients’ sera. An amount of 40 µg secretory proteins from 190/96 and DAYA strains of A. fumigatus was resolved on pH 3-10 and

research articles pH 4-7 linear IPG strips, followed by 2DE Western Blotting with pooled ABPA patients’ sera and silver staining (Figure 1A-D). Separation of proteins in pH 3-10 range showed that a large number of spots clustered in a pI range from 4.5 to 6.5 (Figure 1A and C). Therefore, in order to obtain better resolution of these spots, pH 4-7 IPG strips were employed (Figure 1B and D). The spot patterns from 2DE gels of two geographically distinct Aspergillus strains were significantly different from each other, even after growing under similar conditions (Figure 1). For the identification of immuno-reactive proteins of A. fumigatus, serum samples were obtained from the clinically diagnosed ABPA patients (n ) 13) having high titers of Aspergillus specific IgG, IgE and total serum IgE, and from apparently healthy individuals (n ) 12). The ELISA results from the ABPA patients and controls are shown in Table 1. Positive and negative sera were pooled separately for use in immunoblotting experiments. Specific immuno-blots for IgG and IgE from the secreted proteome of A. fumigatus strains were developed at both pH ranges (pH 3-10 and 4-7). Most IgG reactive proteins were concentrated in the high molecular weight (>50 kDa) area with pIs ranging from 4.5 to 6.0 (Figure 3A and B) in the 190/96 strain. The IgE reactive proteins of

Figure 2. 2DE immuno-blots of secreted proteome of A. fumigatus strains developed with pooled sera of ABPA patients and probed with antihuman IgE HRP conjugated antibodies, 190/96 (A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D). Matched immuno-reactive spots identified in 2DE gels were further excised for mass spectrometric analysis. Journal of Proteome Research • Vol. 9, No. 11, 2010 5521

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Figure 3. 2DE immuno-blots from secreted proteome of A. fumigatus strains developed with pooled sera of ABPA patients and probed with antihuman IgG HRP conjugated antibodies, 190/96 (A and B) and DAYA (C and D), in pH ranges 3-10 (A and C) and pH 4-7 (B and D). Matched immuno-reactive spots identified in 2DE gels were further excised for mass spectrometric analysis.

190/96 were discretely located in the blots (Figure 2A and B). However, there were some differences between the reactive spots from DAYA and 190/96 (Figure 3), but a noticeable number of reactive spots appeared to be common to both strains. Eighty-six spots from 2DE silver stain gels, corresponding to the signals of immuno-blots were excised, in-gel digested and identified (Figure 1). In detail, these spots included 44 from 190/96 (12 from 2DE gel of pH 3-10 and 32 from 2DE gel of pH 4-7) and 42 spots (13 from 2DE gel of pH 3-10 and 29 from 2DE gel of pH 4-7) from DAYA. Proteins underlying these spots were identified by liquid chromatography separation coupled with tandem mass spectrometry (Q-TOF MS/MS). Swiss-Prot and NCBInr protein database search identified 35 different A. fumigatus proteins (Table 2) from 86 excised spots. Twenty-four proteins showed immuno-reactivity both with IgG and IgE, whereas 7 reacted only with IgE (Figure 2) and 4 proteins were exclusively IgG reactive (Figure 3). These proteins showed variable IgG and IgE reactivity governed by presence of specific antigenic epitopes. Therefore, we also analyzed amino acid sequences of all 35 proteins by an in-silico analysis tool DNAstar (DNAstar, windows 32 protean 5.07.1989-2003, DNAstar Inc., Madison, WI). This analysis demonstrated their antigenic indices and located the position of antigenic epitopes 5522


within the amino acid sequence. Out of all identified proteins, 7 were already known as allergens (marked bold in Supplementary Figure 3A, Asp f3-pmp20 peroxisomal-like protein, Asp f10-aspergillopepsin, Asp f1-ribonucleoprotein/mitogillin, Asp f2-Hypothetical protein, Asp f4-Hypothetical protein, Asp f9crf1 and Asp f13/15-serine protease). Asp f3, a peroxiredoxin family-2 protein, only appeared to be IgG reactive in our study. Asp f3 is associated with type 1 hypersensitivity and the recombinant Asp f3 is one of the two single proteins that so far have been successfully used to induce a protective immune response in mice.17,33 We have detected this protein as a possible vaccine candidate in immuno-compromised rabbits which acquired protective immunity30 and in a previous A. fumigatus cDNA-expression library screening approach.34 Moreover, 3 other proteins [1,3-beta-glucanosyltransferase-gel1 (Spot No. 15), NAD-dependent formate dehydrogenase-AciA/Fdh (Spot No. 30) and malate dehydrogenase NAD-dependent (Spot No. 31)] along with Asp f3 have also been identified in our previous study.30 These molecules with thorough characterization may serve as possible vaccine candidates in future. Asp f9-probable glycosidase-crf1 protein and Asp f10-aspergillopepsin only reacted with the IgE antibody fraction from ABPA patients’ sera. Remaining four allergens were reactive with both IgG and IgE antibodies. The detection of a limited number of


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Figure 4. 2DE immuno-blots of secreted proteome of A. fumigatus strain 190/96 in narrow pH range (4-7) developed with pooled (2B) and individual sera (S1-S5) of ABPA patients and probed with antihuman IgE HRP conjugated antibodies.

already established allergenic proteins in the present study could be due to limitation associated with the gel-based strategy. In fact, it is not possible to cover all the immunogenic proteins using a single separation technique; therefore we do not claim to have discovered all the ABPA immuno-reactive proteins. Furthermore, the source of antigens and the sensitivity of the patients to different A. fumigatus antigens expressed

during various stages of infection may cause variability in immuno-reactivity pattern with 190/96 and DAYA strains of A. fumigatus. Three of the proteins, (Dipeptidyl-peptidase-5 precursor “DppV”, Nuclear transport factor-2 “NTF-2” and NAD-dependent malate dehydrogenase) can be categorized as predicted allergens on the basis of significant sequence homologies with Journal of Proteome Research • Vol. 9, No. 11, 2010 5523

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Figure 5. 2DE immuno-blots of secreted proteome of A. fumigatus strain 190/96 in narrow pH range (4-7) developed with pooled (3B) and individual sera (S1-S5) of ABPA patients and probed with antihuman IgG HRP conjugated antibodies.

the reported allergens from other fungal species (source, www.aspergillus.org.uk). DppV was reactive to both IgG and IgE and is a glycoprotein (hyphal invasion enzyme) responsible for the protein degradation of the host cell during invasion and characterized for their potential role in clinical applications.9,35 NTF-2 was only recognized by IgG in the present study and is a cross-reactive allergen produced by A. alternata and C. 5524


herbarum, which is recognized by sera from patients sensitized to A. fumigatus.36 NAD-dependent malate dehydrogenase is involved in citric acid cycle and glyoxalate cycle. Anticipated potential of these three proteins as allergens could be the subject of thorough validation studies. All 35 identified immunogenic proteins reacting with IgE and IgG antibodies were subjected to functional annotation in

Q00050 XP_748349

XP_748380 EDP51083 XP_749213 XP_747586

25 26

27 28 29 (a-b) 30

6

8 9 7 2

7 2 11 10

18 3

5 16 5 8 5 15 26 3 2 2 9 5 2

4

4 15

16

6 6 4 2 3 8

8

14

27 12 9 4

12 8 37 22

67 8

20 28 17 15 12 27 22 11 3 3 19 8 12

23

29 29

19

21 15 12 19 12 21

30

215

269 361 249 135

452 122 421 213

489 146

117 291 200 311 179 525 640 103 76 73 475 123 156

117

184 343

194

175 153 193 80 41 196

164

61.3/5.84

35.8/9.08 83.7/6.12 95.0/5.01 61.3/5.55

68.0/6.32 30.4/4.82 33.8/6.23 45.7/8.43

15.1/5.29 44.6/4.90

34.3/7.79 33.7/5.33 41.5/4.85 48.1/4.94 51.6/4.62 46.4/5.21 79.8/5.50 36.0/5.44 67.3/5.39 68.1/4.59 53.8/5.09 47.6/5.10 19.4/4.43

14.2/4.63

21.5/5.76 33.0/5.02

79.7/5.59

32.8/5.34 32.5/6.64 32.3/4.63 15.9/4.61 35.5/6.00 41.8/6.46

19.6/9.23

peptide sequence MASCOT theoretical matches coverage (%) score mass/pI

51/6.2-6.5

34/6.7 79/6.5-6.7 95/5.1 55/5.3-5.7

31/6.0 25/4.8 38/6.2-6.4 44/6.5

18/4.9 38/4.5

36/7.3-8.2 29/5.4-5.7 41/4.4-4.6 48/4.9-5.2 52/4.5 47/5.2-5.4 78/5.5-5.8 39/5.6 75/5.4 80/4.8 55/4.8-5.2 49/5.6 49/4.4

14/4.6

25/6.1-6.5 34/4.6-5.1

80/5.4-6.1

34/5.1-5.4 29/6.3-6.7 32/4.4-4.6 28/4.1-4.6 35/5.8-6.6 42/5.3-6.3

19/9.3

observed mass/pI

No

No No Yes Yes

No No Yes No

No Yes

No Yes Yes Yes Yes Yes Yes No Yes Yes Yes No No

No

No Yes

Yes

Yes No Yes Yes No No

Yes

secretary signal peptide biological function

Cytoplasmic

Cytoplasmic Cytoplasmic Cytoplasmic Cytoplasmic

Cytoplasmic Cytoplasmic Extracellular Cytoplasmic

Cytoplasmic Extracellular

Oxidoreductase Catalase Peroxidase Glycosidase Hydrolase FAD binding Oxidoreductase activity Isomerase

Not known Not known Lyase Oxidoreductase activity

Cytolysis Hemolysis Glycosidase Hydrolase

Non-Cytoplasmic Purine-specific ribonuclease Non-Cytoplasmic Not known Extracellular Not known Extracellular Glycosidase Hydrolase Extracellular Not known Unknown Not known Cytoplasmic Aminotransferase Transferase Unknown Hydrolase Protease Serine protease Cytolpasmic Chitinolysis Extracellular Glycosidase Hydrolase Transferase Unknown Transcription factor Transporter Extracellular Sugar binding Unknown Ligand binding Extracellular Aspartyl protease Extracellular Transferase activity Unknown Transferase activity Cytolpasmic Glycosidase Hydrolase Cytoplasmic Heme binding Non-Cytoplasmic Reductase Non-Cytoplasmic Lipolysis Extracellular Lipolysis Unknown Glycosidase Hydrolase Extracellular Glycosidase Extracellular Cell-redox homeostasis

cellular localization

2B

2B 2B 2B and 3B 2B and 3B

2C, D and 3C, D 3D 2A, B and 3A 2B

3D 2D and 3D

2C and 3C 2D and 3D 2D 2D and 3D 2B, D and 3D 2D and 3D 2B, D and 3D 2D 2B, D and 3D 2B and 3B 2B, D and 3B, D 2D and 3D 3B, D

3C

2A-D and 3B-D 2C, D and 3C, D

2A, B and 3A, B

2B-D and 3B-D 2C, D and 3C, D 2B 2C and 3C, D 2A, B and 3A, B 2A, B and 3A

2C and 3A, C

appeared signal in blots of figures

a Spot No. ) spot numbers correspond to silver stained gels, Western blot images, and nitrocellulose mambrene. MASCOT score ) Probability based Mowse score by homology based MASCOT search (all p< 0.05). pI ) isoelectric point. Peptide matches ) number of matched peptides by MS/MS analysis. Biological function ) predicted biological function from http://www.uniprot.org.

35 (a-b) EDP54506

31 XP_748936 32 (a-b) XP_747039 33 XP_750327 34 (a-b) XP_747715

Q4WW81 XP_750162 P41748 BOXT72 Q9P8U4 XP_001481609 Q92405 XP_750863 Q6U819 Q6U820 XP_752825 AAP23218 CAA12162

12(a-c) 13(a-b) 14 (a-b) 15 (a-b) 16 17 (a-b) 18 (a-b) 19 20 21 22 (a-b) 23 24

Fucose specific lectin, FleA FG-GAP repeat protein Asp f10-aspergillopepsin-F 1,3-beta-glucanosyltransferase,gel1 1,3-beta-glucanosyltransferase, gel2 Class V Chitinase Catalase-B, Cat-B Thioredoxin reductase, GliT Lysophospholipase-3, Plb3 Lysophospholipase-1, Plb1 Mannosidase, MsdS Chitinase, Chi-B IgE binding protein/Asp f3/putative peroxiredoxin-pmp20 Asp-hemolysin, Asp-HS GPI-anchored cell wall beta-1,3endoglucanase EglC Hypothetical protein AFUA_3G00600 Conserved hypothetical protein Pectate lyase A NAD-dependent formate dehydrogenase, AciA/Fdh Malate dehydrogenase, NAD-dependent Bifunctional catalase-peroxidase, Cat2 Beta-glucosidase FAD/FMN-containing isoamyl alcohol oxidase, MreA Glucose-6-phosphate isomerase

Nuclear transport factor 2, NTF-2

Q4WXR8

Dipeptidyl-peptidase-V precursor, DppV

11

O13479

8 (a-b)

Asp f2-hypothetical protein Asp f4-hypothetical protein Asp f9-probable glycosidase, crf1 Asp f13/15-hypothetical protein Hypothetical protein Aminotransferase-class V, putative

Chitosanase 1,3-beta-glucanosyltransferase, Bgt1

P79017 Q4WV60 CAA11266 O60022 Q4WEM3 Q4WTF6

2 (a-c) 3 4 (a-b) 5 (a-b) 6 (a-c) 7(a-d)

Asp f1-ribonuceoprotein

name

9 (a-b) AAD26111 10 (a-c) XP_752511

A46497

1

spot no.

accession no.

Table 2. Immuno-Reactive Proteins Analyzed by Mass Spectrometry (MS/MS) from Secreted Proteome of A. fumigatusa


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research articles accordance with the uniprot database (www.uniprot.org) and kognitor.32 Based on the functions, these proteins could be divided into 10 categories. Functional annotation of known and predicted A. fumigatus allergens also was performed (Supplementary Figure 3A and B, Supporting Information). It was observed that several proteins not belonging to any particular functional category may be involved in various important functions in fungal survival and virulence. Majority of the 35 proteins identified in present study belonged to carbohydrate transport and metabolism. Single candidate proteins were identified from the functional group of lipid transport and metabolism and energy production and conservation. The in-silico analysis of all 35 identified proteins by SIGPred for detection of signal peptides revealed the presence of signal peptides in 19 proteins. The remaining 16 proteins which did not have secretory signal peptides included 4 hypothetical proteins (Table 2). It has now been accepted that the presence of signal peptide may not be essential for the secretion of proteins as certain nonclassical secretory pathways also exist.37 In a study, Schwienbacher et al. (2005)38 analyzed in vitro the major secreted proteins of A. fumigatus and detected presence of antimitogilin and antichitosanase antibodies in the sera of patients of invasive aspergillosis and aspergilloma. They proposed mitogilin (Asp f1/ribonucleoprotein39) and chitosanase to be potentially relevant for diagnosis of aspergillosis. They also identified three more proteins (aspergillopepsin, Chitinase-B and 1, 3-endoglucanase) which were secreted by A. fumigatus in different growth media. The results of present study also showed the presence of these five proteins, identified from the Spot Nos. 1, 9, 14, 23, and 26. It is thus considered that the fraction used in the present study contained major secreted proteins of A. fumigatus. To determine the cellular localization of all identified proteins, their amino acid sequences were analyzed by PSORTb. The analysis showed that out of 35 proteins, 12 were extracellular, 4 were noncytoplasmic, 12 localized in cytoplasm and 6 had unknown cellular localization with equal probability of being extracellular, noncytoplasmic or cytoplasmic (Table 2). In the host, A. fumigatus secretes several proteins which induce an allergic response. The candidates for such responses could be the proteins that react specifically with IgE antibodies (Figure 2) such as, Asp f2, Asp f15, a 19 kDa allergenic ceratoplatinin, and rAsp f3 (a pmp20, peroxisomal like protein peroxiredoxin family-2).40,41 Cell wall glycosylated proteins of A. fumigatus are divided into cell wall biosynthetic proteins and cell wall degrading molecules. rAsp f9 (probable glycosidase, crf1) is a cell wall biosynthesis associated protein, whereas 1,3-beta-glucanosyltransferase is described to be involved in the cell wall morphogenesis as an anchoring molecule by incorporating 1,3-beta linkages in between 1,3-beta-glucan molecule.20,42,43 Mannosidase is an essential fungal cell wall glycoprotein.44 Moreover, second category contains chitin dissolving molecules such as, chitosanase, class-V Chitinase and Chitinase.45-47 These chitin dissolving glycosidases/hydrolases are abundant in the cell wall which underlines their importance in maintaining appropriate cell wall integrity. These cell wall linked molecules have been considered to be targets for antifungal drug development. Lectins are also considered to be important cell wall associated molecules due to their specific sugar recognition properties. Presence of a fucose specific lectin on the conidial surface reflects a potential role of laminin and fibrinogen in the adherence of A. fumigatus conidia.48 5526


Singh et al. A. fumigatus secretes a large number of proteases and lipases. The role of proteases in conferring virulence still remains controversial.49 The proteases identified in present study included DppV35 a glycoprotein (hyphal invasion enzyme), aspergillopepsin-F (Asp f10) a secreted aspartic protease of the pepsin family and asp-hemolysin.50 The immunoreactivity of these proteases with ABPA patients’ sera provided promising targets for the further investigation of these proteins as potential allergens. These proteins are known for their proteolytic, elastinolytic, hemolytic and cytotoxic activity and collectively may contribute to the degradation of host lung tissue during invasive diseases. Along with proteolytic enzymes A. fumigatus also secretes lipolytic enzymes like, lysophospholipases-3 and lysophospholipases-1. Considering metabolic processes of A. fumigatus, sugar metabolism is the major process by which energy is provided to the fungus to grow in the relatively oxygen deprived environment of the host body. Certain enzymes of sugar metabolism reacted with the patient’s sera, such as, NAD-dependent malate dehydrogenase, beta glucosidase, FAD/FMN-containing isoamyl alcohol oxidase MreA and glucose-6-phosphatase isomerase. The presence of these enzymes of sugar metabolism and energy production in secretome may be due to the fact that they are in abundance in the fungus. The reactivity of these molecules with IgG and IgE antibodies of ABPA patients’ sera may substantiate their role as a panel of proteins for more specific diagnosis of ABPA. Stress proteins are also considered to be an important category of pathogen defense proteins. Molecules of this category play an important role in disease progression and are considered as pathogenic factors.51 Many proteins identified in this study like, DppV,35 Asp f10-aspergillopepsin-F,50 GliTthioredoxin redutase,52,53 rAsp f3-pmp20,17 Cat2-bifunctional catalase peroxidase54 are already extensively studied for their biological role. Catalases secreted by A. fumigatus are considered to be important fungal defense enzymes, responsible for partial escape from the phagocytolytic activity. There have been reports that showed the catalases of A. fumigatus to be insufficient in providing protection against macrophage killing. The immunogenic catalases such as catalase-B (Spot No. 18) and bifunctional catalase peroxidase cat2 (Spot No. 32) may be involved in the protection of the pathogen against host defense by partially inactivating H2O2.54-56 A. fumigatus produced diverse pathogenic molecules which made its survival easier in the host and also have been found to be responsible for development of ABPA.8 Biological functions of most A. fumigatus proteins so far have been predicted from its genomic sequences. There may be some molecules with unknown biological functions which still have potential to play a role in host pathogen interaction and development of disease. We indeed identified three such proteins (Spot No. 6, 27 and 28) without any known experimental or putative functional elucidation. The characterization of these 3 immunogenic, so far, hypothetical proteins of unknown function may reveal their possible role in development of Aspergillus-induced infections. We identified 35 proteins using immunoreactivity of pooled sera of ABPA patients with the secreted fraction of two different A. fumigatus strains (190/96 and DAYA) in broad (pH 3-10) and narrow pH (pH 4-7) ranges. In a next step, we investigated whether these proteins were consistently reacting with the IgG/ IgE from individual ABPA patient’s sera. For this purpose, we randomly selected sera of 5 individual ABPA patients from the positive pool and developed their IgE and IgG immunoblots with the secreted fraction of 190/96 in pH range 4-7. The

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A. fumigatus Vaccine Candidates and Diagnostic Tools a

Table 3. Secreted Proteins of A. fumigatus Strain 190/96 Reactive with Sera of Individual ABPA Patients spot no.

identifications in A. fumigatus

2 4 6 7 8 9 16 18 20 21 22 24

Asp f2-hypothetical protein Asp f 9-probable glycosidase, crf1 Hypothetical protein Aminotransferase-class V, putative Dipeptidyl-peptidase-V precursor, DppV Chitosanase 1,3-beta-glucanosyltransferase, gel2 Catalase-B, Cat-B Lysophospholipase-3, Plb3 Lysophospholipase-1, Plb1 Mannosidase, MsdS IgE binding protein/Asp f3/putative peroxiredoxin-pmp20 Pectate lyase A NAD-dependent formate dehydrogenase, AciA/Fdh Malate dehydrogenase, NAD-dependent Bifunctional catalase-peroxidase, Cat2 Beta-glucosidase FAD/FMN-containing isoamyl alcohol oxidase, MreA Glucose-6-phosphate isomerase

29 30 31 32 33 34 35

accession no.

immuno-reactivity

pooled sera

S1

S2

S3

S4

S5

P79017 CAA11266 Q4WEM3 Q4WTF6 O13479 AAD26111 Q9P8U4 Q92405 Q6U819 Q6U820 XP_752825 CAA12162

IgE and IgG IgE IgE and IgG IgE IgE and IgG IgE and IgG IgE IgE IgE IgE and IgG IgE and IgG IgG

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

+ + + + + + + + + + -

+ + + + + + + + + +

+ + + + + + + + + + -

+ + + + + + + + + -

+ + + + + + + + + -

XP_749213 XP_747586 XP_748936 XP_747039 XP_750327 XP_747715 EDP54506

IgE IgE IgE IgE IgE and IgG IgE and IgG IgE

+ + + + + + +

+ + + + + + +

+ + + + + + +

+ + + + +

+ + + + + +

+ + + +

a Spot No. ) spot numbers correspond to silver stained gels, Western blot images, and nitrocellulose membrane. S1-S5 ) sera from ABPA patient number 1 to 5.

individual ABPA patients’ immunoblots were compared with blots of pooled serum to identify consistently reactive common proteins. The IgE immunoblots of pooled serum showed presence of 18 antigens, whereas 10 immunoreactive proteins were present in the IgG blots. The reactivity of individual sera revealed 8 proteins (Spot No. 2, 6, 8, 9, 21, 22, 33 and 34) with both IgE and IgG reactivty, 7 of which reacted consistently with all five individual patients’ sera (Table 3). There were 10 proteins that selectively reacted with IgE and only 1 (Spot No. 24) was IgG reactive. The remaining proteins out of 35 reactive to pool sera, did not appear in the list. The major reason for the observed discrepancy may be due to the fact that the DAYA strain was not tested and we used limited pH range (only narrow pH). Further studies on these proteins seem to be warranted. In conclusion, the present study describes new properties of these 25/35 A. fumigatus proteins with respect to their IgG and IgE binding capability. In testing individual sera from ABPA patients, we identified a panel of proteins which were consistently reacting with IgG and IgE. Recombinant expression and thorough characterization of such consistently IgE reactive proteins from this panel hold potential to replace the present crude extract by recombinant proteins in diagnostic tools for the detection of aspergillosis. The proteins identified from Spot Nos. 6, 29, and 35 corresponding to a hypothetical protein (Accession No. Q4WEM3), pectate lyase A (Accession No. XP_749213) and Glucose-6-phosphate isomerase (Accession No. EDP54506) are patent protected (Patent No. 1278/DEL/2010, IPMD, Delhi, India).

Acknowledgment. This work was conducted under the Indo-German collaborative project funded by Bundesministerium fu ¨ r Bildung und Forschung, Federal Republic of Germany and Department of Biotechnology, Government of India and Council of Scientific and Industrial Research, Government of India. We are also thankful to Dr. H. R. Das for giving access to the instruments of her laboratory at IGIB, CSIR, Delhi, India and Christina Weise of Proteomics Lab, UMG, Goettingen, Germany for technical support.

Supporting Information Available: Control immunoblots of both A. fumigatus strains (190/96 and DAYA) developed using secretory fraction and pooled sera of apparently healthy individuals. Pie charts showing functional categorization of known predicted allergens and all 35 identified proteins into different functional groups according to kognitor.36 Detailed analysis table (MS/MS) showing identification of all proteins with number of matched peptides, percentage of sequence coverage, MASCOT score, molecular mass and isoelectric point. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Pfaller, M. A.; Diekema, D. J. Rare and Emerging Opportunistic Fungal Pathogens: Concern for Resistance beyond Candida albicans and Aspergillus fumigatus. J. Clin. Microbiol. 2004, 42, 4419– 4431. (2) Ronning, C. M.; Fedorova, N. D.; Bowyer, P.; Coulson, R.; Goldman, G.; Kim, H. S.; Turner, G.; Wortman, J. R.; Yu, J.; Anderson, M. J.; Denning, D. W.; Nierman, W. C. Genomics of Aspergillus fumigatus. Rev. Iberoam. Micol. 2005, 22, 223–228. (3) Shah, A.; Panjabi, C. Allergic bronchopulmonary aspergillosis: a review of a disease with a worldwide distribution. J. Asthma 2002, 39, 273–289. (4) Kurup, V. P.; Banerjee, B.; Hemmann, S.; Greenberger, P. A.; Blaser, K.; Crameri, R. Selected recombinant Aspergillus fumigatus allergens bind specifically to IgE in ABPA. Clin. Exp. Allergy 2000, 30, 988–993. (5) Ruhnke, M.; Maschmeyer, G. Management of mycoses in patients with hematologic disease and cancer-Review of the literature. Eur. J. Med. Res. 2002, 7, 227–235. (6) Wiebe, V.; Karriker, M. Therapy of systemic fungal infections: a pharmacologic perspective. Clin. Tech. Small Anim. Pract. 2005, 20, 250–257. (7) Ward, O. P.; Qin, W. M.; Dhanjoon, J.; Ye, J.; Singh, A. Physiology and biotechnology of Aspergillus. Adv. Appl. Microbiol. 2006, 58, 1–75. (8) Nierman, W. C.; Pain, A.; Anderson, M. J.; Wortman, J. R.; Kim, H. S.; Arroyo, J.; Berriman, M.; Abe, K.; Archer, D. B.; Bermejo, C.; Bennett, J.; Bowyer, P.; Chen, D.; Collins, M.; Coulsen, R.; Davies, R.; Dyer, P. S.; Farman, M.; Fedorova, N.; Fedorova, N.; Feldblyum, T. V.; Fischer, R.; Fosker, N.; Fraser, A.; Garcia, J. L.; Garcia, M. J.; Goble, A.; Goldman, G. H.; Gomi, K.; Griffith-Jones, S.; Gwilliam, R.; Haas, B.; Haas, H.; Harris, D.; Horiuchi, H.; Huang, J.; Humphray, S.; Jimenez, J.; Keller, N.; Khouri, H.; Kitamoto, K.;


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