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Proteomic Analysis Reveals a Synergistic Mechanism of Fluconazole and Berberine against Fluconazole-Resistant Candida albicans: Endogenous ROS Augmentation Yi Xu, Yan Wang,* Lan Yan, Rong-Mei Liang, Bao-Di Dai, Ren-Jie Tang, Ping-Hui Gao, and Yuan-Ying Jiang* Department of Pharmacology, School of Pharmacy, Second Military Medical University, 325 Guo He Road, Shanghai 200433, China Received June 9, 2009

Our previous study showed that concomitant use of berberine (BBR) and fluconazole (FLC) provided a synergistic action against FLC-resistant Candida albicans (C. albicans) clinical strains in vitro. To clarify the mechanism underlying this action, we performed a comparative proteomic study in untreated control cells and cells treated with FLC and/or BBR in 2 clinical strains of C. albicans resistant to FLC. Our analyses identified 16 differentially expressed proteins, most of which were related to energy metabolisms (e.g., Gap1, Adh1, and Aco1). Functional analyses revealed that FLC + BBR treatment increased mitochondrial membrane potential, decreased intracellular ATP level, inhibited ATP-synthase activity, and increased generation of endogenous reactive oxygen species (ROS) in FLC-resistant strains. In addition, checkerboard microdilution assay showed that addition of antioxidant ascorbic acid or reduced glutathione reduced the synergistic antifungal activity of FLC + BBR significantly. These results suggest that mitochondrial aerobic respiration shift and endogenous ROS augmentation contribute to the synergistic action of FLC + BBR against FLC-resistant C. albicans. Keywords: berberine • synergistic mechanism • fluconazole resistance • Candida albicans • proteomics

Introduction Candida albicans, the most prevalent fungal pathogen of humans, causes superficial mycoses, invasive mucosal infections, and disseminated systemic disease.1-4 There are a limited number of antifungal drugs available for clinical use at present, of which fluconazole (FLC) is used most widely because of its high bioavailability and low toxicity.5,6 However, recurrent use of current antifungal drugs has led to the rapid development of drug resistance.7-12 High mortality of invasive Candida infections in immunocompromised patients and limited availability of highly efficacious and safe antifungal agents call for the development of new antifungal therapeutics.13-15 Berberine (BBR), widely distributed in plant families such as Hydrastis canadensis (goldenseal), Coptis chinensis (Coptis or goldenthread) and Berberis vulgaris (barberry), is an alkaloid with a long history of medicinal use in traditional Chinese medicine. Recently, we found that FLC-BBR combination was highly efficacious to kill FLC-resistant C. albicans, with the fractional inhibitory concentration index (FICI) less than 0.1.16 However, the synergistic mechanism of FLC and BBR remains unknown. The development of proteomics offers a powerful method for large-scale analysis of protein expression and investigation of the synergistic mechanism of drugs. In this study, we used * To whom correspondence should be addressed. E-mail: jiangyuanying@ 126.com (Y.Y.J.), [email protected] (Y.W.). Tel: 86-021-8187-1201 (Y.Y.J.), 86-021-8187-1280(Y.W.). Fax: 86-021-6549-0641 (Y.Y.J, Y.W.).

5296 Journal of Proteome Research 2009, 8, 5296–5304 Published on Web 09/14/2009

two-dimensional (2-D) gel electrophoresis to analyze the protein expression profiles of FLC-resistant C. albicans cell strains treated or untreated with FLC and/or BBR, and found that a series of differentially expressed proteins were involved in energy metabolisms, oxidoreduction, and other biological functions. In addition, measurements of mitochondrial membrane potential, intracellular ATP concentration, ATP-synthase activity, endogenous reactive oxygen species (ROS) generation and the effect of antioxidant ascorbic acid (AA) or reduced glutathione (GSH) on the synergistic antifungal activity of FLC + BBR suggested that the synergistic action of FLC + BBR against FLC-resistant C. albicans might be related to mitochondrial aerobic respiration shift and endogenous ROS augmentation.

Materials and Methods Candida Strains, Growth Conditions and Agents. C. albicans 0304103, 01010 (two clinical strains both with FLC MIC80 > 64 µg/mL and BBR MIC80 > 32 µg/mL) were used in this study. C. albicans cells were propagated in yeast-peptone-dextrose (YPD) medium (1% (w/v) yeast extract, 2% (w/v) peptone, 2% (w/v) dextrose). FLC (Pfizer-Roerig Pharmaceuticals, New York, NY) and BBR (Sigma-Aldrich, St. Louis, MO) were prepared in dimethyl sulfoxide (DMSO). Drug Exposure and Protein Sample Preparation. C. albicans cell strains (1 × 106 cells/mL) were treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h at 30 °C. The dosage and exposure time were determined according to the 10.1021/pr9005074 CCC: $40.75

 2009 American Chemical Society

Synergistic Mechanism of FLC and BBR 17,18

literature and the results from our preliminary experiments. Cells were 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 in 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 1 min and cooling on ice for 1 min 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 according to the manufacturer’s directions. 2-D Gel Electrophoresis. Proteins containing 300 µg were rehydrated 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 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/2 gels constant current for 6 h using a Protean II gel tank (Bio-Rad). Gels were silverstained according to Concha Gil and co-workers.19 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 ferricyanide 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/ µL 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)20 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

research articles the GPS Explorer software (Applied Biosystems) to identify the proteins. The search parameters were as follows: NCBI database, fungi, mass ranging 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 the criterion for correct identification. Real-Time RT-PCR. Total RNA samples from C. albicans strains were extracted using the hot-phenol method,21 and 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 a 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.), which are 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 cells exposed to FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h relative to that of the control cells was calculated using the formula 2-∆∆CT, where ∆CT was the CT value of genes of interest minus that of the internal control, and ∆∆CT was the mean ∆CT value of the cells exposed to FLC and/or BBR minus that of the control cells. Assay for Mitochondrial Membrane Potential (∆Ψm). C. albicans cell strains treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h were washed and adjusted to 1 × 106 cells/mL with PBS buffer. After a treatment with 10 µg/ mL 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide22 (JC-1; Molecular Probes, Inc., Eugene, OR) at 30 °C for 15 min, the 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). ∆Ψm was determined by the ratio of red to green fluorescence intensity (FI). Measurement of Intracellular ATP Level. C. albicans cell strains treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h were adjusted to 1 × 107 cells/mL with YPD broth. A total of 100 µL of cell suspension was mixed completely with the same volume of BacTiter-Glo reagent (Promega Corparation, Madison, WI) and incubated for 10 min at room temperature. Luminescent signals were determined on a TD 20/20 luminometer (Turner Biosystem, Sunnyvale, CA) with 1 s integration time per sample. The control tube without cells was used to obtain a value for background luminescence. The signal-to-noise ratio (S/N) 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. Isolation of Mitochondria. C. albicans cell strains were treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/ Journal of Proteome Research • Vol. 8, No. 11, 2009 5297

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mL) for 6 h and a total of 5 × 10 cells of each sample were prepared. Mitochondria were isolated using the Yeast Mitochondria Isolation Kit (GenMed Scientifics, Inc.) and according to the manufacturer’s directions. The isolated mitochondria were lysed in EDTA buffer (2 mM, pH 9.0) by ultrasound (20 s × 3 cycle, 50% power) and the mitochondrial protein concentration was determined by the Bradford method.23 Assay for ATP-Synthase (Complex V) Activity. ATP-synthase activity was determined by colorimetry using the Mito Complex V Activity Assay Kit (GenMed Scientifics, Inc.) and according to the manufacturer’s directions. Optical density (OD) was measured on the POLARstar Galaxy at a wavelength of 340 nm. One unit of ATP-synthase activity is defined as the amount of enzyme activity that oxygenizes 1 µmol NADH/min at 30 °C and pH 7.5. 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) at 30 °C for 30 min. After being washed and resuspended in PBS buffer, cells were treated or untreated with FLC (64 µg/ mL) and/or BBR (16 µg/mL) for 6 h. FI values were detected on the POLARstar Galaxy with excitation wavelength at 488 nm and emission wavelength at 525 nm. ROS production was calculated by subtracting the FI value of cells treated or untreated with FLC and/or BBR without DCFH-DA from that of cells treated or untreated with FLC and/or BBR with DCFHDA. Checkerboard Microdilution Assay. Assays were performed according to the methods of the CLSI (formerly NCCLS) (M27-A).44,45 Briefly, RPMI 1640 medium was adjusted to pH 7 at 25 °C using 3-[N-morpholino]-propanesulphonic acid (MOPS). The initial concentration of the fungal suspension in RPMI 1640 medium was 103 CFU/mL, and the final concentrations ranged from 0.125 to 64 µg/mL for FLC and 1 to 32 µg/ mL for BBR in the 96-well microtiter plates. A drug-free medium with fungi and a fungus-free medium were used as the positive and negative controls, respectively. Plates were incubated at 35 °C for 24 h. OD was measured at 630 nm, and background OD was subtracted from that of each well. MIC80 was determined as the lowest concentration of the drugs (alone or in combination) that inhibited cell growth by 80%, compared with the cell growth of the drug-free wells. The fractional inhibitory concentration (FIC) index is defined as the sum of the MIC of each drug used in combination divided by the MIC of the drug used alone. Synergy and antagonism were defined by FIC indices of e0.5 and >4, respectively. An FIC index >0.5 but e4 was considered indifferent.46 To investigate the effect of antioxidant AA or GSH on the synergistic antifungal activity of FLC + BBR, freshly prepared AA or GSH solution was added to RPMI 1640 medium, and the AA-containing (5 and 10 mmol/ L) or GSH-containing (5 and 10 mmol/L) medium was used in the checkerboard microdilution assay for testing antifungal susceptibility of FLC + BBR. 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. 8

Results Protein Expression Profiles. To obtain stable and comparable 2-D gel map images, total protein samples from C. albicans cell strains treated or untreated with FLC and/or BBR were prepared in parallel for three independent experiments. 5298

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Each protein extract was then separated in triplicate by 2-D PAGE. Typical high-resolution silver-stained 2-D maps of 2 strains (C. albicans 0304103 and 01010) with protein differential expression areas are shown in Figure 1. The results were reproducible due to the repeated results of the same protein sample and of samples from independent cell extracts. Differences in protein expression profiles among the protein samples were analyzed based on the presence or absence of the expression of the protein spot. In addition, the spot must have coincident differential expression in both strains (0304103 and 01010). Twenty spots were absolutely differentially expressed in 4 maps of each strain (Figure 1). Identification of Differentially Expressed Proteins. Differentially expressed proteins were identified based on MALDITOF MS/MS analysis. A protein score greater than 70 means confidence limits >95% in the present study and was considered statistically significant. Of the 20 spots, 16 were identified with a Mascot score over 150, among which 10 were identified with a Mascot score over 300. High Mascot scores for most protein spots reflect a high number of peptides matching to the theoretical database.25 The proteins identified in this study were named according to the CandidaDB database (http://genolist.pasteur.fr/CandidaDB/) and divided into six groups according to their biological functions defined by the Candida Genome Database (Table 1). Interestingly, most of them were found to be involved in energy metabolisms including glycolysis (e.g., Fba1, Tpi1, Gap1, and Eno1), fermentation (e.g., Adh1), and tricarboxylic acid cycle (e.g., Aco1 and Idp2); the remainder functioned in oxidoreduction (e.g., IPF20104, IPF12147), vitamin metabolisms (e.g., Snz1), lipid metabolisms (e.g., Pot14), cell wall organization (e.g., Dit1) and others (e.g., IPF13836, Ino1, IPF15672, IPF19726). It was noticed that the expressions of glycolysisrelated proteins and fermentation-related proteins were decreased, and tricarboxylic acid cycle-related proteins were overexpressed after FLC-BBR combination treatment (Figure 2). According to the Pasteur effect that aerobic oxidation could inhibit glycolysis (alcohol-facient fermentation) under aerobic circumstance, it can be assumed that the efficiency of mitochondrial aerobic respiration might be increased after the combined treatment of FLC and BBR in FLC-resistant C. albicans. Validation of Proteomic Results by Real-Time RT-PCR Analysis. To validate differential expression of the identified proteins, real-time RT-PCR analysis was performed in both 0304103 and 01010 strains. Total RNA samples from the cells treated or untreated with FLC and/or BBR were 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 of gene expression and protein expression (Figure 3). Mitochondrial Membrane Potential (∆Ψm). To evaluate the efficiency of mitochondrial aerobic respiration, mitochondrial membrane potential, a direct indicator of mitochondrial aerobic respiration efficiency, was measured. In both 0304103 and 01010 strains, single FLC treatment resulted in a marked decrease in ∆Ψm (P < 0.05), and single BBR treatment did not result in any significant change in ∆Ψm. FLC + BBR treatment resulted in a significant increase in ∆Ψm (P < 0.05) (Figure 4). These results suggest that FLC + BBR treatment promoted the efficiency of mitochondrial aerobic respiration. Intracellular ATP Content. Since intracellular ATP generation is positively correlated with ∆Ψm in C. albicans under

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Figure 1. 2-D SDS polyacrylamide gel of whole cell extract of C. albicans strains. Molecular weight (MW, kDa) and isoelectric point (pI) are indicated along the y- and x-axis, respectively. Selected regions show significant differences in protein expression profiles among cells treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL). Arrows indicate proteins that are differentially expressed absolutely. (A) Strain 0304103; (B) strain 01010.

normal culture conditions, the intracellular ATP concentration in the cells treated or untreated with FLC and/or BBR was further measured. In both strains, the intracellular ATP content in cells treated with FLC alone was significantly decreased, which was consistent with the results for ∆Ψm (P < 0.01). Single BBR treatment also decreased the intracellular ATP content slightly but significantly (P < 0.05). FLC + BBR treatment caused a very significant decrease in the intracellular ATP concentration (P < 0.01), which was converse to ∆Ψm results (Figure 5). ∆Ψm and normal oxidative phosphorylation of mitochondria are both required for ATP generation, and dysfunction of oxidative phosphorylation can disrupt the positive correlation between ∆Ψm and intracellular ATP content. Therefore, it could

be assumed that the oxidative phosphorylation function of mitochondria may be inhibited after FLC-BBR combination treatment. ATP-Synthase (Complex V) Activity. Given the fact that oxidative phosphorylation is catalyzed by ATP-synthase (complex V), ATP-synthase activity was further examined. As expected, ATP-synthase activity in both strains decreased markedly in FLC + BBR treated cells (42.4% of the control cells for strain 0304103 and 35.6% for strain 01010). FLC treatment alone did not cause significant change in ATP-synthase activity, and BBR treatment alone resulted in a slight decrease in ATPsynthase activity (80.3% of the control cells for strain 0304103 and 75.2% for strain 01010) (Figure 6). These results demonJournal of Proteome Research • Vol. 8, No. 11, 2009 5299

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Table 1. Identified Proteins spot

proteinsa

accession numbera

functions

MWb (kDa)

pIb

controlc

FLCc

BBRc

FLC + BBRc

Energy Metabolism 5 16 17 8 18 19

Gap1 Gap1 Gap1 Tpi1 Eno1 Fba1

1. Glycolysis Glyceraldehyde-3-phosphate dehydrogenase CA5892 Glyceraldehyde-3-phosphate dehydrogenase CA5892 Glyceraldehyde-3-phosphate dehydrogenase CA5892 Triose phosphate isomerase CA5950 Enolase I (2-phosphoglycerate dehydratase) CA3874 Fructose-bisphosphate aldolase CA5180

36 36 36 27 47 39

6.61 6.61 6.61 5.74 5.54 5.69

+ + + + + +

+ + + + + +

+ + + + + +

-

10 14 20

Adh1 Adh1 Adh1

Alcohol dehydrogenase Alcohol dehydrogenase Alcohol dehydrogenase

2. Fermentation CA4765 CA4765 CA4765

46 46 46

8.26 8.26 8.26

+ + +

+ + +

+ + +

-

9 15

Aco1 Idp2

Aconitate hydratase Isocitrate dehydrogenase

3. Tricarboxylic Acid Cycle CA3546 CA0643

84 49

5.96 5.84

-

-

-

+ +

4 13

IPF20104 IPF12147

Alcohol dehydrogenase Oxidoreductase

Oxidoreduction CA2520 CA3008

40 37

6.51 5.89

-

-

+ +

+ +

7

Snz1

Stationary phase protein by homology

Vitamin Metabolism CA4184

32

5.76

-

-

-

+

Lipid Metabolism CA0290

42

6.47

-

+

+

+

Cell Wall Organization CA0437

22

6.16

-

+

+

+

6.94 5.35 5.83 6.10

+ -

+ + + -

+ -

+ + +

11

Pot14

Acetyl-CoA acetyltransferase

12

Dit1

Spore wall maturation protein

2 6 1 3

IPF13836 Ino1 IPF15672 IPF19726

Not Classified and Unknown-Function Proteins Probable heat shock protein CA2342 37 Myo-inositol-1-phosphate synthase CA5986 58 Unknown function CA2392 49 Unknown function CA1269 6

a Protein names and accession numbers according to the C. albicans genomic database (CandidaDB). using the Melanie 3.0 program). c +, expressed; -, not expressed.

strate that FLC + BBR treatment could inhibit ATP-synthase activity and further decrease the ATP level. Endogenous ROS Production. Since intracellular oxygen radicals are mainly generated in mitochondria and involved in the efficiency of mitochondrial aerobic respiration, endogenous ROS generation in cells treated or untreated with FLC and/or BBR were further measured. In both strains, FLC treatment alone did not influence the concentration of endogenous ROS, and BBR treatment alone resulted in a slight increase in endogenous ROS production (2.62 times in strain 0304103 and 2.55 times in strain 01010 as compared with that in the control cells). FLC + BBR treatment resulted in marked augmentation of endogenous ROS (5.74 times in strain 0304103 and 6.04 times in strain 01010 as compared with that in the control cells) (Figure 7). These results indicate that FLC + BBR treatment had a strong ability to augment endogenous ROS in FLC-resistant strains, which might be involved in the synergistic mechanism of FLC and BBR against FLC-resistant C. albicans. Checkerboard Microdilution Assay. To validate that the synergistic effect of FLC and BBR against FLC-resistant C. albicans was mainly due to endogenous ROS augmentation, checkerboard microdilution assay was performed to investigate the effect of antioxidant AA or GSH on the synergistic antifungal activity of FLC + BBR (Table 2). Without antioxidants, FLC + BBR exhibited a highly synergistic action against FLC-resistant C. albicans (FICIs in both strains ) 0.07). In the presence of 5 mmol/L AA, the synergistic 5300

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b

Experimental MW and pI values (calculated by

Figure 2. Central carbon metabolism in C. albicans during aerobic growth on glucose. The gray ellipses indicate that metabolic enzymes of low expression participated in glycolysis in cytoplasm after treatment with FLC + BBR. The black ellipses indicate metabolic enzymes of high expression participated in tricarboxylic acid cycling in mitochondria after treatment with FLC + BBR. G-6-P, glucose-6-phosphate; F-1,6-2P, fructose1,6-biphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde- 3-phosphate; 1,3-DPGA, 1,3-biphosphoglycerate; 3PGA, 3-phosphoglycerate; 2PGA, 2-phospho-glycerate; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.

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Figure 5. Intracellular ATP content in strain 0304103 and 01010 cells treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h. ATP levels represent the mean ( standard deviations for three independent experiments. *P < 0.05 vs control cells; **P < 0.01 vs control cells.

Figure 3. Gene expression changes in 16 proteins identified as differentially expressed by proteomic analysis in cells treated with FLC (64 µg/mL) and/or BBR (16 µg/mL) relative to those in untreated cells. (A) Strain 0304103; (B) strain 01010. All the genes were examined by real-time RT-PCR with gene-specific primers.

Figure 4. Mitochondrial membrane potential in strain 0304103 and 01010 cells treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h. The ratios represent the mean ( standard deviations for three independent experiments. *P < 0.05 vs control cells.

antifungal activity of FLC + BBR reduced markedly (FICIs rose to 0.28 in strain 0304103 and 0.31 in strain 01010), and in the presence of 10 mmol/L AA, no significant synergistic antifungal action of FLC + BBR was observed (FICIs rose to 0.75 in strain 0304103 and 1.25 in strain 01010). With addition of 5 mmol/L GSH, the synergistic antifungal activity of FLC + BBR decreased significantly (FICIs rose to 0.31 in strain 0304103 and 0.56 in strain 01010), and with addition of 10 mmol/L GSH, FLC + BBR failed to show any synergistic antifungal action (FICIs rose to 1.00 in strain 0304103 and

Figure 6. ATP-synthase activity in strain 0304103 and 01010 cells treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h. The ATP-synthase activity in control cells was presented as 100%. The percentages represent the mean ( standard deviations for three independent experiments. *P < 0.05 vs control cells; **P < 0.01 vs control cells.

Figure 7. Intracellular ROS generation in strain 0304103 and 01010 cells treated or untreated with FLC (64 µg/mL) and/or BBR (16 µg/mL) for 6 h. ROS levels represent the mean ( standard deviations for three independent experiments. *P < 0.05 vs control cells; **P < 0.01 vs control cells.

1.50 in strain 01010). These results show that the addition of antioxidants could reduce the synergistic antifungal activity of FLC + BBR significantly, which further indicates that endogenous ROS augmentation is the major mechanism of the synergistic action of FLC + BBR against FLC-resistant C. albicans. Journal of Proteome Research • Vol. 8, No. 11, 2009 5301

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Table 2. Interaction of FLC and BBR against C. albicans Strains 0304103 and 01010 by Checkerboard Microdilution Assay MIC80 (µg/mL) 0304103 used alone FLC

without antioxidants plus ascorbic acid (AA) 5 mmol/L 10 mmol/L plus reduced glutathione (GSH) 5 mmol/L 10 mmol/L

BBR

64

16

64 >64

16 32

64 >64

16 32

FLC

0.5

FIC index

FLC

BBR

0.07

64

16

2 32

4 16

0.28 0.75

64 >64

16 32

4 64

4 16

0.31 1.00

64 >64

16 32

In this study, we investigated the synergistic mechanism of FLC and BBR against FLC-resistant C. albicans. Comparative proteomic study was performed in FLC-resistant C. albicans cell strains treated or untreated with FLC and/or BBR and the results were confirmed by real-time RT-PCR. It was found that differentially expressed proteins were involved in multiple biochemical functions. Our particular interest was the striking changes of energy-metabolism-related proteins after FLC + BBR treatment: the down-regulated glycolysis-related proteins and fermentation-related proteins, and the up-regulated tricarboxylic acid cycle-related proteins. According to the Pasteur effect that aerobic oxidation could inhibit glycolysis (alcoholfacient fermentation) under aerobic circumstances, the results of comparative proteomic study in this research suggest that the efficiency of mitochondrial aerobic respiration might be increased after FLC + BBR treatment. To test this hypothesis, functional analysis was carried out to measure mitochondrial membrane potential, intracellular ATP concentration, ATPsynthase (complex V) activity and endogenous ROS production in FLC-resistant C. albicans cell strains treated or untreated with FLC and/or BBR. To determine whether endogenous ROS augmentation was the major mechanism of the synergistic action of FLC + BBR against FLC-resistant C. albicans, we performed checkerboard microdilution assay to investigate the effect of antioxidant AA or GSH on the synergistic antifungal activity of FLC + BBR. Taken together, our results suggest that the synergistic mechanism of FLC + BBR against FLC-resistant C. albicans is involved in the augmentation of endogenous ROS by affecting aerobic respiration in mitochondria. Endogenous ROS are normal byproducts of energy production in respiring cells. They have strong oxidant activity and may attack large molecules, causing oxidative damage to the organism. It was reported recently that ROS augmentation was involved in the mechanism of several antifungal agents. FLC toxicity is dependent on active oxidative phosphorylation in Saccharomyces cerevisiae, and increased intracellular ROS production is one of the antifungal activities of miconazole.24,26 In addition, decreased endogenous ROS generation contributes to drug resistance of C. albicans. It was found in our previous study that decreased efficiency of mitochondrial respiration in FLC-resistant strains reduced the ability of endogenous ROS to protect cells from being insulted by the antifungal agents, ultimately leading to resistance to FLC.27 These findings are well consistent with the results of the present study: both ∆Ψm and intracellular ATP level were low in the FLC-resistant strains, even after exposure to FLC. However, FLC + BBR treatment could augment endogenous ROS and overcome the drug Journal of Proteome Research • Vol. 8, No. 11, 2009

BBR

used alone

1

Discussion

5302

01010

used in combination

used in combination FLC

0.5

BBR

FIC index

1

0.07

4 32

4 32

0.31 1.25

4 64

8 32

0.56 1.50

resistance through the mechanism of metabolism shift, indicating that ROS augmentation might be a major synergistic mechanism of FLC + BBR against the FLC-resistant C. albicans. The primary source of endogenous ROS is the leakage of electrons from the mitochondrial respiratory chain (electron transport chain).28,29 According to the chemiosmotic hypothesis proposed by Peter Mitchell, coupled hydrogen atoms from tricarboxylic acid cycle provided protons (H+) and electrons to the respiratory chain in mitochondria (2H a 2H+ + 2e). During the process of electron transportation, the proton is transported from the matrix to the intermembrane space by complex I, III and IV, leading to electrochemical gradient (H+ concentration gradient and mitochondrial membrane potential (∆Ψm)) so as to store energy for producing ATP. Normally, the higher ∆Ψm is accompanied by the more efficient electron transportation, which would lead to leakage of more electrons to generate ROS. Previous studies have indicated that the generation of ROS is exponentially dependent on ∆Ψm,30 which is consistent with the result in this study: both ∆Ψm and endogenous ROS were obviously augmented in FLC-resistant C. albicans after FLC + BBR treatment. It was found in this study that FLC + BBR treatment increased ∆Ψm and decreased ATP level. The results of further investigation showed that FLC + BBR inhibited the ATPsynthase (complex V) activity. It is therefore presumable that it was inhibition of the ATP-synthase activity that led to differences between ∆Ψm and ATP level. In normal conditions, protons return to the mitochondrial matrix through the F0 proton channel following the concentration gradient. Meanwhile, ADP was phosphorylated to generate ATP in F1 domain of ATP-synthase. Once the ATP-synthase activity is inhibited and blocks the return way of protons, ADP phosphorylation could not progress, thus, diminishing the generation of ATP. High ∆Ψm is coupled with a low level of ATP when ATPsynthase activity is inhibited,31 which is consistent with the result of this study. In addition, blockage of the proton return way may aggravate electron leakage, further resulting in more production of ROS.32 In summary, FLC + BBR treatment led to ROS augmentation via two ways: (1) promoting electron transportation in the respiratory chain by enhancing the tricarboxylic acid cycle, increasing ∆Ψm to elevate the production of ROS; and (2) preventing protons from returning to the mitochondrial matrix by inhibiting ATP-synthase activity, aggravating electron leakage, and finally leading to augmentation of endogenous ROS (Figure 8). AA and GSH are common antioxidants in the organism. Interaction of AA or GSH with ROS may attenuate the oxidant effect of ROS and alleviate the damage caused by ROS to the

research articles

Synergistic Mechanism of FLC and BBR

Figure 8. FLC + BBR treatment promoted generation of ROS in mitochondria of C. albicans. Stimulation of the tricarboxylic acid cycle by FLC + BBR treatment enhances electron flow into the respiratory chain, and inhibition of ATP-synthase activity by FLC + BBR treatment aggravates electron leakage. These events enhance ROS generation. Complex II is omitted from this diagram for clarity. Cx I-V, complexes I-V; Q, Q cycle; C, cytochrome c; ∆Ψm, membrane potential; TCA cycle, tricarboxylic acid cycle.

organism.47 In this study, addition of AA or GSH reduced the synergistic activity of FLC + BBR against FLC-resistant C. albicans, indicating that the antioxidants could alleviate the oxidative damage caused by endogenous ROS to the organism, surviving the FLC-resistant C. albicans to the combination treatment with FLC and BBR. These results also demonstrate that endogenous ROS augmentation is the major mechanism of the synergistic action of FLC + BBR against FLC-resistant C. albicans. Apart from the energy-metabolism-related proteins, some other differentially expressed proteins were also identified in this study. IPF20104 is a putative alcohol dehydrogenase and has been reported to be involved in oxidative stress response by overexpression in C. albicans treated with hydrogen peroxide or diamide.33,34 In this study, the overexpression of IPF20104 was observed in BBR-treated cells and FLC + BBR-treated cells, which was consistent with the result of ROS generation measurement, indicating that oxidative stress occurred in the cells after treatment with BBR alone or FLC + BBR. Snz1 is a putative stationary phase protein, and has been reported to be exclusively expressed in the stationary phase, a phase caused by nutrient limitation.35,36 In the nutrientexhaustion niche, both generation and consumption of intracellular ATP decreased markedly, and the vital movement became very slow in C. albicans. Snz1 was assumed to be involved in growth limitation in C. albicans. In this study, Snz1 was only expressed in cells treated with FLC + BBR, which was consistent with the phenomenon that intracellular ATP and growth state were much lower in the cells after FLC + BBR treatment. In addition, the expressions of IPF12147, IPF13836 and IPF19726 were also altered after FLC + BBR treatment. However, the roles of these proteins in the synergistic action remain unclear and need further study. Besides, Ino1, Pot14, Dit1 and IPF15672 were expressed differentially not only in cells treated with FLC + BBR, but in cells treated with FLC alone or cells treated with BBR alone, suggesting that the altered expression of these proteins may be induced by multiple surrounding stimuli.37-40 In this study, a slight alteration in aerobic respiration was also observed in cells treated with BBR alone. Treatment with

BBR alone did not cause significant change in ∆Ψm in FLCresistant strains but decreased the intracellular ATP level and increased ROS generation. The results of assay for ATP-synthase activity performed in this study seem to imply that alteration in ATP production and endogenous ROS level might be caused by inhibition of ATP-synthase activity after treatment with BBR alone. Berberine is a kind of cytotoxic drug with extensive and effective bioactivity. However, the antifungal activity of BBR is very weak (MIC > 128 µg/mL).41-43 It is reasonable to assume that there might be a natural BBR-resistant mechanism in C. albicans, which could be disrupted in the presence of FLC. To test this hypothesis, a study on the BBR-resistant mechanism in C. albicans and the role of FLC in disrupting this mechanism is under way in our laboratory. In this proteomic study, a total of 16 different proteins were identified. The limited number of identified proteins was due to the strict inclusion criteria, that is, whether or not the protein spot was expressed in 2-D maps, and if it was, the spot must have coincident differential expression in both strains. Frankly speaking, such an experimental design might cause missing detection of some relevant proteins, but it also effectively avoided appearance of proteins with false differential expressions. In conclusion, the results of the present study demonstrate that FLC-BBR combination treatment could augment the production of endogenous ROS through two different ways in FLC-resistant C. albicans: (1) by enhancing the tricarboxylic acid cycle, and (2) by inhibiting the ATP-synthase activity. Increased ROS production contributes to the antifungal effect by means of strong oxidative damage to the organism. This biochemical process might be involved in the mechanism of the synergistic action of FLC and BBR against FLC-resistant C. albicans.

Acknowledgment. This work was supported by the National Natural Science Foundation of China (30825041, 30500628 and 30630071), Shanghai Educational Development Foundation (2007CG51), National Basic Research Project (2005CB523105), and Shanghai Natural Science Foundation (07ZR14142). The authors thank professor Gu Jun from Changhai Hospital (Shanghai, China) for providing C. albicans strains. 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 among the cells of FLC-resistant strains treated or untreated with FLC and/ or BBR are shown in Table S2. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Fridkin, S. K.; Jarvis, W. R. Epidemiology of nosocomial fungalinfections. Clin. Microbiol. Rev. 1996, 9, 499–511. (2) Gudlaugsson, O.; Gillespie, S.; Lee, K.; Vande Berg, J.; Hu, J.; Messer, S.; Herwaldt, L.; Pfaller, M.; Diekema, D. Attributable mortality ofnosocomial candidemia. Clin. Infect. Dis. 2003, 37, 1172–1177. (3) Pfaller, M. A. Nosocomial candidiasis: emerging species, reservoirs, and modes of transmission. Clin. Infect. Dis. 1996, 22 (Suppl. 2), S89–S94. (4) Wilson, L. S.; Reyes, C. M.; Stolpman, M.; Speckman, J.; Allen, K.; Beney, J. The direct cost and incidence of systemic fungal infections. Value Health 2002, 5, 26–34. (5) Kohli, A.; Smriti, Mukhopadhyay, K.; Rattan, A.; Prasad, R. In vitro low-level resistance to azoles in Candida albicans is associated with changes in membrane lipid fluidity and asymmetry. Antimicrob. Agents Chemother. 2002, 46, 1046–1052.

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