Proteomic Analysis Reveals a Metabolism Shift in a Laboratory

Proteomic Analysis Reveals a Metabolism Shift in a Laboratory Fluconazole-Resistant Candida albicans Strain Lan Yan, Jun-Dong Zhang, Yong-Bing Cao, Ping-Hui Gao, and Yuan-Ying Jiang* Department of Pharmacology, College of Pharmacy, Second Military Medical University, 325 Guo He Road, Shanghai 200433, People’s Republic of China Received December 8, 2006

Multifactorial and multistep alterations are involved in acquired fluconazole (FLC) resistance in Candida albicans. In this study, a FLC-resistant C. albicans strain was obtained by serial cultures of a FLC-susceptible C. albicans strain in incrementally increasing concentrations of FLC. The comparative proteomic study, confirmed by real-time RT-PCR, was performed with the susceptible parental strain and the resistant daughter strain to identify proteins altered during the development of FLC resistance. Our analysis of the differentially expressed proteins identified 22 different proteins, most of which were related to energy metabolisms (e.g., Pgk1, Fba1, and Adh1), and some of which have been previously identified as being involved in FLC resistance in C. albicans (e.g., Ald5, Cdc19, and Gap1). Functional analysis revealed lower intracellular ATP level and mitochondrial membrane potential, less endogenous reactive oxygen species generation in response to antifungal agents, and identical susceptibility to exogenous hydrogen peroxide, heat, and hyperosmotic shock in the resistant strain compared with the susceptible strain. Our results suggest that a metabolism shift might contribute to FLC resistance in C. albicans. Keywords: Candida albicans • fluconazole resistance • mass spectrometry • two-dimensional polyacrylamide gel electrophoresis

Introduction Candida albicans is the major systemic fungal pathogen of human beings. The triazole fluconazole (FLC) is the most widely used antifungal drug to treat Candida infections. Unfortunately, widespread uses of FLC have led to the rapid development of drug resistance which has severely hindered antifungal therapy.1 The specific mechanisms of acquired resistance in C. albicans have been extensively studied over the past decades, including alteration in the sterol biosynthesis pathway,2 overexpression of the gene encoding the target of the azoles, lanosterol demethylase (ERG11),3 mutations in the ERG11 gene,4,5 reduced intracellular drug accumulation due to the overexpression of genes that encode membrane transport proteins such as energy-dependent ATP-binding cassette (ABC) transporters, and the proton motive force-dependent major facilitators superfamily,6-8 as well as the formation of biofilm.9 Recently, DNA microarray studies have shown that a large number of genes are associated with drug resistance in C. albicans in both clinical10,11 and laboratory12,13 isolates, such as genes related to ergosterol biosynthesis pathway (ERG2, ERG10),10-12 oxidative stress response (CRD2, GPX1),10,11 and glutathione peroxidase (GRE99, HSP12),13 among others. Furthermore, proteomic approaches have also identified several * To whom correspondence should be addressed. E-mail, [email protected]; tel, 86-021-2507-0371; fax, 86-021-6549-0641.

2248

Journal of Proteome Research 2007, 6, 2248-2256

Published on Web 04/14/2007

differentially expressed proteins such as Grp2, Ifd1, Ifd4, and Ifd5 in clinical C. albicans isolates resistant to FLC.14-16 This study was designed to gain insight into alterations in protein expression associated with FLC resistance in laboratory C. albicans strains. Specifically, exposure of the susceptible strain SC5314S to incrementally increasing concentrations of FLC resulted in the resistant daughter strain SC5314R. Analysis of the protein expression profile of this matched pair has revealed that several differential proteins were involved in energy metabolisms, cell stress, macromolecule biosynthesis, chaperones, and others. Furthermore, the measurements of intracellular ATP concentration, mitochondrial membrane potential, and endogenous reactive oxygen species (ROS) generation were carried out, showing that a metabolism shift occurred during the development of FLC resistance and thereby protected the resistant C. albicans strain from the insult of antifungal agents.

Materials and Methods Antifungal Agents. Standard antifungal powders of FLC, ketoconazole (KTC), itraconazole (ITC), miconazole (MCZ), terbinafine (TRB), and amphotericin B (AMB) were obtained from Sigma (St. Louis, MO). Stock solutions were prepared in distilled water (FLC) or dimethyl sulfoxide (KTC, ITC, MCZ, TRB, and AMB), sterilized by filter, and stored at -70 °C. Organism and Media. C. albicans collection strain SC5314 was kindly provided by Dr. William A. Fonzi.17 Media utilized 10.1021/pr060656c CCC: $37.00

 2007 American Chemical Society

Metabolism Shift in a Lab FLC-Resistant C. albicans Strain

in this study included yeast nitrogen base (YNB) broth (Difco, Detroit, MI) supplemented with 2% glucose, yeast extract pentose dextrose (YPD; Difco), sabouraud dextrose agar (SDA; Difco), and RPMI 1640 medium (American Bioorganics, Niagara Falls, NY). Induction of FLC Resistance. The strategy for the induction of FLC resistance was performed as previously described18 with some modifications. A single colony of the strain SC5314 was first inoculated into 10 mL of YNB broth and incubated overnight at 30 °C in a shaking (200 rpm) incubator. An aliquot of the culture containing 106 cells was then transferred to 10 mL of YNB broth containing twice their most recently measured minimal inhibitory concentration (MIC) of FLC, and the cells were incubated overnight at 30 °C with shaking. When the cultures reached a density of 108 cells/mL, an aliquot containing 106 cells was transferred into fresh YNB broth also containing twice their most recently measured MIC of FLC and incubated as described above. At each passage, the broth microdilution method for in vitro antifungal sensibility testing of C. albicans was performed as standard method.19 Protein Sample Preparation. The early logarithmic phase cultures of the resultant FLC-resistant strain and its susceptible parental strain were obtained by transferring of a single colony of the strains to 100 mL of YPD broth for 16 h culture at 30 °C with shaking (200 rpm) and subculturing of 300 µL of the above suspensions for another 6 h in 100 mL of fresh YPD broth. Cells were then harvested and washed with phosphate-buffered saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium chloride, and 140 mM sodium chloride, pH 7.4) buffer. Next, the cell pellet was lysed with 10 mL of lysis buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, 65 mM dithioerythritol (DTT), 1 mM PMSF, and 0.5% v/v Phamalyte 3-10) and an equal volume of 0.5 mm diameter glass beads (Biospec, Bartlesville, OK) by vortexing for 30 s and cooling on ice for 30 s repeatedly in a Mini Bead-beater (Biospec) until at least 80% of the cells had been lysed as determined by phase-contrast microscopic examination. Cell debris and glass beads were removed by centrifugation at 5000g for 15 min at 4 °C. After centrifugation at 13 000g for 30 min, the clarified protein supernatant was determined using the RC DC Protein Assay Kit (Bio-Rad, Herclues, CA) and manufacturer’s directions. Two-Dimensional Gel Electrophoresis. Proteins containing 300 µg were rehyrated in sample buffer (5 M urea, 2 M thiourea, 2% CHAPS, 2% SB3-10, 65 mM DTT, and 0.5% v/v Phamalyte 3-10) and were then applied onto pH 3-10 nonlinear IPG Strips (17 cm long, Bio-Rad). Isoelectric focusing was carried out on a PROTEAN IEF unit (Bio-Rad) at 20 °C with the following program: passive rehydration for 12 h, 500 V for 1 h, 1000 V for 1 h, 2000 V for 1 h, and 8000 V for 6 h. After focusing, IPG strips were reduced (2% DTT) and then alkylated (2.5% iodoacetamide) in equilibration buffer (6 M urea, 0.375 M TrisHCl (pH 8.8), 20% glycerol, and 2% sodium dodecyl sulfate (SDS)). For the second dimension run, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on homogeneous 10% polyacrylamide gels at 40 mA/2gels constant current for 6 h using a Protean II gel tank (Bio-Rad). Gels were silverstained according to Concha Gil and co-workers.20 Gel images were captured by scanning the 2-D PAGE gels using a GS-690 imaging densitometer (Bio-Rad) and analyzed with the MELANIE 3.0 software. In-Gel Tryptic Digest. The protein spots were excised from the gels and placed into a 96-well microtiter plate. Gel pieces were destained with a solution of 15 mM potassium ferricya-

research articles nide and 50 mM sodium thiosulfate (1:1) for 20 min at room temperature, washed twice with deionized water, shrunk by dehydration in acetonitrile (ACN), and swollen in a digestion buffer containing 20 mM ammonium bicarbonate and 12.5 ng/ mL trypsin at 4 °C for 30 min. After digestion for more than 12 h at 37 °C, peptides were finally extracted from the gel pieces twice using 0.1% trifluoroacetic acid (TFA) in 50% ACN. Mass Spectrometry and Database Search. The obtained peptides were spotted onto a matrix-assisted laser desorption/ ionization (MALDI) target and overlaid with 0.8 µL of matrix solution (R-cyano-4-hydroxycinnamic acid (Sigma) in 0.1%TFA and 50%ACN). Then the samples were analyzed on an Applied Biosystems 4700 TOF-TOF Proteomics Analyzer (Framingham, MA)21 in the positive reflection mode with a 200 Hz UV laser operating at a wavelength of 355 nm. Positive ion mass spectra were recorded on a home-built linear time-of-flight mass spectrometer. For MS/MS, selected peptide peaks of the proteins from the first mass spectra were fragmented by collision-induced dissociation with a floating collision cell, atmosphere as the collision gas at a pressure of 1 × 10-6 Torr, and a 1 keV acceleration voltage. Myoglobin digested by trypsin was used to calibrate the mass instrument with internal calibration mode. Both MS and MS/MS spectra were searched using the MASCOT engine (Matrix Science, London, U.K.) with the GPS Explorer software (Applied Biosystems) to identify the proteins. The search parameters were as follows: NCBI database, fungi, mass ranged from 700 to 3200 Da, trypsin digest with one missing cleavage, peptide tolerance of (0.2 Da, MS/ MS ion mass tolerance of (0.6 Da, and possible oxidation of methionine. The Mascot score calculated by the software was used as criterion for correct identification. Real-Time RT-PCR. Total RNA samples from the resultant FLC-resistant strain and its susceptible parental strain were extracted using the hot-phenol method,22 and were treated with DNase I (TaKaRa, Biotechnology, Dalian, P. R. China) to remove genomic DNA contamination. Reverse transcription was performed in a total volume of 20 µL with Avian Myeloblastosis Virus Reverse Transcripase (TaKaRa), Random Primer (6-mer) (TaKaRa), 1 µg of total RNA, followed by the condition of 30 °C for 10 min, 45 °C for 15 min, and 99 °C for 2 min, as recommended by the manufacturer. Real-time PCR reactions were performed with SYBR Green I (TaKaRa), using LightCycler Real-Time PCR system (Roche Molecular Biochemical). Gene-specific primers were designed using Discovery Studio Gene software (Accelrys, Inc.) and were shown in supplementary Table S1 of Supporting Information. The thermal cycling conditions comprised an initial step at 95 °C for 10 s, followed by 40 cycles at 95 °C for 10 s, 62 °C for 20 s, and 72 °C for 15 s. The change in fluorescence of SYBR Green I in every cycle was monitored by the system software, and the threshold cycle (CT) was measured. 18S rRNA was used as an internal control, and the gene expression level of the resistant strain relative to that of the susceptible strain was calculated using the formula 2-∆∆CT, where ∆CT was the CT value of genes of interest minus that of internal control and ∆∆CT was the mean ∆CT value of the resistant strain minus that of the susceptible strain. Measurement of Intracellular ATP Level. The resultant FLCresistant strain and the parental susceptible C. albicans strain in middle logarithmic phase were adjusted to 1 × 108 cells/mL and serial 10-fold dilutions with YPD broth. A total of 100 µL from the cell suspensions and a same volume BacTiter-Glo reagent (Promega Corparation, Madison, WI) were mixed Journal of Proteome Research • Vol. 6, No. 6, 2007 2249

research articles

Yan et al.

completely and incubated for 10 min at room temperature. Then the luminescent signals were determined on a TD 20/20 luminometer (Turner Biosystem, Sunnyvale, CA) with an integration time of 1 s per sample. The control tube without strains was to obtain a value for background luminescence. The signal-to-noise ratio was calculated: S/N ) [mean of signal mean of background]/standard deviation of background. A standard curve for ATP increments (from 1 µM to 10 pM) was constructed. Signals represented the mean of three separate experiments, and the ATP content was calculated from the standard curve. Assay for Mitochondrial Membrane Potential. C. albicans strains in middle logarithmic phase were treated with 60 mM azide sodium (Sigma) and harvested at each hour after the treatment. Cells were washed and adjusted to 1 × 106 cells/ mL with PBS buffer. After treatment with 10 µg/mL of 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide23 (JC-1; Molecular Probes, Inc., Eugene, OR) at 30 °C for 15 min, cells were washed twice with PBS buffer and analyzed on a POLARstar Galaxy (BMG, Labtech, Offenburg, Germany) at 485 nm excitation wavelength and emission wavelength shifting from green (∼525 nm) to red (∼590 nm). Mitochondrial membrane potential was determined by the ratio of red to green fluorescence intensity (FI). Measurement of Endogenous ROS Production. ROS production was assessed as reported before.24 In brief, C. albicans strains (1 × 107 cells/mL) were incubated with 20 µg/mL of 2,7-dichlorofluorescin diacetate (DCFH-DA; Molecular Probes) for 30 min at 30 °C. After being washed and resuspended in PBS buffer, cells were treated with FLC (64 µg/mL), MCZ (12.5 µg/mL), AMB (32 µg/mL), and TRB (64 µg/mL) separately for the indicated times. Control groups were treated with PBS containing equal volume of solvents (e.g., distilled water for FLC or dimethyl sulfoxide for MCZ, AMB, and TRB). FI values were detected on the POLARstar Galaxy with excitation wavelength at 488 nm and emission wavelength at 525 nm. The kinetic measurements of ROS were continued for 48 h after the treatments. ROS production was calculated by subtracting the FI value of cells treated with antifungal agents alone from that of cells treated with antifungal agents in the presence of DCFH-DA. Stress Susceptibility Testing. The susceptibility to hydrogen peroxide (H2O2) in C. albicans was tested by spotting serial 10fold dilutions of middle logarithmic phase cells on YPD agar plates in the presence or absence of 4, 6, 8, 16, 32, 64, and 128 mM H2O2 and monitoring growth at 30 °C for 48 h. The heat shock and the hyperosmotic shock susceptibility assays were performed as previously described.25 In brief, strains in the middle logarithmic phase were transferred from 23 to 37 °C within 1.5 min by immersing the samples in a water bath to create the heat shock. For the hyperosmotic shock assay, a prewarmed NaCl solution was added to the culture to obtain a 0.3 M final concentration. The same volume of PBS buffer at 30 °C was added to the control cultures. For each assay, at 60 min after the initiation of the stress, cells of the control and stress samples were harvested and resuspended in fresh YPD broth. Aliquots of these cultures were spotted in serial 10-fold dilutions on YPD agar plates and grown at 30 °C for 48 h. Statistics. Experiments were performed at least three times. Data are presented as mean ( standard deviations, and analyzed using the Student’s t test where indicated. 2250

Journal of Proteome Research • Vol. 6, No. 6, 2007

Figure 1. Variations of MIC values of FLC, KTC, and ITC for the C. albicans strain SC5314 grown in medium containing twice their most recently measured MIC of FLC. Each datum point represents one passage.

Results Generation of the FLC-Resistant Strain. To obtain a FLCresistant C. albicans strain in the laboratory, the susceptible wild strain SC5314S (FLC MIC, 0.5 µg/mL) was serially cultured in incrementally increasing concentrations of FLC. FLC resistance was developed in a stepwise fashion. Reduced sensibility to FLC was detected after 5 to 8 passages; the high-level resistant strain, SC5314R (FLC MIC, 128 µg/mL) was generated after about 65 passages and retained the resistant phenotype for more than 15 passages (Figure 1). When the resultant SC5314R cells were cultured in drug-free YNB broth, they kept the stability of high-level resistance to FLC (MIC 128 µg/mL) for nearly 40 passages (data not shown). Sensibility testing also showed that the development of resistance to both ITC and KTC paralleled that of FLC, indicating that the strain SC5314R was cross-resistant to both ITC and KTC (Figure 1). Protein Expression Profile. Total protein samples from the susceptible strain SC5314S and the resistant strain SC5314R were prepared in parallel for three independent experiments. Each protein extract was then separated in triplicate by 2-D PAGE. Figure 2 showed the typical high-resolution silver-stained 2-D map of the strain SC5314R and the strain SC5314S with the number of the proteins differentially expressed. This pattern of protein was reproducible due to the repeated results of the same protein sample and of samples from independent cell extracts. Differences in protein expression profile between this matched pair were analyzed based on the criterion that FLC-resistantassociated changes were greater than 2-fold by using MELANIE 3.0 software. In the strain SC5314R, 25 spots (1-28, with the exception of 2, 7, and 26) were observed up-regulated in comparison with the strain SC5314S, while 3 spots (2, 7, and 26) were down-regulated (Figure 2). Identification of FLC-Resistant-Associated Proteins. The differentially expressed proteins were identified based on MALDI-TOF MS/MS analysis, and protein score greater than 74 was over the 95% confidence limit considered of significance in this study. Of the 28 spots, 24 were identified with a Mascot score over 150, among which 18 were with Mascot score over 300; 4 spots (12, 21, 23, and 26) were not identified due to their MS spectra yielding short amino acid sequences. The high Mascot scores for a majority of protein spots reflect a high number of peptides matched to the theoretical database.26 Summary of peptide sequence data was shown in Supporting Information Table S2.


research articles

Figure 2. Proteomic analysis of FLC-resistant-related changes with 2-D PAGE separation of whole cell extracts of C. albicans strains. Molecular weight (MW, kDa) and isoelectric point (pI) are indicated along the y- and x-axis, respectively. Selected regions are showning the significant differences in protein expression profile between the FLC-resistant strain SC5314R (R) and the -susceptible strain SC5314S (S). Arrows indicate proteins which have a difference greater than 2-fold at protein expression level.

The proteins identified in this study were named according to the CandidaDB database (http://genolist.pasteur.fr/CandidaDB/) and were divided into five groups according to their biological functions defined by the Candida Genome Database and the Saccharomyces Genome Database (Table 1). Interestingly, most of them were found to be involved in energy metabolisms including glycolysis (e.g., Pgk1, Fba1, and Gpm1), fermentation (e.g., Adh1), glyoxylate cycle (e.g., Mls1), tricar-

boxylic acid cycle (e.g., Idh1), respiration (e.g., Qcr7), and transport (e.g., Mir1); the remainder functioned in cell stress (e.g., Ssa4), macromolecule biosynthesis (e.g., Rps5), chaperoning (e.g., Zuo1), and others (e.g., Pot14). Validation of Proteomic Results by Real-Time RT-PCR Analysis. To validate the differential expression of identified proteins, we performed real-time RT-PCR analysis. Total RNA samples from the strain SC5314S and the strain SC5314R were Journal of Proteome Research • Vol. 6, No. 6, 2007 2251

research articles

Yan et al.

Table 1. Differentially Expressed Proteins between the C. albicans SC5314S Strain and the SC5314R Strain spot

protein namea

accession no.a

function description

MWb (kDa)

pI b

SC5314R vs SC5314S c

55 45 45 45 39 27 27 36

6.54 6.07 6.07 6.07 5.69 5.79 5.74 7.0

+ + + + + + + +

46 54

8.26 5.56

+ -

62 36

8.74 5.25

+ +

21

8.65

+

14

5.71

+

33

9.25

+

70 38 22

5.06 6.04 4.98

+ + +

25 17

8.81 6.15

+ +

48

8.49

+

42 38 30

6.47 5.53 5.11

+ + -

Energy Metabolism (I) Glycolysis 5 9 10 11 14 15 16 18

Cdc19 Pgk1 Pgk1 Pgk1 Fba1 Gpm1 Tpi1 Gap1

22 2

Adh1*d Ald5

3 8

Mls1 Mdh11

24

Idh1

28

Qcr7

17

Mir1

1 13 25

Ssa4 Grp2 IPF2431

20 27

Rps5 Ynk1

4

Zuo1

19 6 7

Pot14*d IPF4328 IPF16253

Pyruvate kinase CA3483 Phosphoglycerate kinase CA1691 Phosphoglycerate kinase CA1691 Phosphoglycerate kinase CA1691 Fructose-bisphosphate aldolase CA5180 Phosphoglycerate mutase CA4671 Triose phosphate isomerase CA5950 Glyceraldehyde-3-phosphate dehydrogenase CA5892 (II) Fermentation Alcohol dehydrogenase CA4765 Aldehyde dehydrogenase (NAD+) CA4159 (III) Glyoxylate cycle Malate synthase CA4748 Malate dehydrogenase CA5826 (IV) Tricarboxylic Acid Cycle Isocitrate dehydrogenase (NAD+) subunit 1 CA4753 (V) Respiration Ubiquinol-cytochrome-c reductase subunit 7 CA2764 (VI) Transporter Mitochondrial phosphate transport protein CA1513 Stress Response Cahsp70 mRNA for heat shock CA1230 Reductase CA2644 Thiol-specific antioxidant-like protein CA5714 Macromolecule Biosynthesis Ribosomal protein S5.e CA0632 Nucleoside diphosphate kinase CA2645 Chaperones Zuotin, a putative Z-DNA binding protein CA1289 Others and Unknown-Function Proteins Acetyl-CoA acetyltransferase CA0290 Unknown function CA0210 Unknown function CA0782

a Protein names and accession numbers according to the C. albicans genomic database (CandidaDB). b Experimental MW and pI values (calculated by using the Melanie 3.0 program). c +, up-regulated; -, down-regulated. d * Protein fragment.

prepared in parallel for three separate experiments. Real-time RT-PCR reactions were performed in triplicate with independent RNA isolations. In general, there was a good correlation between changes in gene expression and those in protein expression (Figure 3). Since Cdr1, Cdr2, Mdr1, or Erg11 have been found upregulated in several clinical and laboratory FLC-resistant C. albicans isolates,3,6-8 real-time RT-PCR analysis was also designed to detect their alternations in our resistant strain. As shown in Figure 3, CDR1-mRNA and CDR2-mRNA significantly increased by 9.2-fold and 2.5-fold, respectively, in the strain SC5314R, while no change of MDR1-mRNA and ERG11-mRNA was found. These results further demonstrate the FLC resistance in the strain SC5314R with enhanced activity of drug efflux pump mediated by ABC transporters. Intracellular ATP Content and Mitochondrial Membrane Potential. In view of the marked changes of energy-metabolismrelated proteins in the FLC-resistant strain, we measured intracellular ATP concentration in the susceptible parental strain and the resistant daughter strain. As shown in Figure 4, the level of intracellular ATP in the SC5314R strain was lower than that in the SC5314S strain. Since the fact that oxidative phosphorylation of the mitochondria is the main source of intracellular ATP supply and that a substantial proton gradient indicated by mitochondrial membrane potential is required for active oxidative phosphorylation, we further measured mitochondrial membrane potential in both strains. As shown in 2252


Table 2, the SC5314R strain had a lower mitochondrial membrane potential compared with the SC5314S strain. Membrane potential of both strains decreased after the treatment of sodium azide for 4 h, while that of the SC5314S strain decreased more dramatically. These results suggest that a lower mitochondrial membrane potential resulting in a less efficient oxidative phosphorylation in the resistant strain may be the major cause of its lower ATP level and therefore change its energy supply pathway from mitochondria to cytoplasm. Endogenous ROS Production. Since intracellular oxygen radicals are mainly generated in mitochondria and are involved in the mechanisms of several antifungal agents, we examined endogenous ROS generation in both strains stimulated with the agents either increasing or decreasing ROS production. The alterations of ROS production over time were shown in Figure 5. Without exposure to antifungal agents, there was no significant difference in the amount of endogenous ROS generation between the two strains at each time-point measurement. And the ROS generation increased time-dependently in both strains, which could be explained by the lack of nutrients in PBS medium resulting in stress within the cells. With the treatment of ROS-induction agents (FLC, MCZ, and AMB), ROS production was dramatically augmented in the SC5314S strain, while in the SC5314R strain to a less extent (Figure 5A-C); similar phenomena were observed in both strains with the treatment of ROS-reduction agent, TRB (Figure 5D). These data indicate


research articles

Figure 3. Gene expression changes in 22 proteins identified as differentially expressed by proteomic analysis and in 4 known FLCresistant-specific genes in the SC5314R strain relative to those in the SC5314S strain. All the genes were examined by real-time RT-PCR with gene-specific primers. Relative fold change was calculated with CT value (see details in Materials and Methods). Results are the mean ( standard deviations for three independent experiments.

that FLC-resistant phenotype decreases susceptibility to chemicals that generate oxidative stress in C. albicans. Stress Susceptibility. We further examined the sensibility of both strains to exogenous H2O2, heat, and hyperosmotic stress. As shown in Figure 6, both strains survived gentle oxidative treatments (4, 6, and 8 mM H2O2), and the degree of cell survival was roughly equivalent in both strains; nevertheless, with a strong oxidative exposure (>16 mM H2O2), neither of the strains survived (data not shown). In addition, there was still no significant difference in the sensibility to heat shock or hyperosmotic shock between the two strains (data not shown). These results imply that the FLC-resistant phenotype does not cause resistance to exogenous H2O2, heat, and hyperosmotic stress.

Discussion To investigate biological mechanisms of FLC resistance in C. albicans, a FLC-resistant C. albicans strain was obtained by serial cultures of a FLC-susceptible C. albicans strain in inhibitory concentrations of FLC. This resistant strain possessed high-level and stable resistant characteristic, as well as crossresistance to two other azole antifungal agents. The comparative proteomic study, confirmed by real-time RT-PCR, was performed with the susceptible parental strain and the resistant daughter strain. The differentially expressed proteins were found to be involved in multiple biochemical functions. Our particular interest was the striking changes of energy-metabolism-related proteins. Functional analysis was carried out to measure intracellular ATP concentration, mitochondrial membrane potential, endogenous ROS production, and stress susceptibility. Taken together, our results suggest that a metabolism shift might contribute to FLC resistance in C. albicans. Our analysis of the differentially expressed proteins between the susceptible and the resistant strains identified 22 different proteins that had diverse functions including energy metabolisms, stress response, macromolecule biosynthesis, chaper-

Figure 4. Intracellular ATP content in C. albicans strains. ATP levels represent the mean of three separate experiments. A linear correlation between ATP concentration and fungi cell numbers was detected in both strains SC5314S (S) and SC5314R (R).

ones, and others. It is reasonable to assume that many of the observed alternations are somewhat related to FLC resistance in C. albicans. Among them, Ald5, Cdc19, Gap1, Grp2, Mir1, Pot14, and Ynk1 have been known to have changed expression at mRNA or protein levels in both experimentally induced and clinically acquired FLC-resistant C. albicans isolates.10,11,14,27 The remaining identified proteins were, to our knowledge, found for the first time as being altered in the FLC-resistant strain. For example, the enzyme Pgk1 was more highly expressed in the resistant strain which catalyzes the reversible conversion of 1,3-diphosphoglycerate to 3-phosphoglycerate with generation of one molecule of ATP. Overexpression of Pgk1 is also found in the methicillin-resistant Staphylococcus aureus in comparison to the methicillin-sensitive strains.28 Particularly, in human, Pgk1 induces a multidrug-resistant phenotype in paclitaxel cancer through an MDR1-independent mechanism.29 Journal of Proteome Research • Vol. 6, No. 6, 2007 2253

research articles

Yan et al.

Figure 6. Susceptibility to H2O2 in C. albicans strains. Serial 10fold dilutions of the strains were spotted on YPD agar plates containing H2O2 with the indicated concentrations and incubated at 30 °C for 48 h. The first and the third row of panels show growth of the FLC-susceptible strain SC5314S in parallel. The second and the fourth row of panels show growth of the FLCresistant strain SC5314R in parallel.

Table 2. Mitochondrial Membrane Potential in the C. albicans SC5314S Strain and the SC5314R Straina ratio (red fluorescence/green fluorescence)

Figure 5. Effects of antifungal agents on intracellular ROS generation in the strain SC5314S (S) and the strain SC5314R (R). The level of endogenous ROS production was measured for the indicated times after the treatment of FLC (A), MCZ (B), AMB (C), and TRB (D) with the indicated concentrations. Each datum point represents the mean ( standard deviations for three independent experiments.

Thus, the up-regulation of Pgk1 in this study suggests this protein may have the similar function in C. albicans involved in FLC resistance. Another example is the cytosolic ribosomeassociated chaperone Zuo1. It forms a heterodimer with Ssz1 and functions on the ribosome with the Hsp70 for the production of nascent polypeptide chains.30 ZUO1 has been reported to induce pleiotropic drug resistance in Saccharomyces cerevisiae when up-regulated.30 Overexpression of Zuo1 in our results associates the function of Zuo1 with FLC resistance in C. albicans. Further explanation for the observed alternations of these newly identified proteins in the resistant strain warrants functional analysis to ascertain the relationship between the FLC resistance and such alternations. Since the striking changes in the FLC-resistant strain detected by proteomic analysis were a group of energy-metabolismrelated proteins, we speculate that such alternations might be associated with FLC resistance. Mitochondria provide a majority of cellular ATP, and a substantial mitochondrial membrane potential is required for mitochondrial ATP synthesis. Thus, it could be inferred that the altered expression of a group of metabolic enzymes in the FLC-resistant strain might have functions that compensate for the mitochondrial respiration deficiency and ATP supply. The measurement of ATP content and the mitochondrial membrane potential revealed lower intracellular ATP level and even lower mitochondrial mem2254


Time (h)b

SC5314S

SC5314R

0 4 24

11.15 ( 0.6 4.50 ( 0.244c 0.99 ( 0.144c

8.10 ( 0.4**c 7.12 ( 0.3 0.91 ( 0.122c

a Results are the mean ( standard deviations for three independent experiments. b Time after sodium azide treatment. c **, P < 0.01 vs SC5314S at the same time point; 44, P < 0.01 vs SC5314S without sodium azide treatment; 22, P < 0.01 vs SC5314R without sodium azide treatment.

brane potential in the resistant strain, suggesting a deficiency in oxidative phosphorylation and lower mitochondria-based source of ATP supply. Under this circumstance, it seems possible for the resistant strain to survive in that an additional source of ATP may be provided by an increased rate of substrate level phosphorylation in cytoplasm as a result of feedback regulation. Strongly supporting our hypothesis, the comparative results in this study showed that the resistant strain overexpressed enzymes involving in glycolysis and glyoxylate cycle, decreased Ald5 protein expression in mitochondria (Figure 7). On the other hand, since the primary source of endogenous ROS, normal byproducts of energy production in respiring cells, is leakage of electron from mitochondrial respiratory chain,31,32 and FLC toxicity is dependent on the active oxidative phosphorylation in S. cerevisiae,33 and the increase of intracellular ROS production is one of the antifungal activities of MCZ,24 we speculated that in the FLC-resistant strain the outcome of the decreased efficiency of mitochondrial respiration might reduce the ability of endogenous ROS generation to protect cells from the insult of the antifungal agents, ultimately leading to the drug resistance. As expected, the endogenous ROS generation in the FLC-resistant strain was actually less sensitive to antifungal agents’ stress than that in the susceptible strain. Consequently, the high tolerance of the FLC-resistant strain to antifungal agents may result partly from changed capability of intracellular ROS generation due to oxidative phosphorylation deficiency.

research articles


thank Prof. Jun-Ping Zhang for critical reading of the manuscript; and Yan Wang and Xin-Ming Jia for technical assistance.

Supporting Information Available: Gene-specific primers used in real-time RT-PCR analysis are listed in Table S1. Peptide sequence data of the proteins identified by MALDITOF MS/MS as being differentially expressed between the strain SC5314S and the strain SC5314R are shown in Table S2. This material is available free of charge via the Internet at http:// pubs.acs.org. References

Figure 7. Central carbon metabolism in C. albicans during aerobic growth on glucose. Black elliptical highlighting metabolic enzymes which were highly expressed in the SC5314R strain participate in cytosolic glycolysis and glyoxylate cycle, with the exception of Idh1. In mitochondria, Ald5 expressed at a level lower in the SC5314R strain than that in the SC5314S strain. G-6P, glucose-6-phosphate; F-1,6-2P, fructose-1,6-biphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; 1,3DPGA, 1,3-biphosphoglycerate; 3PGA, 3-phosphoglycerate; 2PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.

Furthermore, there was no difference in the sensibility of both strains to either extracellular H2O2 stress similar to that seen in the three pairs of matched FLC-susceptible and -resistant clinical C. albicans isolates,16 or heat shock and hyperosmotic shock stress. These findings may be of significant clinical importance. In human immunological systems, it is thought that invading pathogen of C. albicans is attacked by phagocytic cells such as neutrophils or mononuclear macrophages through the release of ROS as killing agents during antifungal defense.34 According to our results that no difference existed between the FLC-resistant strain and the -susceptible strain in the sensibility to extracellular H2O2 stress, the identical chance could be expected for both strains to be killed by ROS generated by phagocytic cells; however, the resistant strain may coordinate itself with the host in a better manner due to its lower intracellular ROS generation once antifungal agents are employed. In summary, the comparative proteomic analysis has identified 22 proteins altered during the development of FLC resistance in C. albicans in this study. The identification of these proteomic changes and the further functional analysis have clarified a metabolic shift in the FLC-resistant C. albicans strain. The data presented in this study may therefore provide a more detailed understanding of the evolution of drug resistance in C. albicans.

Acknowledgment. We thank Dr. William A Fonzi for kindly providing the isolate C. albicans SC5314 used in this study. This work was supported by grants from the National Natural Science Foundation of China (No. 30200012, 30200353) and from the Key Programs of Science and Technique Foundation of Shanghai (No. 03JC14006, 04JC14003). We

(1) Almirante, B.; Rodriguez, D.; Park, B. J.; Cuenca-Estrella, M.; Planes, A. M.; Almela, M.; Mensa, J.; Sanchez, F.; Ayats, J.; Gimenez, M.; Saballs, P.; Fridkin, S. K.; Morgan, J.; RodriguezTudela, J. L.; Warnock, D. W.; Pahissa, A.; and the Barcelona Candidemia Project Study Group. J. Clin. Microbiol. 2005, 43, 1829-1835. (2) Kelly, S. L.; Lamb, D. C.; Kelly, D. E.; Manning, N. J.; Loeffler, J.; Hebart, H.; Schumacher, U.; Einsele, H. FEBS. Lett. 1997, 400, 80-82. (3) White, T. C.; Holleman, S.; Dy, F.; Mirels, L. F.; Stevens, D. A. Antimicrob. Agents Chemother. 2002, 46, 1704-1713. (4) White, T. C. Antimicrob. Agents Chemother. 1997, 41, 1488-1494. (5) Sanglard, D.; Ischer, F.; Parkinson, T.; Falconer, D.; Bille, J. Antimicrob. Agents Chemother. 2003, 47, 2404-2412. (6) Jha, S.; Dabas, N.; Karnani, N.; Saini, P.; Prasad, R. FEMS Yeast Res. 2004, 5, 63-72. (7) Coste, A. T.; Karababa, M.; Ischer, F.; Bille, J.; Sanglard, D. Eukaryotic Cell. 2004, 3, 1639-1652. (8) Wirsching, S.; Michel, S.; Kohler, G.; Morschhauser, J. J. Bacteriol. 2000, 182, 400-404. (9) Mukherjee, P. K.; Chandra, J.; Kuhn, D. M.; Ghannoum, M. A. Infect. Immun. 2003, 71, 4333-4340. (10) Rogers, P. D.; Barker, K. S. Antimicrob. Agents. Chemother. 2002, 46, 3412-3417. (11) Rogers, P. D.; Barker, K. S. Antimicrob. Agents Chemother. 2003, 47, 1220-1227. (12) Liu, T. T.; Lee, R. E. B.; Baker, K. S.; Lee, R. E.; Wei, L.; Homayouni, R.; Rogers, P. D. Antimicrob. Agents Chemother. 2005, 49, 22262236. (13) Cowen, L. E.; Nantel, A.; Whiteway, M. S.; Thomas, D. Y.; Tessier, D. C.; Kohn, L. M.; Anderson, J. B. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9284-9289. (14) Hooshdaran, M. Z.; Barker, K. S.; Hilliard, G. M.; Kusch, H.; Morschhauser, J.; Rogers, P. D. Antimicrob. Agents Chemother. 2004, 48, 2733-2735. (15) Hooshdaran, M. Z.; Hilliard, G. M.; Rogers, P. D. Methods Mol. Med. 2005, 118, 57-70. (16) Kusch, H.; Biswas, K.; Schwanfelder, S.; Engelmann, S.; Rogers, P. D.; Hecker, M.; Morschhauser, J. Mol. Genet. Genomics 2004, 271, 554-565. (17) Fonzi, W. A.; Irwin, M. Y. Genetics 1993, 134, 717-728. (18) Cowen, L. E.; Sanglard, D.; Calabrese, D.; Sirjusingh, C.; Anderson, J. B.; Kohn, L. M. J. Bacteriol. 2000, 182, 1515-1522. (19) Espinel-Ingroff, A.; Rodriguez-Tudela, J. L.; Martinez-Suarez, J. V. J. Clin. Microbiol. 1995, 33, 3154-3158. (20) Pitarch, A.; Sanchez, M.; Nombela, C.; Gil, C. Mol. Cell. Proteomics 2002, 1, 967-982. (21) Yergey, A. L.; Coorssen, J. R.; Backlund, P. S., Jr.; Blank, P. S.; Humphrey, G. A.; Zimmerberg, J.; Campbell, J. M.; Vestal, M. L. J. Am. Soc. Mass Spectrom. 2002, 13, 784-791. (22) Schmitt, M. E.; Brown, T. A.; Trumpower, B. L. Nucleic Acids Res. 1990, 18, 3091-3092. (23) Salvioli, S.; Ardizzoni, A.; Franceschi, C.; Cossarizza, A. FEBS Lett. 1997, 411, 77-82. (24) Kobayashi, D.; Kondo, K.; Uehara, N.; Otokozawa, S.; Tsuji, N.; Yagihashi, A.; Watanabe, N. Antimicrob. Agents Chemother. 2002, 46, 3113-3117. (25) Enjalbert, B.; Nantel, A.; Whiteway, M. Mol. Biol. Cell. 2003, 14, 1460-1467. (26) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Electrophoresis 1999, 20, 3551-3567. (27) Karababa, M.; Coste, A. T.; Rognon, B.; Bille, J.; Sanglard, D. Antimicrob. Agents Chemother. 2004, 48, 3064-3079.

Journal of Proteome Research • Vol. 6, No. 6, 2007 2255

research articles (28) MacKenzie, F. M.; Greig, P.; Morrison, D.; Edwards, G.; Gould, I. M. J. Antimicrob. Chemother. 2002, 50, 689-697. (29) Duan, Z.; Lamendola, D. E.; Yusuf, R. Z.; Penson, R. T.; Preffer, F. I.; Seiden, M. V. Anticancer Res. 2002, 22, 19331941. (30) Eisenman, H. C.; Craig, E. A. Mol. Microbiol. 2004, 53, 335-344. (31) Staniek, K.; Gille, L.; Kozlov, A. V.; Nohl, H. Free Radical Res. 2002, 36, 381-387.

2256


Yan et al. (32) Danley, D. L.; Hilger, A. E.; Winkel, C. A. Infect. Immun. 1983, 40, 97-102. (33) Kontoyiannis, D. P. J. Antimicrob. Chemother. 2000, 46, 191-197. (34) Helmerhorst, E. J.; Stan, M.; Murphy, M. P.; Sherman, F.; Oppenheim, F. G. Mitochondrion 2005, 5, 200-211.

PR060656C

Proteomic Analysis Reveals a Metabolism Shift in a Laboratory

Recommend Documents