Candida albicans strain SC5314/ATCC® MYA-2876™ (American Type Culture Collection, Manassas, VA, USA) was maintained on yeast extract-peptone-dextrose (YPD) medium (1% w/v yeast extract, 2% w/v glucose, and 2% w/v peptone) and incubated at 30°C. J774.1 murine macrophages (RIKEN BioResource Center, Ibaragi, Japan) were maintained in complete culture medium [DMEM media containing 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Carlsbad, CA, USA)] at 37°C in a humidified atmosphere with 5.0% CO2. Cells were maintained at low densities (75% confluence) and passaged until reaching the confluent state, usually every 3–4 days on 90 mm cell culture dishes. For phagocytic experiments, macrophage cells were plated at 1.0 × 105 cell/well for 16 h prior to the experiments.
Interaction of macrophages with C. albicans
A total of 5.0 × 106 macrophage cells were plated in complete culture media in culture dishes for 16 h prior to the experiments. C. albicans was pre-cultivated in 10 ml YPD media for 12 h. These C. albicans cells were washed with complete culture medium, counted with a hemocytometer and diluted to 5.0 × 106 cell/ml in 50 ml of complete culture medium. A total of 5.0 × 106
C. albicans cells were added per macrophage dish to obtain a fungus-macrophage ratio of 10:1 and incubated for the indicated times at 37°C and under 5.0% CO2.
Measurement of tumor necrosis factor-α (TNF-α)
Fifty microliters of the supernatant of C. albicans-macrophage interaction cultures were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 12, and 24 h to determine TNF-α levels. The amount of TNF-α was determined using a mouse TNF-α ELISA kit (R&D systems, Minneapolis, MN, USA), according to the manufacturer’s instructions.
Isolation of proteins
The macrophage cells were incubated with C. albicans cells for 3 h for proteome analysis. After three washing with ice-cold phosphate-buffered saline (PBS; pH 7.4, 1.4 M NaCl, 81 mM Na2HPO4, 27 mM KCl, 15 mM KH2PO4), the macrophages and C. albicans cells were dislodged by scraping the dish with rubber scrapers in ice-cold wash buffer [20 mM Tris-HCl, pH 7.8, containing 1.0% protease inhibitor cocktail for use with mammalian cell and yeast extract (Life Technologies)]. Collected samples were frozen quickly using liquid nitrogen and preserved at −80°C until use. Proteins were extracted as described previously with minor modifications (Aoki et al. 2013). Briefly, each sample was centrifuged for 5 min at 3,000×g, and the resulting cell pellets were suspended in 400 μl of lysis buffer [4% w/v 3-(3-cholamidepropyl)dimethylammonio-1-propanesulfonate, 1% w/v dithiothreitol, 1% v/v protease inhibitor cocktail for mammalian and yeast cells, 7 M urea, and 2 M thiourea in 20 mM Tris-HCl, pH 7.8]. The solution was mixed with 200 mg of 0.5 mm beads (TOMY SEIKO, Tokyo, Japan), and the cells were mechanically disrupted 10 times using a BeadSmash 12 (Wakenyaku, Kyoto, Japan) at 4°C, 4,000 oscillations per minute for 1 min. The solution was centrifuged at 3,000×g for 15 min and the supernatant was collected. Two hundred microliters of 200 mM triethyl ammonium bicarbonate (TEAB; Sigma-Aldrich, St. Louis, MO, USA) was added to each pellet and centrifuged at 3,000×g for 15 min. The supernatant was combined with the previously collected supernatant and the solutions were concentrated using Amicon Ultra YM-10 (Millipore, Bedford, MA, USA) with buffer exchange into 200 mM TEAB. The concentrated samples were dissolved in 100 μl of 200 mM TEAB.
Reduction, alkylation, and digestion
The sample solutions were mixed with 135 µl of 200 mM TEAB and 30 μl of 200 mM tris-(2-carboxyethyl) phosphine. The solutions were incubated at 55°C for 1 h for reduction. After the reaction, 60 μl of 375 mM iodoacetamide was added to the solutions and incubation was continued for 30 min at room temperature, with protection from light. The reactants were mixed with 1 ml of ice-cold acetone and incubated at −20°C for 2 h to precipitate the proteins. The precipitated proteins were suspended with 250 μl of 200 mM TEAB and mixed with 2 μl of sequencing grade modified trypsin (1 µg/μl) (Promega, Fitchburg, WI, USA). The mixture was incubated at 37°C for 12 h. Peptide concentration was determined using the Bicinchoninic acid assay kit (Nacalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions.
The peptide solutions were labeled using the TMT sixplex Isobaric Label Reagent Set (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s protocol. The TMT-labeling reagents were dissolved in 41 µl acetonitrile and mixed with 0.75 µg of each digest. In brief, a total of 0.75 µg of the proteins from the monoculture and co-culture were mixed with TMT-126 and -127, respectively. In addition, an equal amount of a mixture containing all of the samples types was labeled with TMT-131 as an internal control for quantification. The reactions were quenched by the addition of 8 µl of 5% hydroxylamine, followed by combining and lyophilizing the solutions. The dried samples were dissolved in 200 µl of 0.1% formic acid for calibration of TMT labeling. To measure the precision of protein quantification with the nanoLC–MS/MS system, the standard sample was separated into three tubes at a ratio of 0.5:1:2 by volume and the samples were labeled with TMT-128, TMT-130, and TMT-131, respectively. After quenching the reaction, these samples were combined in a single tube and injected into the nanoLC–MS/MS and the relative intensities of reporter ions of each identified protein were calculated.
Proteome analyses were performed using a nanoLC (Ultimate 3000®; Thermo Fisher Scientific)-MS/MS (LTQ Velos orbitrap mass spectrometer®; Thermo Fisher Scientific) system. Tryptic digests were injected and separated by reversed-phase chromatography using a long monolithic silica capillary column, which was prepared from a mixture of tetramethoxysilane and methyltrimethoxysilane (500-cm long, 0.1-mm ID) as described previously (Motokawa et al. 2002; Aoki et al. 2013), at a flow rate of 500 nl min−1. A gradient was established by changing the mixing ratio of the two eluents; A, 0.1% (v/v) formic acid; and B, 80% acetonitrile containing 0.1% (v/v) formic acid. The gradient was started with 5% B, increased to 45% B for 600 min, further increased to 95% B to wash the column and then returned to the initial condition and held for re-equilibration. The separated peptides were detected on the MS with a full-scan range of 350–1,500 m/z (resolution 60,000) in the positive mode followed by 10 data-dependent high-energy C-trap dissociation (HCD) MS/MS scans to acquire TMT reporter ions. For data-dependent acquisition, the method was set to automatically analyze the top 10 most intense ions observed in the MS scan. An ESI voltage of 2.4 kV was applied directly to the LC eluent distal to the chromatography column. Normalized collision energy of 40% in HCD with 0.1 ms activation time was used. The dynamic time exclusion was 180 s. The ion-transfer tube temperature on the LTQ Velos ion trap was set to 300°C. Triplicate analyses were done for each sample of four biological replicates.
The mass spectrometry data of each biological replicate was used for protein identification and quantification. Analysis was performed using Proteome Discoverer 1.2 (Thermo Fisher Scientific). Protein identification was performed using MASCOT (Matrix Science, London UK, USA) against the Assembly 21 Candida genome database (6,198 sequences) for C. albicans and against the Mus musculus database (25,530 sequences) from the common part of NCBI (http://www.ncbi.nlm.nih.gov/) and IPI (http://www.webcitation.org/getfile?fileid=ccad550bc21e5bcf0f4b8763a56240fcb7058693) database with a precursor mass tolerance of 50 ppm, a fragment ion mass tolerance of 20 mmu and strict specificity allowing for up to one missed cleavage. For trypsin digestion, carbamidomethylation of cysteine, TMT sixplex of N-term (+229.1629 Da) and TMT sixplex of lysine (+229.1629 Da) were set as fixed modifications. The data were then filtered at a q value ≤0.01 corresponding to a 1% false discovery rate (FDR) on a spectral level. Protein quantification was performed by Reporter Ions Quantifier with the TMT sixplex method on Protein Discoverer. Four independent biological experiments were performed and proteins identified in every replicate were considered.
Calculation of false positive proteins rates
Candida albicans monoculture, macrophage monoculture, and complete culture medium were incubated for 3 h and the proteins as a control were extracted, reduced, alkylated, and digested with trypsin. Tryptic digests were analyzed by nanoLC–MS/MS system with a long monolithic silica capillary column under the same conditions. Triplicate analyses were performed for each sample of three biological replicates. The mass spectrometry data were used for protein identification using MASCOT, working on Proteome Discoverer with a peptide tolerance of 1.2 Da, MS/MS tolerance of 0.8 Da, and maximum number of missed cleavages of two. For trypsin digestion, cysteine carbamidomethylation (+57.021 Da) and methionine oxidation (+15.995 Da) were set as a variable modification. The data were then filtered at a q value <0.01 corresponding to 1% false discovery rate on a spectral level. To identify false positive proteins, proteins derived from each organism and complete culture medium were analyzed with C. albicans, M. musculus database for macrophage, and Bos taurus database for complete culture medium from NCBI (http://www.ncbi.nlm.nih.gov/genome?term=bos%20taurus). Proteins identified by detection of at least two peptides in any of three biological replicates or by a single peptide at all three biological replicates were considered as ‘false positive protein’ and are listed in Additional file 1.
Extraction of differentially produced proteins
After removing false positive proteins from the total set of quantified proteins, a global median normalization was carried out to normalize the amount of tryptic digest injected into the nanoLC–MS/MS. Proteins identified in this study are listed in Additional file 2. To select the proteins that showed significant fold-change under the co-culture condition as compared with monoculture, an empirical Bayes moderated t test was performed and p values were adjusted with the Benjamini–Hochberg method to avoid the problem of multiple testing. Volcano plots were generated to visualize differentially produced proteins for co-culture. The criteria of differentially produced proteins used an FDR-adjusted p value <0.01 and fold-change of protein ratio (log2) >0.5.
An annotation tool, KEGG pathway of DAVID (Huang et al. 2007) (http://david.abcc.ncifcrf.gov/) was used for functional annotation and pathway analysis of the protein sets. The threshold was set to enrichment score >1.6.