Proteomic Analysis of Intracellular and Membrane Proteins From Voriconazole-Resistant Candida glabrata

2.1 C. glabrata strains and growth conditions

A total of 56 C. glabrata strains collected from tertiary and nontertiary hospitals were used in this study. We previously reported the results of an antifungal susceptibility test [17]. We selected three C. glabrata strains according to voriconazole susceptibility for a comparative proteomic study. All strains were stored at –80 °C, and prior to the experiment each strain was subcultured twice on sabouraud dextrose agar to ensure viability and purity. For the proteomic experiment, an aliquot of glycerol stock from each strain was diluted in yeast peptone dextrose (YPD; 1% yeast extract, 2% peptone, 1% dextrose) and grown overnight at 30 °C in a shaking incubator. The cultures were diluted to an optical density 0.2 at OD₆₀₀ in 0.5 L of YPD and grown to the exponential phase of growth.

2.2 Cellular protein extraction

To isolate the cellular proteins, C. glabrata cells were cultured in YPD broth at 30 °C to the exponential phase of growth. Cells were harvested in centrifugation 4000 rpm for 15 minutes. The pellet cells were pooled and washed twice using 50 mM Tris-HCl pH 7.6 buffer solution. The cells were disrupted using 0.45-μm glass beads (Sigma, St. Louis, MO, USA) on ice. After homogenization, the solution was centrifuged twice at 14,000 rpm for 20 minutes. The supernatant was harvested carefully without contaminant similar to a lipid component, and it was freeze dried for further experiment.

2.3 Membrane protein extraction

After an exponential phase of growth, cells were harvested, washed with distilled water, and resuspended in homogenizing buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 1 mM phenylmethylsulfonylfluoride). After disruption of the cell using the glass bead, cell debris and unbroken cells were removed by centrifugation at 5000 g for 10 minutes. A crude membrane fraction was isolated from the cell-free supernatant by second centrifugation at 30,000 g for 30 minutes. The pellet was washed in GTE buffer (10 mM Tris-HCl, pH 7.0, 0.5 mM EDTA, 20% glucose), resuspended in GTE buffer, and stored at –80 °C. The protein concentration was determined by a micro-Bradford assay using a protein assay kit II (Bio-Rad, Hercules, CA, USA).

2.4 Sample preparation and 2-Dimentional Gel Electrophoresis

The harvested samples were suspended in 0.5 mL of 50 mM Tris buffer containing 7 M urea, 2 M thiourea, 4% [weight/volume (w/v)] CHAPS, and 16 μL protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA). The lysates were homogenized and centrifuged at 12,000 × g for 15 minutes. Fifty units of Benzonase (250 units/μL; Sigma) was added to the mixture and suitably stored at −80 °C until use after quantitation by the Bradford method. For 2-DE analysis, pH 3–10 immobilized pH gradient (IPG) strips (Amersham Biosciences, UK, Ltd) were rehydrated in swelling buffer containing 7 M urea, 2 M thiourea, 0.4% (w/v) Dithiothreitol, and 4% (w/v) CHAPS. The protein lysates (500 μg) were cup-loaded into the rehydrated IPG strips using a Multiphor II apparatus (Amersham Biosciences, UK, Ltd) for a total of 57 kVh. The two-dimensional separation was performed on 8–16% (v/v) linear gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels. Following fixation of the gels for 1 hour in a solution of 40% (v/v) methanol containing 5% (v/v) phosphoric acid, the gels were stained with Colloidal Coomassie Blue G-250 solution for 5 hours. The gels were destained in 1% (v/v) acetic acid for 4 hours and then imaged using a GS-710 imaging calibrated densitometer (Bio-Rad).

Protein spot detection and two-dimensional pattern matching were carried out using ImageMasterTM 2D Platinum software (Amersham Biosciences, UK, Ltd). For comparison of protein spot densities between control and treated samples, more than 20 spots throughout all gels were correspondingly landmarked and normalized. The quantified spots of candidate proteins were compared with the aid of histograms. For ensuring the reproducibility of 2DE experiments, each sample was analyzed in duplicate.

2.5 In-gel protein digestion

Protein bands of interest were excised and digested in-gel with sequencing grade, modified trypsin (Promega, Madison, WI, USA). In brief, each protein spot was excised from the gel, placed in a polypropylene tube, and washed four to five times with 150 μL of 1:1 acetonitrile/25 mM ammonium bicarbonate, pH 7.8. The gel was dried in a Speedvac concentrator, and then rehydrated in 30 μL of 25 mM ammonium bicarbonate, pH 7.8, containing 20 ng of trypsin. After incubation at 37 °C for 20 hours, the liquid was transferred to a new tube. Tryptic peptides remaining in the gel matrix were extracted for 40 min at 30 °C with 20 μL of 50% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid. The combined supernatants were evaporated in a Speedvac concentrator and dissolved in 8 μL of 5% (v/v) aqueous acetonitrile solution containing 0.1% (v/v) formic acid for mass spectrometric analysis.

2.6 Identification of proteins by liquid chromatograph/tandem mass spectrometry

The resulting tryptic peptides were separated and analyzed using reversed phase capillary high-performance liquid chromatography (HPLC) directly coupled to a Finnigan LCQ ion trap mass spectrometer [liquid chromatography-tandem mass spectrometry (LC-MS/MS)]. A 0.1 × 20 mm trapping and a 0.075 × 130 mm resolving column were packed with Vydac 218 MS low trifluoroacetic acid C18 beads (5 μm in size, 300 Å in pore size; Vydac, Hesperia, CA, USA) and placed in-line. Next, the peptides were bound to the trapping column for 10 minutes with 5% (v/v) aqueous acetonitrile containing 0.1% (v/v) formic acid, then the bound peptides were eluted with a 50-minute gradient of 5-80% (v/v) acetonitrile containing 0.1% (v/v) formic acid at a flow rate of 0.2 μL/min. For tandem mass spectrometry, a full mass scan range mode was m/z = 450–2000 Da. After determination of the charge states of an ion on zoom scans, product ion spectra were acquired in MS/MS mode with relative collision energy of 55%. The individual spectra from MS/MS were processed using the TurboSEQUEST software (Thermo Quest, San Jose, CA). The generated peak list files were used to query either the MSDB database or National Center for Biotechnology Information (NCBI) using the MASCOT program (http://www.matrixscience.com). Modifications of methionine and cysteine, peptide mass tolerance at 2 Da, MS/MS ion mass tolerance at 0.8 Da, allowance of missed cleavage at 2, and charge states (+1, +2, and +3) were taken into account. Only significant hits as defined by MASCOT probability analysis were initially considered.

3.1 Strains

Among the C. glabrata strains, voriconazole susceptible strain [C. glabrata I-49, minimum inhibitory concentration (MIC) 0.5 μg/mL], S-DD strain (C. glabrata D-54, MIC 2 μg/mL) and resistant strain (C. glabrata D-91, MIC 4 μg/mL) were selected. All strains were isolated from blood specimen of patients.

3.2 Expression of intracellular proteins and identification

The two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D-SDS PAGE) gels are shown in Figure 1. The profiling of 459 intracellular proteins was detected in three strains. Of the total proteins, 38 proteins having abundance ratios of twofold or more showed continuous increase of expression from susceptible and S-DD to resistant strain. In addition, 34 proteins were identified by LC-MS/MS (Table 1). The 15 proteins showing decrease of expression from susceptible and S-DD to resistant strain were also identified. Among the identified proteins, aldehyde dehydrogenase family, serine hydroxymethyltransferase, acetolactate synthase, heat shock protein, pyruvate kinase, potassium efflux protein, isocitrate dehydrogenase, and other proteins showed increased expression. Expression was decreased in proteins such as glycerol-3-phosphate dehydrogenase, ATP synthase, acetyl-coA hydrolase, oxidoreductase, and malate dehydrogenases (Table 1). Among the proteins for which expression was decreased, phosphoglycerate kinase protein showed the largest decreased expression, at 9.09 times reduction of expression. The identified proteins, classified according to their function, are summarized in Table 2. The functional category showed that the identified proteins were cell regulation, energy production, carbohydrate transport, amino acid transport, and various metabolism-related proteins.

Figure 1

Cellular and membrane protein spot of C. glabrata strains resolved by 2D gel electrophoresis. Spots representing differentially expressed proteins that were identified by LC-Ms/Ms peptide mass fingerprinting. (a) cellular protein spot of voriconazole susceptible strain, (b) cellular protein spot of voriconazole SDD strain, (c) cellular protein spot of fluconazole susceptible strain, (d) membrane protein spot of voriconazole susceptible strain, (e) membrane protein spot of voriconazole SDD strain, (f) membrane protein spot of voriconazole resistant strain.

Table 1

Differentially expressed intracellular proteins (by twofold or more), as identified by liquid chromatography-tandem mass spectrometry between voriconazole susceptible, susceptible dose-dependent, and voriconazole resistant strains

Table 2

Functional classification of identified intracellular proteins from voriconazole susceptible, susceptible dose-dependent, and resistant strains

3.3 Expression of membrane proteins and identification

A total of 329 membrane proteins were resolved by 2D gel electrophoresis. Of the 17 spots (differential ratio twofold or more) for which expression was increased, 12 proteins were identified. The identified proteins showed enolase, heat shock protein 70, pyruvate kinase, cysteine synthase, pyruvate decarboxylase, pyrophosphate requiring enzyme, regulatory modules in signal transduction, and phosphoglycerate kinase (Table 3). Among the identified proteins, phosphate requiring enzymes showed the most increased expression (3.66 times). Enolase and phosphoglycerate kinase proteins also showed 2.69 and 2.77 times increased expression, respectively. Thirty-seven spots showed decreased expression in the order of susceptible, S-DD, and resistant strains. Among the 37 spots, 31 proteins were identified. The identified membrane proteins included heat shock protein 70, aldehyde dehydrogenase, nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase, phosphoglycerate mutase I, glutamine aminotransferase, superoxide dismutase, Stm1 protein, phosphoglycerate kinase, and others. A total of 12 heat shock proteins were observed and heat shock protein 70 was 11. In addition, 9 heat shock protein 70 showed the deceased expression in resistant strain compared to susceptible and S-DD strain. The identified membrane proteins were classified into carbohydrate metabolism, amino acid synthesis, and response to stress-related proteins (Table 4).

Table 3

Differentially expressed membrane proteins (by twofold or more), as identified by liquid chromatography-tandem mass spectrometry between voriconazole susceptible, susceptible dose-dependent, and voriconazole resistant strains

Table 4

Functional classification of identified membrane proteins from voriconazole susceptible, susceptible dose-dependent, and resistant strains