Proteomic Characterization of Aspergillus fumigatus Treated with an

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Proteomic Characterization of Aspergillus f umigatus Treated with an Antifungal Coumarin for Identification of Novel Target Molecules of Key Pathways Seema Singh,†,§ Shilpi Gupta,‡ Bharat Singh,†,∥ Sunil K. Sharma,‡ Vijay K. Gupta,∥ and Gainda L. Sharma*,† †

Division of Diagnostics and Biochemistry, CSIR-Institute of Genomics and Integrative Biology, University Campus, Mall Road, Delhi-110007, India ‡ Department of Chemistry, University of Delhi, Delhi-110007, India § Department of Biotechnology, University of Pune, Pune-411007, India ∥ Department of Biochemistry, Kurukshetra University, Kurukshetra-136119, India S Supporting Information *

ABSTRACT: A synthetic coumarin, N,N,N-triethyl-11-(4-methyl-2-oxo2H-chromen-7-yloxy)-11-oxoundecan-1-aminium bromide (SCD-1), having potent activity against pathogenic Aspergilli (MIC90 15.62 μg/mL), was investigated to identify its molecular targets in the pathogen. The proteome of Aspergillus fumigatus was developed after treatment with sublethal doses of compound and analyzed. The results demonstrated 143 differentially expressed proteins on treatment with SCD-1. The expression of four proteins, namely cell division control protein, ubiquitin-like activating enzyme, vacuolar ATP synthase catalytic subunit A, and UTP-glucose-1phosphate uridylyltransferase of A. f umigatus, was completely inhibited, whereas there were 13 newly expressed and 96 overexpressed proteins, mainly belonging to stress pathway. The treatment of A. f umigatus with SCD-1 also led to attenuation of proteins involved in cell replication and other important biosynthetic processes, including riboflavin biosynthesis, which has been pathogen-specific. In addition to key enzymatic players and antioxidants, nine hypothetical proteins were also identified, seven of which have been novel, being described for the first time. As no cellular functions have yet been described for these hypothetical proteins, their alteration in response to SCD-1 provides significant information about their putative roles in pathogen defense. KEYWORDS: Aspergillus f umigatus, therapy, coumarin, antifungal, proteome, molecular targets

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enzyme systems, thus inhibiting the cell growth.5 The echinocandins have been reported to target the fungal cell wall by inhibiting β, 1−3 glucan synthesis and, hence, depleting glucans, which are necessary to maintain its stability.6 The drugs presently used for treating aspergillosis have been found to be highly toxic and immunosuppressive.7 The development of resistance in the pathogen against most antifungals has been another major reason for the limited therapeutic success of these drugs.8 Owing to the limitations associated with current antifungals, the identification of new molecules to develop improved therapeutic formulations with better efficacy and less toxicity has been emphasized. This can be accomplished by identifying molecules which use pathways different than those used by current drugs to exhibit pathogen-specific activity. Therefore, development of ideal antifungals having novel and specific

INTRODUCTION A. fumigatus has been a highly evolved saprophytic mold bestowed with numerous adaptations which enable it to survive in a multitude of extreme environmental conditions.1,2 The morbidity and mortality caused by A. f umigatus infection has been significant, as successful management of the disease often becomes complicated as a result of the delay in establishing diagnosis and lack of effective drugs. Current therapeutic options for aspergillosis have been limited to only a few classes of antifungal agents, such as polyenes (amphotericin B and its liposomal formulations), azoles (fluconazole, voriconazole, itraconazole), and echinocandins (caspofungin and anidulafungin).3 Most of these drugs have been known to target the fungal cell wall and cell membrane. The polyenes bind to the ergosterol and form transmembrane channels leading to efflux of monovalent cations to the exterior, thus disrupting the membrane function.4 Azoles inhibit the ergosterol biosynthesis by targeting demethylation of lanosterol, leading to accumulation of 14α-methylsterols. This leads to impairment of other © 2012 American Chemical Society

Received: January 3, 2012 Published: April 25, 2012 3259

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USA). The germinating conidia were examined microscopically after 16 h of incubation, and the growth was seized by placing the culture flasks on ice for 30 min. (ii) Protein Preparation. The total cytosolic proteins from germinating conidia of control and SCD-1 treated cultures were isolated by the method established in our laboratory by Singh et al.24 Briefly, A. f umigatus was cultured in L-asparagine broth and fungal cells were pelleted by centrifugation at 6500g using a centrifuge (5810R eppendorf, Hamburg, Germany). The pellet was washed with 10 mM sodium phosphate buffer (pH 6.0) and subjected to mechanical grinding with a 0.4 volume of glass beads (0.5 mm) suspended in 50 mM Tris HCl buffer (pH 7.0) in a pestle mortar for 1 h at 4 °C. Soluble proteins were separated by centrifugation at 18,000g, filtered through a 0.2 μm membrane, aliquoted, and stored at −70 °C until use. (iii) Protein Quantification. The concentration of protein in the sample was estimated by the Bradford method using bovine serum albumin as standard.25 (iv) Gel Electrophoresis. The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed to fractionate A. f umigatus proteins. Equal amounts of protein prepared from SCD-1 treated and untreated cultures of A. f umigatus were run on 12.5% SDS-polyacrylamide gels and subjected to silver staining by the modified method of Blum et al.26 (v) Development of Proteomes. Two dimensional gel electrophoresis (2DE) was carried out to develop differentially expressed proteomes of A. f umigatus.24 The protein samples were precipitated using a 2DE clean up kit (catalogue no. 806484-51, GE Healthcare Biosciences Corporation, Piscataway, NJ), as per instructions from the supplier. The clean proteins were solubilized in rehydration buffer (RB) containing 7.0 M urea, 2.0 M thiourea, 20 mM dithiothreitol (DTT), 0.5% b i o a mp h o l y t e s , a n d 2% 3- [ ( 3 - c h o l a m i d o pr o p y l ) dimethylammonio]-1-propanesulfonate. A total of 300 μg of protein in RB (300 μL) was applied to 17 cm nonlinear (pH 3−10 and 4−7) IPG strips (BioRad, CA, USA) and left overnight for rehydration after overlaying with mineral oil (BioRad, CA, USA). Following incubation, the strips were transferred to the focusing tray. After adding mineral oil over the strips, the separation of proteins in the first dimension was performed in an IEF cell (BioRad, CA, USA) by using the standard program: 250 V for 30 min, 10,000 V for 3 h, and 10,000 V for an additional 40,000 V·h. After focusing, a twostep equilibration was performed in buffers containing SDS. In step-I, the strips containing immobilized proteins were reduced in buffer containing 6.0 M urea, 2% SDS, 30% glycerol, 2% DTT, and 0.375 M Tris-HCl (pH 8.8) for 20 min. Step-II involved alkylation of reduced proteins in the buffer of same composition except DTT, which was replaced by 2.5% iodoacetamide, again for 20 min. After equilibration the strips were placed over 1 mm thick 12.5% vertical acrylamide gel and held in position with molten 0.4% agarose containing bromophenol dye. The second dimension separation was performed in a Protean II XL assembly (BioRad, CA, USA) using Tris-glycine-SDS buffer (250 mM glycine, 25 mM Tris, and 0.1% SDS) until the dye front reached near the bottom edge of the gel. After electrophoresis, the gels were subjected to mass compatible silver staining.26 (vi) Gel Imaging and Excision of Spots. For the detection of differentially expressed proteins, the images were acquired as 16 bit gray scale tiff images using Gel doc XR (BioRad, CA, USA). The images were carefully aligned in

targets in pathogens for molecules obtained from synthetic and natural sources has been of prime importance.9,10 Among various classes of chemical moieties, the coumarins (2H-1-benzopyran-2-ones) have been reported to possess a wide range of biological properties.11−14 Although a few reports have suggested that coumarins may have antifungal potential,15,16 there has been no effort to study their anti-Aspergillus properties, cytotoxicity, and the mechanism by which they inhibit the growth of pathogen. The proteome based studies on A. f umigatus have provided crucial insight into molecular changes that occur in the pathogen due to alterations in the environment.17 Gautam et al.18 and Cagas et al.19 used proteomic tools to study the global response of A. f umigatus arising from stress generated by known antifungal drugs. We earlier screened a panel of coumarin derivatives,20 to identify and characterize novel coumarin(s) having potential against microbial pathogens. As a result, a synthetic coumarin derivative, SCD-1, was found to have significant activity against pathogenic species of Aspergillus. The present study deals with the identification of molecules targeted by SCD-1 to elucidate the probable mechanism of action by which the compound elaborates lethal effects on A. f umigatus.

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MATERIALS AND METHODS

Aspergillus Strain

The A. fumigatus strain used in the present study was a clinical isolate obtained from an ABPA patient and characterized at Vallabhbhai Patel Chest Institute, Delhi, India. It was further typed (ITCC6604) at Indian Type Culture Collection, Indian Agriculture Research Institute, New Delhi, India. Culture of Aspergillus

The culture of A. fumigatus was maintained aseptically on Sabouraud dextrose agar (Himedia, Mumbai, India) plates in a BOD incubator (Calton, NSW, India) at 37 °C. The spores/ conidia were harvested from 72 h cultures using 10 mM sodium phosphate buffer containing 0.9% NaCl (pH 6.0). The tubes were vortexed gently for 3−4 min, and spores were allowed to swell for 20−30 min at room temperature for use in subsequent experiments. Compound

The coumarin used was SCD-1, a well characterized quaternary ammonium alkyl ester with chemical formula N,N,N-triethyl11-(4-methyl-2-oxo-2H-chromen-7-yloxy)-11-oxoundecan-1aminium bromide. It was synthesized in the laboratory according to the procedure described in our earlier report.20 The compound was found to have MIC90 15.62 μg/mL determined by microbroth dilution and percent spore germination inhibition assays against pathogenic strain of Aspergillus.21,22 Molecular Target Identification

The protein molecules of A. f umigatus targeted by SCD-1 were identified by analyzing the differentially expressed proteomes of compound treated and untreated normal culture of A. f umigatus. (i) Treatment of A. f umigatus with SCD-1. A. f umigatus was cultured in the absence or presence of a sublethal concentration of SCD-1 (half of MIC90, i.e. 7.81 μg/mL) prepared in sterilized L-asparagine broth.23 Both control and treated cultures were incubated at 37 °C with continuous shaking at 200 rpm using a shaker incubator (C25KC, incubator shaker, New Brunswick Scientific, Edison, NJ, 3260

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proteins were identified on the basis of their two or more peptide matches whose ion scores exceeded the threshold, p < 0.05, which indicated the 95% confidence level for the peptides. The 2DE gels for control and SCD-1 treated proteins of A. f umigatus were run thrice in both pH ranges (pH 3−10 and pH 4−7) using samples prepared from two biological replicates. As it was not feasible to characterize all differentially expressed spots from one single gel because of slight clustering of proteins in the gels of broad pH range (pH 3−10), separation of proteins was achieved in the narrow pH range (pH 4−7) also, and well resolved matching spots were excised from more than one replicate gel for accurate identification.

uniform settings to minimize differences in protein separation and migration between gels. The spots of 2DE gels were analyzed by PDQuest software (version 8.0.1, BioRad, CA, USA) using an automated matching program, and any mismatch was corrected by the use of a manual matching feature available in the software. The proteins which disappeared, newly appeared, overexpressed, and underexpressed in A. f umigatus on treatment with SCD-1 were processed further for mass spectrometric characterization. Of the overexpressed and underexpressed proteins, the spots that showed at least 1.5-fold change in expression level (Student’s t test, p < 0.05) were selected for excision.27,28 (vii) In Situ Tryptic Digestion of Proteins and Peptide Extraction. The proteins of interest were subjected to in-gel tryptic digestion for their mass spectrometric analysis. The gel pieces were washed with 100 μL of ultrapure water for 10 min and then destained in 100 μL of destaining solution (15 mM potassium ferricyanide and 50 mM sodium thiosulfate in water). The destained gel pieces were again washed and dehydrated in 100 μL of acetonitrile (ACN) at room temperature for 5 min. The ACN was removed, and the gel pieces were air-dried for 5 min at room temperature. The proteins in the gels were reduced by incubating them with 150 μL of 10 mM DTT and 100 mM of ammonium bicarbonate (NH4HCO3) at 56 °C for 30 min. The reducing solution was discarded, and washing was performed again. The gel pieces were again dehydrated using ACN and air-dried. The dried pieces of gel were rehydrated in proteomic grade trypsin (20 ng/μL, Sigma, USA) in 25 mM NH4HCO3 and further incubated at 37 °C for 16 h. The peptides were then extracted in 20 μL of 1% trifluoroacetic acid. The mixture was sonicated in a water bath sonicator (Life Clear Equipments Pvt. Ltd., Mumbai) for 5 min, prior to mass spectrometric analysis. (viii) NanoLC MS-MS. The peptide solution (6 μL) was injected into an Agilent nanoLC-1100 (Agilent, Palo Alto, CA, USA) system coupled with a microwell-plate sampler and a thermostatted column compartment for preconcentration (LC Packings, Agilent). The samples were loaded onto the nano column, Zorbax 300SB-C18 (150 mm × 75 μm, 3.5 μm) using a preconcentration step in a microtrap column cartridge Zorbax 300SB-C18 (5 mm × 300 μm, 5 μm). The solvent system used was water/ACN, each containing 0.1% HCOOH, and the flow rate was maintained at 5 μL/min. After 5 min of equilibration, the precolumn was connected with the separating nanocolumn and multistep gradient of ACN (3% for 5 min, 15% for 5−8 min, 45% for 8−50 min, 90% for 50−70 min, then again 3% for 70−85 min) was employed to elute the peptides. An LC/MSD Trap XCT with a nanoelectrospray interface (Agilent) operating in the positive ion mode was used. The ionization of peptides was carried out using a 1.5 kV potential with a liquid junction and a noncoated capillary probe (New Objective, Cambridge, MA, USA). The peptide ions were analyzed by the data-dependent method as follows: full MS scan, the scan sequence consisted of 1 full MS scan followed by 4MS/MS scans of the most abundant ions. The data was analyzed using Agilent ion trap analysis software (version 5.2), and proteins were identified by MASCOT search (Matrix Science, London, U.K.; http://www.matrixscience.com) against the NCBInr database. The sequences obtained were searched with the following parameters: enzyme, trypsin; allowance of up to one missed cleavage peptide; peptide mass tolerance, (±)1.2 Da; fragment mass tolerance, (±)0.6 Da; fixed modification, carboamidomethylation; variable modification, oxidation. The

Functional Annotation and Cellular Localization

All the identified proteins were functionally annotated by using information obtained from Uniprot (www.uniprot.org) and Kognitor (www.ncbi.nlm.nih.gov/COG/grace/kognitor) protein databases. The cellular localization of the identified proteins was determined by using the online in silico analysis tool “Sub-cellular Localization Prediction Tool, version 3.0”, PSORTb (http://www.psort.org/psortb/). Protein−Protein Interaction and Pathway Prediction

The functional interaction and association between the identified proteins of A. f umigatus was examined by the precomputed database STRING,29 Version 9.0, http://stringdb.org/, with the following analysis parameters: species, A. f umigatus; confidence level, 0.400; active prediction methods, all; and using a combined list of Uniprot accession IDs as input. The biological pathways of the identified proteins were deduced from the Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.jp/kegg/), a resource of about 20 databases. The molecular interaction between the networks was integrated and visualized by using cytoscape (http://www. cytoscape.org/).30

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RESULTS AND DISCUSSION The present study demonstrated the effect of a well characterized antifungal coumarin, SCD-1, on the proteome of A. fumigatus. The subminimum inhibitory concentration (half of MIC90, i.e. 7.81 μg/mL) of SCD-1 was used for treating the cultures of A. f umigatus. Subsequently, the differential proteomics coupled with mass spectrometry was employed to characterize the proteomic signatures of A. fumigatus and identify the key molecules which showed altered expression on antifungal treatment. The cytosolic proteins prepared from normal and SCD-1 treated cultures of A. f umigatus were subjected to 1D SDSPAGE and 2DE gel electrophoresis to study their expression profiles. The 1D SDS gels clearly demonstrated the difference in the protein profile of SCD-1 treated cultures of A. f umigatus (Figure 1). The treatment resulted in altered expression of proteins in A. f umigatus (marked with arrows in Figure 1). To get a comprehensive view of differentially expressed proteins, high resolution proteomes of control and treated cytosolic proteins of A. fumigatus in a wide pH range (pH 3−10) were developed. The silver stained wide pH range gels demonstrated slight clustering of protein spots in the middle area of gels (Figure 2a and b). Therefore, narrow pH range (pH 4−7) gels were employed for better resolution and to have a clear view of the global effect of SCD-1 on the protein profile of A. f umigatus (Figure 2c and d). The 2DE gels were scanned to obtain the images and analyze the protein spots using PDQuest software. The analysis of 2DE proteomes clearly showed the effect of 3261

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with N. f ischeri and have not been described yet, whereas, of the six remaining hypothetical proteins, two, having Accession Nos. XP_748110 and XP_752513, were identified in A. f umigatus by Cagas et al.35 in a recent proteome based study. These proteins may be considered as important biomarkers and, therefore, merit further characterization. As the biological functions of these hypothetical proteins have not been reported until now, the proteomic studies focusing on their presence and sensitivity to drug treatment must provide useful insight into their biological roles and also help in designing new drugs. As the functional properties of proteins have been closely related to their cellular arrangement, we determined the subcellular localization of the identified proteins using PSORTb. The analysis demonstrated that the majority of identified proteins (113/143) were localized in the cytoplasm (Figure 4a). Such a pattern of cellular localization of proteins was indeed expected because cytosolic fractions of A. f umigatus were used to develop the 2DE proteomes. The functional categorization of all identified proteins was carried out by in silico analysis using Uniprot and Kognitor databases. Out of 143 identified proteins, 120 were alienated in the following major categories: (1) hypothetical proteins with unknown functions and those having predicted functions only, (2) proteins involved in : (a) posttranslational modification, turnover and folding, (b) metabolism of amino acid, (c) carbohydrate metabolism, (d) energy production and conversion. The remaining 23 proteins were distributed into other minor categories (Figure 4b). As the identified proteins belonged to diverse functional categories, it could be inferred that the SCD-1 treatment affected different biological processes of A. fumigatus by attenuating and inducing several molecules. The proteins targeted by SCD-1 included 32 allergenic molecules of A. fumigatus which were identified in our earlier studies.24,36 Four proteins of A. f umigatus whose expression was completely inhibited by SCD-1 were identified as cell division control protein (Cdc48), ubiquitin-like activating enzyme (UlaA), vacuolar ATP synthase catalytic subunit-A (V-ATPase), and UTP-glucose-1-phosphate uridylyltransferase (Ugp1). The altered ubiquitylation37 has been the characteristic of a variety of pathologic conditions, and its inhibition by SCD-1 by targeting Cdc48 and UlaA must have contributed to anti-A. f umigatus activity. A recent study by Okoli et al.38 on C. albicans provided supportive evidence to our finding that the inhibition of vacuolar ATP synthase/V-ATPase activity has been an important determinant in exerting antifungal activity. In addition, the suppressed expression of fungal Ugp1 as demonstrated in the present study holds significant therapeutic utility for A. f umigatus induced infections, as bacterial Ugp1 has already been reported as a target for drug design.39 It could thus be suggested that Cdc48, Ugp1, V-ATPase, and UlaA of A. f umigatus, which had not been expressed in response to treatment with SCD-1, may be potential targets for further evaluation. Interestingly, the expression of 30 proteins in A. f umigatus was found to be attenuated upon treatment with SCD-1 (Supporting Information Table 1). The most significant decrease (3.07−6.42-fold) was observed in the expression of six proteins, namely proliferating cell nuclear antigen (PCNA), mitochondrial cochaperone (GrpE), cobalamin-independent methionine synthase (MetH/D), methylene tetrahydrofolate dehydrogenase, adenosine kinase, and a conserved hypothetical protein. We observed more than 6-fold reduction in the

Figure 1. Polyacrylamide gel demonstrating the effect of SCD-1 on the expression profile of cytosolic proteins of A. f umigatus. The proteins from untreated cultures or those treated with a sublethal concentration of SCD-1 were isolated and fractionated by SDS-PAGE using 12.5% gel. Proteins affected by the treatment of SCD-1 have been marked with arrows. Lane C, A. f umigatus proteins from control culture; lane T, A. f umigatus proteins from SCD-1 treated culture; lane M, marker.

SCD-1 treatment on the expression profile of proteins in A. f umigatus (Figure 2). Although some limitations of 2DE, such as poor solubility of membrane and resolution of highly acidic and basic proteins, especially of the fungal proteins, are known, it still remains the most powerful tool for proteome analysis.31 The effect of amphotericin B and caspofungin on the proteome of A. f umigatus has been investigated by Gautam et al.18 and Cagas et al.,19 respectively. There also have been studies which reported drug induced changes in the proteome of Candida albicans.32,33 In the present study, high resolution proteomes were used to pick the functional molecules of A. f umigatus targeted by SCD1. A total of 208 differentially expressed protein spots were excised from pH 3−10 and pH 4−7 gels, and analyzed. The mass spectrometric analysis of excised spots led to the identification of 143 proteins of A. f umigatus in the molecular weight range of 120 to less than 20 kDa, and only one protein was identified from each spot. This could be attributed to the fact that only well separated protein spots were sliced out for spectrometric analysis. There were few proteins, especially in the low molecular weight region, which were excised for analysis but could not be characterized. The putative biological function and relative fold change in the expression of the proteins so identified as targets of SCD-1 has been provided in Supporting Information Table 1. Of the 143 characterized proteins of A. f umigatus, the expression of 96 increased and that of 30 decreased as compared to the case for solvent treated controls. It was important to observe that the expression of 4 proteins corresponding to Spot Nos. 3, 9, 10, and 15 was completely inhibited (Figures 2 and 3a) while 13 protein molecules identified from Spot Nos. 22, 24, 30, 31, 64, 95, 96, 107, 118, 125, 129, 130, and 143 (Figures 2 and 3b) were found to be newly expressed in A. f umigatus after treatment with SCD-1. Out of 143 characterized proteins, 134 were identical to those already reported in A. f umigatus whereas 9 had homology with proteins of Neosartorya f ischeri. Such homology may be associated with the fact that N. f ischeri has been the closest sexual relative, showing high degree of similarity, and has a close evolutionary relationship with A. f umigatus.34 The results also revealed nine hypothetical proteins targeted by SCD-1 in A. f umigatus, out of which three showed homology 3262

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Figure 2. Silver stained 2DE polyacrylamide gels of control and SCD-1 treated A. f umigatus cytosolic proteins, in the pH ranges 3−10 (a and b) and 4−7 (c and d). The cytosolic proteins were prepared from a 16 h culture, with or without treatment with SCD-1, by mechanical grinding of cells with glass beads at 4 °C. An amount of 300 μg clean protein was loaded on nonlinear pH gradient strips and focused, followed by a second dimension separation on 12.5% SDS polyacrylamide gels.

link between oxidative stress, methionine availability, and levels of methionine synthases. Therefore, significantly decreased expression of MetH/D in A. f umigatus after SCD-1 treatment indicated that oxidative stress occurred in response to the antifungal treatment. This observation holds importance, as the methionine−cysteine biosynthetic pathway has been considered as a target for novel antifungal drugs, since molecules of this pathway are essential for survival of pathogen in the host.45 It was further observed that there was decreased expression of methylene tetrahydrofolate dehydrogenase by SCD-1, which might suggest that there was dysregulation of processes involved in one-carbon metabolism and, hence, the antifungal effect was observed. A supportive report on the enzymes of folate-dependent one-carbon metabolism being potential pharmaceutical targets was provided by Schimdt el al.46 A 3.30-fold decrease in adenosine kinase expression in A. f umigatus on SCD-1 treatment was observed. Adenosine kinase

expression of PCNA after SCD-1 treatment. The PCNA has been reported to be involved in DNA replication and repair processes,40 and disrupting the replication machinery by targeting the PCNA sliding clamp has been indicated in the antibacterial drug discovery,41 but a similar strategy in treating aspergillosis has not been explored. This makes PCNA an attractive molecule for developing antifungal therapies. The molecular chaperones or heat shock proteins (Hsps) have been highly abundant (1−2%) proteins in eukaryotic cells which are found to increase by 4−6% under stress conditions.42 The homologue of cochaperone GrpE (Mge1) which has been reported to catalyze nucleotide exchange for Hsp70 in preprotein translocation and folding in Saccharomyces cerevisae was reported to be crucial for cellular functions.43 It could, therefore, be assumed that a decrease in abundance of GrpE by SCD-1 was an important factor leading to the inhibitory effect on A. fumigatus. Hondorp and Matthews44 suggested a possible 3263

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Figure 3. PDQuest generated representative images of proteins whose expression was completely inhibited (a) and induced by the treatment with SCD-1 (b).

The riboflavin biosynthesis has been specific to A. f umigatus.48 Kaiser et al.49 highlighted the significance of targeting biosynthetic pathways of riboflavin and tetrahydrofolate, since the corresponding pathways are absent in humans. The knowledge on decreased expression of molecules of the riboflavin biosynthesis pathway by SCD-1, therefore, will be of immense significance to develop less toxic and specific therapeutics for aspergillosis. Apart from proteins which did not express or had decreased expression upon SCD-1 treatment, there were 13 newly appeared and 96 with increased expression. A major biological functional category of these proteins was energy production and conversion, which included glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and pentose phosphate

plays an important role in the maintenance of intracellular and extracellular levels of adenosine, and the entities targeting adenosine kinases have been reported to demonstrate antinociceptive, anti-inflammatory, and anticonvulsant activities in animal models, thus suggesting their potential therapeutic utility.47 We also observed a significant decrease in the synthesis of a conserved hypothetical protein (XP_755445) of A. f umigatus. As this protein has not been reported to be affected by any of the previously described antifungal agents, its impaired expression by SCD-1 may present XP_755445 to be a novel antifungal target. The riboflavin biosynthesis pathway also emerged as an important target of SCD-1 in A. f umigatus, as the riboflavin synthase and riboflavin kinase showed a decrease in abundance. 3264

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Figure 4. (a) Subcellular localization of differentially expressed proteins identified on analysis by using PSORTb. (b) Functional classification of 143 identified differentially expressed proteins by Kognitor upon SCD-1 treatment.

pathway. An increase in the expression of glycolytic enzymes such as phosphoglycerate mutase 2,3-bisphosphoglycerateindependent, phosphoglycerate kinase, fructose-bisphosphate aldolase (class II), and enolase was observed. This could be a defense response in A. f umigatus against SCD-1 induced stress. The increased expression of components of pyruvate dehydrogenase complex, namely Pdx1 and PdbA, as observed in the present study, might be for efficient conversion of pyruvate to acetyl-CoA, which is the intersection point in important pathways of carbon metabolism.50 The acetyl-CoA hydrolase (Ach1), which has been known to catalyze the important step of acetyl-CoA hydrolysis to acetate, was expressed at a highly increased level (>8 fold) upon SCD-1 treatment. Such an increase in concentration of Ach1 could be a regulatory mechanism to maintain cellular acetyl-CoA levels, thus preventing autoacetylation of proteins and production of toxic metabolites.51 In addition to the heightened glycolytic activity, A. f umigatus in response to SCD-1 treatment also showed increased expression of enzymes and molecules of the TCA cycle, such as citrate synthase, aconitate hydratase, isocitrate dehydrogenase, dihydrolipoamide succinyl transferase, and the succinylCoA synthetase β subunit. TCA has been the major pathway

for oxidation of biomolecules, resulting in production of ATP through oxidative phosphorylation. Concomitant with the increase in cellular metabolic processes in A. f umigatus after SCD-1 treatment, the putative ubiquinol cytochrome C reductase complex core protein 2 was newly expressed whereas the expression of ATP synthase subunit E was increased. It was also found that the expression of 6-phosphoglucolactonase, transketolase, transaldolase, and 6-phosphogluconate dehydrogenase of the pentose phosphate pathway was also increased. Change in the abundance of enzymes of glycolysis, the TCA cycle, and the pentose phosphate pathway in response to treatment with different antifungals was also reported by Hoehamer et al.32 Enzymes of nucleotide metabolism, which included xanthine-guanine phosphoribosyl transferase (Xpt1), phosphoribosylglycinamide formyltransferase, formyltetrahydrofolate, and 3′(2′),5′-bisphosphate nucleotidase, were also observed to be increased in abundance on treatment with SCD1. A significant change was also observed in Xpt1 (>5 fold), which has been essential for synthesis of xanthine and guanine. Enzymes of nucleotide metabolism, such as 5-aminoimidazone4-carboxamide ribonucleotide transformylase and IMP cyclohydrolase, were also reported to overexpress in C. albicans by the treatment of mulundocandin.33 3265

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The treatment of A. f umigatus with SCD-1 also led to the increased production of proteins involved in amino acid biosynthesis and metabolism in the pathogen. These proteins included 60S ribosomal protein P0, 40S ribosomal protein S12, 40S ribosomal protein S3, translation elongation factor γ subunit, translation elongation factor 1 subunit putative and translation elongation factor EF2 subunit, carboxypeptidase, 3isopropylmalate dehydrogenase, glutamate/leucine/phenylalanine/valine dehydrogenase, aminopeptidase P, and alanine aminotransferase. The increased expression of molecules of amino acid biosynthesis in response to treatment with antifungal drugs has been reported by several workers.18,19,32 The elevated cellular stress induced by SCD-1 was demonstrated by the increased expression of Hsps and antioxidants. Among the heat shock proteins, Hsp88, Hsp90, and Hsp30/Hsp42 showed increased expression. Hsps have been known to be synthesized by the cells under stress conditions as a protective response.52 There was increased expression of cellular antioxidants such as mitochondrial peroxiredoxin, GliG, thioredoxin reductase, and antioxidant LsfA in A. f umigatus on exposure to SCD-1. Such responses to antifungals were also observed in other proteome based studies in A. f umigatus18 and C. albicans.32 The hydroxymethylglutaryl-CoA synthase, Erg13, has been an important protein of the ergosterol biosynthesis pathway, which showed increased expression in A. f umigatus on treatment with SCD-1. These observations were found to be consistent with the results reported in the literature.18,32 The increase in the enzymes of ergosterol synthetic pathways indicated that this could be a protective response against SCD1 induced depletion of ergosterol.32 The SCD-1 appeared to induce cytoskeletal rearrangement in A. f umigatus, as there was an increase in the expression of p20-ARC (a member of ARP 2/3 complex) and cofilin. Altered expression of cofilin in response to treatment with caspofungin was reported by Hoehamer et al.32 Among the elements showing increased expression of the signal transduction pathway, only crosspathway control protein-B, which has been a transcriptional activator G-protein complex b-subunit, was identified. Cross pathway control (Cpc) in A. f umigatus constitutes a major regulator of amino acid biosynthesis which is activated under stress conditions.53 Therefore, as observed in the present study, increased expression of the components of the Cpc system correlates well with the increase in the expression of proteins involved in the amino acid biosynthesis/metabolism. We observed the appearance of autophagic serine protease, Alp2, in response to the treatment with SCD-1. Mizushima et al.54 suggested that autophagy has been a promising drug target, as it is believed to result in decline of rapid fungal growth. The study of functionally interacting partners has been important to obtain a systemic view of the regulatory networks, where protein−protein interactions are known to play a crucial role in the cell. Since the function of a protein could be often determined by its interacting partner protein(s), an interactome of the molecules of A. f umigatus, which were inhibited completely and those which appeared new upon treatment with SCD-1, were selected to predict the interaction of molecules using the STRING database (Supporting Information Figure 1). There were a total of nine proteins which were found to interact with each other. To organize the findings and draw a complete view of the overall effect of SCD-1, the data set generated was analyzed by the KEGG database and the resulting SCD-1 responsive/targeted pathways were integrated

using cytoscape (Supporting Information Figure 2). The list of KEGG generated pathways has been provided in Supporting Information Table 3. The maximum numbers of interacting partners were observed for enolase, Ugp1, Gnd1, Hsp90, and Hsp88. Of the molecules whose synthesis in A. f umigatus was completely inhibited by SCD-1, Cdc48 and UlaA did not demonstrate interaction with any other protein, indicating them to exhibit toxicity to the pathogen independently. Similarly, of the newly expressed proteins upon treatment, oxidoreductase, glycerol dehydrogenase, farnesyl pyrophosphate synthetase, ubiquinol cytochrome C reductase complex core protein 2, and a conserved hypothetical protein, also did not show any interacting partner. The Gnd1 and Hsp90 were predicted to coexpress and occur in neighborhood. The putative homologues of Gnd1 and Hsp90 have been reported to interact in Saccharomyces cerevisae.55 The enolase and Ugp1 in addition to coexpression have been shown to interact by experimental/ biochemical findings.56 The experimental evidence of interaction between Hsp90 and Ugp1 has also been demonstrated in S. cerevisae by McClellan et al.57 The molecular chaperones Hsp90 and Hsp88 have been predicted to coexpress in Caenorhabditis elegans, Arabidopsis thaliana, and S. cerevisae.29 It has also been demonstrated in one of our previous studies that Gnd1, Hsp90, Hsp88, and enolase are immunodominant allergens and coexpress in A. f umigatus.24 Abad et al. (2010) highlighted Alp2 and Hsp90 as factors important in making A. f umigatus a successful pathogen.1 The pictorial representation of SCD-1 responsive proteins identified in the present study, and their involvement in key metabolic pathways of the pathogen, has been provided in Supporting Information Figure 3. The proteins belonging to minor categories and those with unknown functions were excluded while constructing the pathway chart. A total of 86 proteins of A. f umigatus were found to be distributed over different major biological processes. The proteins, whose expression was completely inhibited by SCD-1, have been marked with the symbol “Ψ”. It was important to note that there were several intermediate molecules which adversely affected the vital processes to enhance the killing pathway, while at the same time there had been induction/potentiation of the fungal defense mechanisms for countering the antifungal stress generated by SCD-1 treatment. In conclusion, the present study demonstrated the effect of SCD-1 on the proteome of A. f umigatus. The treatment of pathogen with the compound resulted in the differential expression of a number of proteins, of which 143 were characterized by tandem mass spectrometry. The SCD-1 completely inhibited the expression of four proteins of crucial metabolic processes and decreased the abundance of two proteins belonging to the pathogen specific riboflavin synthesis pathway in A. f umigatus. Thus, these proteins could be considered as important molecular targets of SCD-1. Further, the molecular imbalance generated as a reslut of altered expression of other proteins in the cell could also be equally important for the killing pathway in A. f umigatus. Although further validation of different target molecules has been warranted, the global responses of A. f umigatus toward SCD1 at the protein expression level have set a panel of molecules for further studies, and the same may be used for designing suitable antifungal compounds, including more effective analogues of SCD-1. 3266

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ASSOCIATED CONTENT

S Supporting Information *

Protein−protein interactions among the differentially expressed proteins using the STRING database (Supplementary Figure 1). The KEGG generated SCD-1 responsive pathways after integration using cytoscape (Supplementary Figure 2). Pictorial representation of the proteins found in the present study showing distribution in different biological pathways of A. f umigatus (Supplementary Figure 3). MS/MS identification of proteins of A. f umigatus expressed differentially on treatment with SCD-1 (Supplementary Table 1). Mass spectral details of identified proteins showing number of matched peptides, percentage of sequence coverage, MASCOT score, molecular mass, and isoelectric point (Supplementary Table 2). List of KEGG generated A. f umigatus pathways targeted by SCD-1 (Supplementary Table 3). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91-11-27667439. Fax: +91-11-27667471. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are thankful to Council of Scientific and Industrial Research, Defence Research and Development Organization, and University Grants Commission, Government of India, for providing research facilities and a financial grant. We also thank Ms. Shikha Chawla of The Centre for Genomic Application for her help provided during mass spectrometry.

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