Microarray

resistance
in | a laboratory
C. ofalbicans
strainand Cell Biology, SIBS, CAS | All Rights Reserved 1672-9145
Acta Biochimanalysis
Biophys of
SinFLC
(2008):
1048-1060
2008 Institute
Biochemistry
http://www.abbs.info; www.blackwellpublishing.com/abbs | DOI: 10.1111/j.1745-7270.2008.00483.x

DNA microarray analysis of fluconazole resistance in a laboratory Candida

albicans strain
Lan Yan1, Jundong Zhang1, Miaohai Li2, Yongbing Cao1, Zheng Xu1, Yingying Cao1, Pinghui Gao1, Yan Wang1, and
Yuanying Jiang1*
1
2

Department of Pharmacology, College of Pharmacy, Second Military Medical University, Shanghai 200433, China
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang 110016, China

Received: June 22, 2008

Accepted: October 13, 2008
This work was supported by the grants from the National Basic Research
Program of China (No. 2005CB523105), the Hi-tech Research and
Development Program of China (No. 2007AA02Z187), the Natural
Science Fou nda tion of Shanghai (No. 07ZR14 142 ), and the Key
Progra ms of Science and Technique Foundation of Shanghai (No.
07JC14064)
*Corresponding author: Tel, 86-21-25070371; Fax, 86-21-65490641;
Email, jiangyysmmu@sina.com

Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1048

albicans isolates.

Keywords
microarray

Candida albicans; fluconazole; resistance;

Can dida a lbic a ns is one of the most common

opportunistic fungal pathogens in humans. Certain strains
are able to cause systemic infections in immunocompromised patients. Azole antifungal agents, especially
fluconazole (FLC), are usually used to treat these
infections. However, the overuse of and long-term
treatment with FLC have resulted in the emergence and
rapid expansion of FLC-resistant C. albicans, which has
become a severe clinical and social problem in treating
Candida infections [1].
Several mechanisms of FLC resistance in C. albicans
have been identified: defective Erg3 function with altered
sterol content [2], overexpression of the drug target
enzyme lanosterol demethylase gene ERG11 [3], point
mutations in ERG11 that result in reduced affinity of Erg11
to azoles [4,5], reduced intracellular accumulation of drugs
correlated with the increased expression of ATP-binding
cassette (ABC) transporters (CDR1 and CDR2) and/or
the major facilitators superfamily efflux pump (MDR1)
[68], and the formation of biofilms [9]. Many investigations have revealed that FLC resistance in C. albicans
results from a combina tion of several of these
mechanisms. Indeed, overexpression of ERG11 has
recently been shown to be controlled by the gain-offunction mutation in the transcription factor gene UPC2
[10]. Overexpression of CDR1 and CDR2 is due to the
similar mutations in both TAC1 alleles [11,12], while
overexpression of MDR1 is due to similar mutations in
MRR1 [13].
Recently, genome-wide gene expression studies by DNA

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Several mechanisms are responsible for the acquired

fluconazole (FLC) resistance in Candida albicans. In this
study, we developed a FLC-resistant C. albicans strain
through serial cultures of a FLC-susceptible C. albicans
strain with inhibitory concentrations of FLC. Complimentary DNA microarray analysis and real-time reverse transcription-polymerase chain reaction were used to investigate gene expression changes during the acquisition of azole
resistance in the susceptible parental strain and the resistant daughter strain. The differentially expressed genes represented functions as diverse as transporters (e.g. CDR1,
PDR17), ergosterol biosynthesis (e.g. ERG2, ERG9), sterol
metabolism (e.g. ARE2, IPF6464), energy metabolism (e.g.
ADH3, AOX2) and transcription factors (e.g. FCR1,
ECM22). Functional analysis revealed that energy-dependent efflux activity of membrane transporters increased and
that ergosterol content decreased with the accumulation of
sterol intermediates in the resistant strain as compared with
the susceptible strain. We found that a point mutation
(N977K) in transcription factor TAC1 that resulted in hyperactivity of Tac1 could be the reason for overexpression of
CDR1, CDR2, and PDR17 in the resistant strain.
Furthermore, a single amino acid difference (D19E) in ERG3
that led to the inactivation of Erg3 could account for both
sterol precursor accumulation and the changes in the expression of ergosterol biosynthesis genes in this resistant
strain. These findings expand the understanding of potential novel molecular targets of FLC resistance in clinical C.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

Materials and Methods

Strains and media
C. albicans strain y0109 (ATCC76615) was obtained from
Changzheng Hospital Strains Collection Center (Shanghai,
China). Media used in this study included yeast nitrogen
base (YNB) broth (Difco, Detroit, USA) supplemented with
2% glucose, yeast extract pentose dextrose (YPD; Difco),
sabouraud dextrose agar (Difco), and RPMI-1640 with
0.165 M morpholinepropanesulfonic acid (Sigma, St. Louis,
USA) adjusted to pH 7.0.
Antifungal agents
Standard antifungal powders of FLC, ketoconazole (KTC),
itraconazole (ITC), amphotericin B (AMB), and nystatin
were obtained from Sigma. Stock solutions were prepared
in distilled water (FLC, AMB) or dimethyl sulfoxide (KTC,
ITC), filter sterilized and then stored at 70 C.
Induction of drug resistance

Drug resistance was induced as described in previous

study [22]. Briefly, a single colony of C. albicans y0109
was inoculated into 10 ml YNB broth and incubated
overnight at 30 C with shaking (200 rpm). An aliquot of
this culture containing 106 cells was then transferred to
10 ml YNB broth containing twice their most recently
measured FLC minimal inhibitory concentration (MIC)
and incubated at 30 C with shaking. When the cultures
reached a density of about 108 cells/ml, aliquots containing
10 6 cells were transferred into fresh YNB broth also
containing twice their most recently measured FLC MIC
and incubated overnight at 30 C with shaking (200 rpm).
At each passage, an antifungal susceptibility test was
performed [23]. All the strains were stored in 1.5 ml 50%
glycerol at 80 C.
RNA preparation
A pair of FLC-susceptible and resistant C. albicans strains
were grown on sabouraud dextrose agar, and a single
colony of cells was transferred to 100 ml YPD broth in a
500-ml Erlenmeyer flask. After 16 h incubation at 30 C
with constant shaking (200 rpm), the suspension (300 l)
was subcultured for 6 h in 100 ml YPD broth to obtain
early logarithmic phase cultures. Then, cells were collected
by centrifugation at 1000 g for 5 min, flash-frozen, and
stored in liquid nitrogen. Total RNA was isolated using the
hot phenol method [24]. Frozen cells were resuspended in
12 ml AE buffer [50 mM sodium acetate (pH 5.2), 10 mM
EDTA] at room temperature, after which 800 l 25%
sodium dodecyl sulphate (SDS) and 12 ml acid phenol
were added. The cell lysate was then incubated for
10 min at 65 C with vortexing each minute, cooled on ice
for 5 min, and subjected to centrifugation at 12,000 g for
15 min. Supernatants were transferred to new tubes
containing 15 ml chloroform, mixed, and subjected to
centrifugation at 200 g for 10 min. RNA was precipitated
from the resulting aqueous layer by transferring that
portion to new tubes containing one volume isopropanol
and 0.1 volume 2 M sodium acetate (pH 5.0), and mixed
well. The mixture was centrifuged at 18,000 g for 35 min
at 4 C. The pellet was resuspended in 10 ml 70% ethanol
and centrifuged at 18,000 g for 20 min at 4 C to collect
RNA. RNA integrity was visualized by subjecting the sample
to electrophoresis through 1% agarose gel. Poly(A) mRNA
was extracted using Oligotex mRNA kit (Qiagen, Hilden,
Germany) and quantitated using the RiboGreen RNA
Quantitation kit (Invitrogen, Eugene, USA).
Gene expression analysis
Microarray preparation, synthesis of fluorescent cDNA
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microarray have shown that many genes, such as those

involved in sterol mechanism (ERG10, ERG25) [1416],
oxidative stress response (CRD2, GPX1, IFD5, IPF4065,
IPF7817) [14,15,17], and carbohydrate mechanism
(ADH4, YPL88) [18], are associated with FLC resistance
in clinical C. albicans isolates. Moreover, proteomic
analyses have also identified several differentially expressed
proteins such as Grp2, Ifd1, and Ifd4 in clinical FLCresistant C. albicans isolates and Pgk1, Qcr7, and Adh1 in
a laboratory resistant strain [1922]. These results have
identified differential genes already known to be involved
in drug resistance, genes whose expression are coordinately up-regulated or down-regulated with the upregulation of known resistant genes (CDR1, CDR2, and
MDR1).
The present study was designed to explore the potential
mechanisms of FLC resistance in a laboratory C. albicans
strain. We developed an in vitro model of FLC-resistant
C. albicans strain y0109 R that resulted from FLCsusceptible strain y0109S being exposed to concentrations
of FLC that increased incrementally. With this model, we
performed complementary DNA (cDNA) microarrays to
examine changes in the expressed-gene expression profile
between this matched pair. We identified the differential
genes involved in diverse biological functions. We also
investigated rhodamine 6G (R6G) efflux and measured
sterol content for further confirmation. Moreover, point
mutations in ERG3, ERG11, and TAC1 genes were further
examined in this pair of C. albicans strains.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1050

balanced differential expression ratio 2.0 or 0.5 for

each spot were considered to be statistically significant
(P<0.05). DNA sequences were annotated on the basis
of results of BLASTN searches using GenBank (http://
www.ncbi.nlm.nih.gov/entrez/query.fcgi), the sequencing
database of the Candida Genome Database (http://www.
candidagenome.org/), and the CandidaDB database (http:/
/genolist.pasteur.fr/CandidaDB/).
Quantitative real-time reverse tr anscriptionpolymerase chain reaction (RT-PCR)
C. albicans strains were cultured in the same conditions
as they were prior to microarray analysis. Briefly, the
suspension (300 l) of a 16 h culture was subcultured for
6 h in 100 ml YPD broth to obtain early logarithmic phase
cultures. Three independent RNA samples from the FLCsusceptible and resistant strains were isolated for three
separate experiments. Genomic DNA contamination was
removed, first-strand cDNA was synthesized, and realtime PCR reactions were performed [22]. In brief, RNA
samples were treated with 10 U DNase I (Takara, Dalian,
China) per 50 l RNA at 37 C for 1 h to remove genomic
DNA contamination. First-strand cDNA were synthesized
from 1 g total RNA in a 20 l reaction volume using a
cDNA synthesis kit (Ta ka ra ) a ccording to the
manufacturers instructions. Real-time PCR reactions
were performed in triplicate with cDNA from independent RNA isolations, using SYBR Green I (Takara) on the
LightCycler Real-Time PCR system (Roche Molecular
Biochemical, Indianapolis, USA). Gene-specific primers
were designed using Discovery Studio Gene software
(Accelrys Inc, San Diego, USA) (Table 1). The CT value
of 18S rRNA was subtracted from that of the gene of
interest to obtain a CT value. The CT value of the FLCsusceptible strain was subtracted from the CT value of
the FLC-resistant strain to obtain a CT value. The gene
expression level of the resistant strain relative to that of
the susceptible strain was expressed as 2CT. Triplicate
independent real-time RT-PCR analyses were performed
for each sample.
Glucose-induced R6G efflux assay
The glucose-induced efflux of R6G (Sigma) from C.
albicans strains was investigated [29]. Cells in the middle
logarithmic phase were collected by centrifugation at 3000 g
for 5 min and washed three times with phosphate buffered
saline (PBS; 10 mM phosphate buffer, 2.7 mM potassium
chloride, 140 mM sodium chloride, pH 7.4) buffer. Then
cells were resuspended at a concentration of 1107 cells/ml
in PBS buffer containing 5 mM 2-deoxy-D-glucose and

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probes, hybridization with C. albicans 3136 cDNA

microarray, and signal analysis were strictly conducted as
reported previously [2528]. In brief, the microarrays
consisted of full-length and partial cDNA sequences
representative of the sequences of expressed genes in
C. albicans, including unknown-in-function, known, and
control genes. The fluorescent cDNA probes were
synthesized from purified mRNA with Cy3- or Cy5-dUTP
(Amersham Pharmacia Biotech, Piscataway, USA) by oligo
(dT)-primed polymerization using Superscript II reverse
transcriptase (Invitrogen). The reaction buffer mixture
contained dNTPs (200 M dATP, dCTP, dGTP, 60 M
dTTP, and 60 M Cy3- or Cy5-dUTP), 2 l Superscript
II reverse transcriptase, and 1 reaction buffer. Reactions
were carried out at 42 C for 2 h. To stop the reaction, 4 l
2.5 M NaOH was added, the mixture was incubated at
65 C for 10 min, and was neutralized with 4 l 2.5 M
HCl. To avoid dye-associated effects on cDNA synthesis,
two independent hybridization experiments were performed
with RNA from the FLC-susceptible strain labeled by
Cy5-dUTP and by Cy3-dUTP separately, and so did RNA
from the FLC-resistant strain.
The probes were dissolved in 20 l hybridization solution
[5sodium chloride sodium citrate (SSC) (0.75 M NaCl
and 0.075 M sodium citrate), 0.4% SDS, 50% formamide].
Microarrays were pre-hybridized with hybridization
solution containing 0.5 mg heat-denatured salmon sperm
DNA at 42 C for 6 h. The fluorescent probe mixtures
were denatured at 95 C for 5 min, and then were applied
to the pre-hybridiz ed chip under a cover gla ss.
Hybridization was performed at 42 C overnight in a
homemade chamber. The slides were then washed for
10 min each at 60 C in solutions of 2 SSC and 0.2%
SDS, 0.1 SSC and 0.2% SDS, and 0.1 SSC. They were
then dried at room temperature.
The slides were scanned using a ScanArray 3000 (GSI
Lumonics, Bellerica, USA) to detect emissions from both
Cy3 and Cy5. The acquired images were analyzed with
ImaGene software (version 3.0; BioDiscovery, Los
Angeles, USA). The intensities of each spot at the two
wavelengths respectively represented the quantities of
Cy3-dUTP and Cy5-dUTP that hybridized to each spot.
The ratios of Cy5 to Cy3 were calculated for each location
on each microarray. To minimize artifacts that arise from
low expression values, only genes with raw intensity values
>800 counts for both Cy3 and Cy5 were chosen for
analysis.
In this study, duplicate independent experiments were
carried out, and RNA samples of each strain were labeled
and hybridized repeatedly. Only features with a mean

Microarray analysis of FLC resistance in a laboratory C. albicans strain

Table 1 Primers used for real-time reverse transcription-polymerase chain reaction

Gene

Primer pairs (53)

Amplicon size (bp)

CDR1

CCAACAATACAAGACCAGCATCTCC (F)/AATCGACGGATCACCTTTCATACG (R)

104

PDR17

TCCTACAGGAGGGATTTTCCCCAG (F)/TGCGGGACAAGATTCATTAGCG (R)

ERG2

CAGCAATTGGGACTGAAGGTCATAC (F)/CGGGAATCAATGCACCAGGATAAG (R)

105

ERG9

TTCCTCAAGTAATGGCAGTGG (F)/TTGACAACCCCTGGTAATGTTC (R)

136

IPF7817

AAGAGAACAGATGAATACGGTGGC (F)/GGATCAGGACTATTTTCAGCAGCAG (R)

140

PDR6
IPF4065

GGAAGACGAGAATGTTAGTGAGCAG (F)/GTTTCATCTCCTCGGCGTCATC (R)

ACCAAAGGGTCATCTACCATTCC (F)/ACCTTCAGCACCACGTTGTC (R)

86
84

EFG1

GCTGCTACTACTACTGCTGCTACTG (F)/CAGACACATTACTGCCACCACTG (R)

94

STE7

CAACAAGCGGTAGCCTAAGGAG (F)/GTTGGAGTGGACGTAGCAGATG (R)

NAG2

AGCCGTGAAAGCTGTTGAGAAAG (F)/TCAAACCCACAACACCAGCATTAC (R)

98

ECM22

AGCAAGCTCAAATCCTTCTGTCC (F)/CGTTGTTGTTGAAGCAACTCCTG (R)

87

PGK1
ADH4

AGAGCCCACTCCTCTATGGTTG (F)/TGTCAGAAACTTTAGCACCACCC (R)

TTGTGCTCCAGGTCCTATG (F)/CCTCCTTCAGTGACCAAAAATC (R)

148
150

GPM1

CCAAGTCGGCGACAGAAGATAC (F)/ACTTTACCAGCCAACAAGTCACC (R)

210

SSE1

GGTACACTGAAAAACAACGTC (F)/CAGCTTTGAAAACACCATAACC (R)

120

AOX2

GTACTCGTTGGGAAATGACTGAAGG (F)/ACGAAGAAATCCTGCAACAGATCC (R)

MCR1

ACTGATAGCAATGAATGGGTGG (F)/GGTCAACAAACATGAAGCAGTG (R)

ZCF20
IPF6235

TCCATTTGCTGTTCAACCACGAG (F)/AGGCAAACTGGGAAAGACAAGAC (R)

AGGTGGAAGAGAAACTTGAGG (F)/GAGATTTGGAAGACTACGGAC (R)

90
80

FCR1

TCTTCTCCTGCTTCATCAACAC (F)/TTTATAGGAGCAACCACACCAG (R)

95

IPF4905

CTTCCAACACCAATGACGAGTACC (F)/GTCGTCTTCTTGTGCTTCATCCTC (R)

104

IPF2857

GCCGAGGCAATTAACCAGAAAAC (F)/ACTGCTTCAGCACCTTTCTTGATG (R)

119

ERG11

ATGGGATACTGCTGCTGCC (F)/CCCCTTTAGAAACTTTCCCAAACC (R)

MDR1
CDR2

GGGTAGTTCCGTGTTGGGTTTC (F)/TGCTCTCAACTTTGGTCCGTTC (R)

CGAGGTGGAGCACTTTTCTTTTCAG (F)/AGCTAATGCATCAGCAGATGGAC (R)

85
141

TAC1

TTTAAATCCGGTAAACCTCCAG (F)/TTGTGTGGCAAACAGGAC (R)

145

18S

GTGCCAGCAGCCGCGGTA (F)/TGGACCGGCCAGCCAAGC (R)

85

133

89

99

F, forward; R, reverse.

10 M R6G. Cell suspensions were incubated at 30 C

with shaking (200 rpm) for 90 min to allow R6G
accumulation under glucose starvation conditions. The
starved cells were washed twice in PBS buffer, and 1 ml
portions were incubated at 30 C for 5 min before the
addition of 1 mM glucose to initiate R6G efflux. At specified
intervals after the addition of glucose, the cells were
removed by centrifugation. Triplicate 100 l volumes of
the cell supernatants were transferred to 96-well flat-bottom
microtiter plates on a Polarstar Optima instrument (BMG
Labtech, Offenburg, Germany). The fluorescence of the
samples was measured with an excitation wavelength at
515 nm and an emission wavelength at 555 nm.
Sterol analysis

C. albicans strains were inoculated from a single colony

and cultured overnight in YPD broth at 30 C with constant
shaking (200 rpm). After 16 h incubation, 2 ml from this
suspension were subcultured for 24 h in 98 ml YPD broth
containing 0, 0.25, 1 and 4 g/ml FLC, respectively. Cells
were then washed, harvested, and weighed. From each
wet cell, a 0.5 g sample was transferred to glass tubes
containing 6 ml 15% NaOH in 90% (V/V) ethanol and
2.5 ml PBS buffer. The samples were heated at 80 C for
60 min and then cooled on ice. Non-saponifiable lipids
were extracted twice with 6 ml mineral ether (3060 C
boiling extent) each time, and the extracts were evaporated
to dryness under N2 at 45 C. The samples were dissolved
in 1 ml cyclohexane per gram of wet cells and stored
at 20 C prior to analysis by gas chromatography-mass
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98
135

Microarray analysis of FLC resistance in a laboratory C. albicans strain

spectrometry (GC-MS).
GC-MS analyses were performed with a Finnigan
Voyager instrument (Finnigan, San Jose, USA). The gas
chromatograph was equipped with a phenylmethylpolysiloxane column (length 30 m, inner diameter
0.25 mm, film thickness 0.25 m). The settings were as
follows: source, positive electron ionization; scan, 50 to
650 Da in 0.9 s; interscan delay, 0.1 s; multiplier voltage,
450 V; solvent delay, 2.0 min; gas chromatograph
temperature ramp, 0 min at 150 C, 10 min at 290 C
(15 C/min); detector temperature, 280 C; sample injection
temperature, 250 C; carrier gas, helium gas, 1 ml/min.
Sterols were identified from their retention times and
specific mass spectrometric patterns.

Statistical analysis
Experiments were performed at least three times. Data are
presented as meanSD and analyzed using the Students
t-test where indicated.

Results
Generation of FLC-resistant C. albicans strain
To obtain a FLC-resistant C. albicans strain in the
laboratory, the susceptible C. albicans strain y0109S (FLC
MIC, 0.5 g/ml) was serially cultured in inhibitory
concentrations of FLC, and developed FLC resistance in
a stepwise fashion (Fig. 1). The MIC rose from 0.5 to 8
g/ml after 20 passages, and this value remained stable
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1052

for 15 passages. Further increases of FLC MIC were

measured: from 8 to 32 g/ml after 40 passages and from
32 to 64 g/ml, after 60 passages. The resultant organism
showed FLC MIC of 64 g/ml stable for up to 20 passages
in drug-containing medium. Meanwhile, the increase of
both ITC and KTC MIC paralleled that of FLC MIC,
indicating that the FLC-resistant strain was cross-resistant
to both ITC and KTC (Fig. 1). The strain at passage 75
(FLC MIC, 64 g/ml) was named y0109R. Interestingly,
the strain y0109R was also resistant to AMB (MIC, 32 g/
ml) and nystatin (MIC, 6.24 g/ml).
Furthermore, the resistant strain y0109R was serially
cultured in FLC-free broth medium. The FLC MIC of
strain y0109R decreased to 32 g/ml after 8 passages and
to 16 g/ml after 18 passages. KTC and ITC MIC similarly
decreased (Fig. 2). After 40 drug-free passages, the strain
reverted to the FLC-susceptible dose-dependent (16 g/ml),
KTC-susceptible (0.0625 g/ml), ITC-susceptible (0.125
g/ml), AMB-susceptible (0.5 g/ml), and nystatinsusceptible (0.78 g/ml) phenotype.
Overview of differentially expressed genes
In order to get insight into the development of FLC
resistance in this laboratory strain, we performed cDNA
microarrays to identify changes in the gene expression
profiles of y0109S and y0109R. In this study, two biological
replicates were performed, and RNA from the FLC-resistant
strain labeled by Cy5-dUTP and by Cy3-dUTP separately.
In each hybridization process, duplicate spots were
measured. Out of 3136 cDNA sequences examined, 278

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PCR amplification and DNA sequencing

Genomic DNA from the C. albicans strains was extracted
with the YeaStar Genomic DNA kit (Zymo Research,
Orange, USA) and was used as templates for amplification
of ERG3, ERG11, and TAC1 genes, respectively. Primer
pairs were chosen to amplify regions spanning the open
reading frame of the C. albicans ERG3 gene (5-CCCATTTCTTTCCCTATTGTGC-3; 5-AGACGACTTTCAAGATTGTCC-3), ERG11 gene (5-GTCAATTTGAGAACGAGAACG-3; 5-CTGAATCGAAAGAAAGTTGCC-3),
and TAC1 gene (5-AGAGCCTTTCTCCTTCTCTC-3; 5CATCGCTTTCACCAATTACAAC-3). PCR reactions
were carried out with high-fidelity Pyrobest DNA
polymerase (Takara). Amplifications were purified with
PCR product purification kit (ZhongDing Biotech
Company, Ningbo, China), and their nucleotide sequences
were determined by primer elongation with an ABI PRISM
3730 automated DNA sequencer (Applied Biosystems,
Foster City, USA) with Taq dye-primer and dyeterminator chemistries (Applied Biosystems).

Fig. 1 Variations of minimal inhibitory concentration (MIC)

of fluconazole (FLC), ketocolazole (KTC), and itroconazle (ITC)
for the Candida albicans strain y0109 S cultured in medium
containing twice its most recently measured MIC of FLC Each
datum point represents one passage.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

were shown to have more than 2-fold difference in

expression level between the pair of strains. Among these,
130 were up-regulated and 148 were down-regulated in
the strain y0109 R compared with those in y0109 S .
According to biological function, they were classified into
membrane transporters, ergosterol biosynthesis, sterol
metabolism, carbohydrate metabolism, lipid, fatty acid
metabolism, morphogenetic transition, and unknown
biological process. Differentially expressed genes are
shown in Table 2.

Table 2 Typically differentially expressed genes between Candida albicans strains y0109 S and y0109 R were identified by complimentary DNA microarray analysis

Gene name

Function description

Transporters
CDR1
Multidrug transporter of ABC superfamily
PDR17
Lipid biosynthesis and multidrug resistance
PDR6
Pleiotropic drug resistance regulatory protein
HXT62
Sugar transporter
HXT5
Sugar transporter
HGT11
Hexose transporter
HGT12
Hexose transporter
STL2
Sugar transporter
SNF3
High affinity glucose transport protein
IPF3277
Hexose transporter
Ergosterol biosynthesis and sterol metabolism
ERG2
C-8 sterol isomerase
ERG9
Squalene synthase
ARE2
Acyl-CoA:sterol acyltransferase
IPF6464
Triacylglycerol lipase
Carbohydrate metabolism
ADH3
Alcohol dehydrogenase
ADH4
Alcohol dehydrogenase
GPM1
Phosphoglycerate mutase
KGD2
2-oxoglutarate dehydrogenase
MCR1
NADH-cytochrome-b5 reductase
MDH12
Mitochondrial malate dehydrogenase
TPI1
Triose phosphate isomerase

Accession No.

y0109 R:y0109 S

CA6066
CA6183
CA4807
CA1067
CA1069
CA1506
CA4038
CA5606
CA6095
CA3914

19.20
3.01
4.06
5.33
3.73
3.34
2.25
2.37
2.48
2.89

CA2154
CA1676
CA3590
CA4480

4.11
2.43
9.35
11.26

CA2334
CA4765
CA4671
CA2997
CA2457
CA5489
CA5950

40.34
9.13
2.15
2.06
2.42
2.19
2.72
(To be continued)

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Fig. 2 Variations of minimal inhibitory concentration (MIC)

of fluconazole (FLC), ketocolazole (KTC), a nd itroconazle
(ITC) for the FLC-resistant Candida albicans strain y0109 R
grown in medium without FLC Each datum point represents
one passage.

Validation of microarray results by real-time RT-PCR

analysis
To validate the differential expression of genes identified
by microarray analysis, we performed real-time RT-PCR
analysis. In general, for the representative genes in each
category in the strain y0109R, the n-fold changes were
almost equal between microarray and real-time RT-PCR

Microarray analysis of FLC resistance in a laboratory C. albicans strain

(Continued)
Gene name

Function description

y0109 R:y0109 S

CA1834
CA1691
CA2764
CA2189
CA3564
CA2009
CA1333

2.68
4.24
2.41
5.24
2.24
2.60
3.19

CA2037
CA1623
CA3361
CA2787
CA1131
CA5066
CA4800
CA4125
CA0188
CA2015
CA3208
CA5894
CA4822
CA0844
CA4259
CA2931
CA4648
CA4857
CA3867

2.95
3.38
2.19
3.93
4.98
2.89
0.48
0.26
0.31
0.41
0.17
0.43
0.39
0.34
0.18
4.23
0.38
0.06
0.35

CA1596
CA4311
CA6220
CA4113
CA5736
CA4117

2.82
2.33
3.00
0.30
0.38
0.46

CA5040
CA5842
CA1586
CA2561

3.10
3.67
2.44
0.45

CA1202
CA2593
CA5682
CA0283
CA5307
CA2636

3.55
2.34
3.06
2.75
4.16
2.83
(To be continued)

Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1054

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PFK1
6-phosphofructokinase, subunit
PGK1
Phosphoglycerate kinase
QCR7
Ubiquinol-cytochrome-c reductase subunit 7
AOX2
Alternative oxidase
IPF7817
NADH-dependent flavin oxidoreductase
IPF1732
Intramitochondrial protein sorting
MRF1
Mitochondrial respiratory proteins
Morphogenesis
IFF3
Putative GPI-anchored protein
STE7
Mitogen-activated protein kinase
SNF1
Serine/threonine protein kinase
EFG1
Enhanced filamentous growth factor
NAG2
N-acetyl-glucosamine-6-phosphate deacetylase
TPS2
Trehalose-6-phosphate phosphatase
PGA4
Putative GPI-anchored protein
PGA62
Putative GPI-anchored protein
PGA52
Putative GPI-anchored protein
PGA16
Putative GPI-anchored protein
PSA1
GDP-mannose pyrophosphorylase
PMT2
O-D-mannosyltransferase
DFG5
N-linked mannoprotein
CDC3
Cell division control protein
CDC10
Cell division control protein
IPF7841
Nuclear envelope protein
IPF9864
Mitotic spindle protein
PHR1
pH responsive glycosyl transferase
PHR2
pH-regulated protein 2
Lipid, fatty acid metabolism
FAA21
Long-chain-fatty-acid-CoA ligase
FAA23
Long-chain-fatty-acid-CoA ligase
TES15
Thiosterase
CHO1
Phosphatidylserine synthase
OPI3
Methylene-fatty-acyl-phospholipid synthase
LAB5
Lipoic acid synthase
Amino acid metabolism
BAT22
Branched chain amino acid aminotransferase
LEU1
3-isopropylmalate dehydratase
ODC1
Ornithine decarboxylase
CAR2
Ornithine aminotransferase
Macromolecular synthesis
DBP3
ATP-dependent RNA helicase
RRP6
Involved in 5.8S rRNA processing
NOG1
Nucleolar G-protein
MDN1
Midasin
IPF2172
Nuclear transport factor
MSW1
Tryptophanyl-tRNA synthetase

Accession No.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

(Continued)
Gene name

Function description

y0109 R:y0109 S

CA1246
CA5842
CA3722
CA4181
CA4802
CA4133
CA3727
CA1580

3.42
3.67
0.30
0.43
0.33
0.30
0.31
0.29

CA1911
CA1216
CA2644
CA5526
CA0386

3.59
0.39
0.37
22.70
3.74

CA5890
CA5671
CA5976
CA3088
CA0471
CA2216
CA4250
CA0899
CA6084
CA2369

0.30
2.67
3.33
7.80
0.07
12.56
5.60
21.21
3.46
0.41

CA2284
CA2473
CA4744

0.46
0.41
2.26

CA2805
CA4155
CA4696
CA5752

0.15
2.39
0.21
3.39

CA4578
CA0124
CA4808
CA0917
CA5495
CA1384
CA3968

0.47
0.33
0.34
4.78
0.34
0.32
0.33
>2.00
<0.50

Gene names and accession numbers according to the Candida albicans genomic database (CandidaDB). Data>2 means genes were up-regulated in
y0109R compared to y0109S; data <0.5 means genes were down-regulated in y0109R compared y0109S. ABC, ATP-binding cassette; tRNA, transfer
RNA; GDP, guanosine diphosphate.; GPI, glycosylphosphatidylinositol
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1055

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IMH3
Inosine-5-monophosphate dehydrogenase
LEU1
3-isopropylmalate dehydratase
PHO13
4-nitrophenylphosphatase
HOM6
Homoserine dehydrogenase
ILV3
Dihydroxyacid dehydratase
CHO1
Phosphatidylserine synthase
HIS5
Histidinol-phosphate aminotransferase
TRP5
Tryptophan synthase
Cell stress
SSE1
Heat shock protein 70
EBP1
NADPH dehydrogenase
GRP2
Reductase
IPF2857
Regulated by cAMP and by osmotic stress
IPF4065
Possible stress protein
Transcription
FCR1
Zinc cluster transcription factor
CTA4
Transcription factor
IPF928
Zinc-finger transcription factor
ZCF20
zinc-finger protein of transcription factor
ECM22
Putative transcription factor
IPF6235
CaTca2 retrotransposon
IPF4292
Bleomycin Hydrolase
IPF4905
GTPase regulators
IPF29
Zinc-finger transcription factor
TBP1
TATA-binding protein
Mitosis
MIF2
Kinetochore protein
CBF1
Putative centromere binding factor 1
IPF4489
Mitotic spindle organization
Chromatin and chromosome structures
RNR1
Ribonucleoside-diphosphate reductase
RNR21
Ribonucleoside-diphosphate reductase
HTA1
Histone H2A
IPF1055
Chromatin binding
DNA replication and DNA repair
RFC1
DNA replication factor C
RFC5
DNA replication factor C
RFA1
DNA replication factor A
RAD16
Nucleotide excision repair protein
SGS1
ATP-dependent DNA helicase
IPF8423
Exonuclease
IPF9378
Subunit of the RNA polymerase II mediator
Others
66 genes of unknown function and others
112 genes of unknown function and others

Accession No.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

data (Fig. 3). As with the microarray data, there was no

fold change in ERG11 and MDR1 in the strain y0109R by
real-time RT-PCR compared with the strain y0109 S .
Nevertheless, we observed less n-fold change for CDR1
in y0109R with real-time RT-PCR than with microarray
analysis. This may reflect the greater dynamic range of
real-time RT-PCR analysis. In addition, since CDR2 has
been shown to be co-regulated with CDR1 and both genes
are regulated by trans-acting factor Tac1 [11] , we performed real-time RT-PCR for CDR2 and TAC1. As
expected, CDR2 and TAC1 were observed to be up-regulated 4.8-fold and 2.8-fold, respectively, in y0109R in comparison with y0109S (Fig. 3).
Glucose-induced R6G efflux
Since CDR1 was observed by microarray analysis to be
up-regulated 19-fold and CDR2 was observed by real-time
RT-PCR analysis to be up-regulated 4.8-fold, we then examined the energy-dependent efflux of R6G in both strains.
Fig. 4 clearly shows that y0109R effluxed more R6G than
y0109S in the presence of glucose. Efflux from deenergized
(by incubation with 2-deoxy-D-glucose), R6G-preloaded
cells of y0109R required the presence of glucose; within
10 min of adding glucose, the extracellular R6G concentration had increased more than 6-fold. In contrast, R6G
efflux from y0109S in the presence of glucose was only 1.
5-fold greater than that in the absence of glucose. These
results provided evidence that overexpression of ABC transporters Cdr1 and Cdr2 in the strain y0109R leads to increased energy-dependent extrusion of their substrates.
Sterol profile analysis
Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1056

Fig. 4 Energy-dependent efflux of rhodamine 6G (R6G) in

Candida albicans strains y0109 S and y0109 R The results are the
meansSD for three independent experiments. R, y0109R; S, y0109S.

In light of the changes to ergosterol biosynthetic and sterol metabolic genes in the FLC-resistant strain, sterol components were extracted from both strains with or without
FLC treatment and then subjected to GC-MS analysis. As
summarized in Table 3, ergosterol was the major sterol in
the strain y0109S, which accounted for 98.9% of the total
sterols extracted from the untreated culture. However, FLC
induced an increase in the lanosterol fraction at the expense of the ergosterol fraction, consistent with the FLC
activity (inhibition of Erg11) in the ergosterol biosynthetic
pathway. Particularly, the augment of 24-methylenelanosterol, the direct substrate of target enzyme of FLC in
C. albicans, was higher than that of lanosterol.

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Fig. 3 Validation of expression profile of genes as differentially expressed by microarray analysis in the strain y0109 R relative to
those in the strain y0109S All the genes were examined by relative quantitative real-time reverse transcription-polymerase chain reaction with
gene-specific primers. Relative fold changes were calculated with CT value. Results are the meanSD for three independent experiments.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

Table 3 Sterol compositions of Candida albicans strains y0109S and y0109R with or without flucoanzole treatment analyzed by gas
chromatography-mass spectrometry
Sterol identified
Y0109

Lanosterol
24-methylene-lanosterol
4,4-dimethyl-zymosterol
4-methyl-zymosterol
Zymosterol
Ergosta-7,22-dienol
4-methylergoster-7,24(28)-dienol
Ergosterol

ND
ND
ND
ND
ND
1.100.23
ND
98.95.77

Y0109 +
F0.25

Y0109 +
F1

Sterol composition (%) (n=3)

Y0109S+
Y0109R
Y0109R+
F4
F0.25

26.02.19
ND
ND
ND
ND
ND
3.290.58
70.84.27

25.72.01
42.13.29
1.000.15
ND
ND
ND
2.300.27
28.93.18

48.12.85
50.23.91
ND
ND
ND
ND
ND
1.720.46

2.780.12
ND
5.100.79
5.021.08
13.12.02
13.51.89
ND
60.53.56

11.81.28
ND
3.200.79
4.200.49
11.31.04
10.51.03
ND
59.03.24

Y0109R+
F1

Y0109R+
F4

35.92.59
ND
2.120.23
2.720.27
5.441.01
2.180.58
0.640.12
51.03.36

37.52.78
3.160.38
1.080.26
1.370.24
3.500.11
3.620.19
0.970.13
48.83.85

The results are the meanSD for three independent experiments. F0.25, 0.25 g/ml fluconazole. ND, not detected.

Mutations in ERG3 and TAC1

To screen for potential mutations that may have resulted
in decreased FLC susceptibility in y0109R, ERG3, ERG11,
and TAC1 genes in both y0109S and y0109R were sequenced
and compared to the ERG3, ERG11, and TAC1 sequences
in the Candida Genome Database. Single fragments of the
expected length were obtained in each strain. For these
two strains, the only one missense mutation in ERG3 gene
was the single amino acid substitution D19E (Table 4).
Meanwhile, both strains harbored one nucleotide change
at base position 304 (ACC) in ERG3 which did not affect
the amino acid sequence. There were no mutations in TAC1
in the strain y0109S. In contrast, the TAC1 gene from

y0109R had a total of two missense mutations (L47K and

N977K) (Table 4). In addition, there was no amino acid
difference in Erg11 in the two strains.

Discussion
Using in vitro conditions simulating chronic FLC exposure,
we established the FLC-resistant C. albicans strain for the
purpose of examining the potential drug resistance mechanisms in this FLC-resistant model. High-density oligonucleotide microarray analysis, confirmed by real-time RT-PCR,
was performed with the susceptible parental strain and
the resistant daughter strain to explore alteration on gene
expression profile during the acquisition of FLC resistance.
The differentially regulated genes were found to be involved in multiple biochemical functions. Our particular
interest was the overexpression of ABC transporter genes,
ergosterol biosynthetic genes, and sterol metabolic genes.
Functional analysis was carried out to measure R6G efflux
and sterol components. One single point mutation (D19E)
in ERG3 and two point mutations (L47K and N977K) in
TAC1 were identified in the strain y0109R , which could
contribute to its FLC-resistant phenotype.
Particularly remarkable transcriptional expression change
was the up-regulation of the membrane transporter CDR1
by greater than 19 folds and CDR2 by 4.8 folds in the
strain y0109R , while no MDR1 expression change was
detected. With the glucose-induced R6G efflux assay, we
further demonstrated that the activity of energy-dependent efflux pump was greater in the resistant y0109R than
that in the susceptible y0109S. Recent studies have reported
that CDR1, CDR2, and PDR17 contain a common drugActa Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1057

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In contrast, the types of sterol and the sterol composition in y0109R significantly differed from those in y0109S.
Ergosterol accounted for only 60.5% of the total sterols
extracted from the untreated culture in y0109R, while lanosterol was increased to 2.78%. Moreover, in y0109R, sterol
intermediates, 4,4-dimethyl-zymosterol, 4-methylzymosterol and zymosterol, were identified. Interestingly,
ergosta-7,22-dienol was increased to 13.5% of the total
sterols. Furthermore, with the treatment of FLC, lanosterol and 24-methylene-lanosterol increased, while 4,4-dimethyl-zymosterol, 4-methyl-zymosterol, zymosterol, and
ergosta-7,22-dienol all decreased. However, the degree of
the ergosterol decrease was significantly lower than that
of the lanosterol increase and the sterol intermediates decrease in the strain y0109R. In general, the sterol profile of
this pair of strains was largely distinct, and the effect of
FLC in sterol composition appeared to be dose dependent
and proportional to the susceptibility of the individual strain.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

Table 4 Sequence divergence of ERG3 and TAC1 present in the Candida albicans strains y0109 S and y0109 R

Base position

55
304

139
2929

ERG3 alleles in each strain

y0109 R
y0109 S

Amino acid position

Deduced amino acids

y0109 S
y0109 R

GAT
ACC

19
102

D (Asp)
T (Thr)

TAC1 alleles in each strain

y0109 R
y0109 S

Amino acid position

Deduced amino acids

y0109 S
y0109 R

TTA
AAT

47
977

L (leu)
N (Asn)

GAA
ACC

AAA
AAG

E (Glu)
T (Thr)

K (lys)
K (lys)

Position represents the open reading frame of the first base of the strain y0109S. Underlining shows the base that differs between the strains
y0109S and y0109R. The bases differ from the nucleotide sequence (ACT) in the Candida Genome Database which have no effect in the amino
acid sequence between the strains y0109S and y0109R.

Acta Biochim Biophys Sin (2008) | Volume 40 | Issue 12 | Page 1058

tected in y0109S, as well as ergosta-7,22-dienol; ergosterol was the predominant sterol. Among these, 4,4-dimethyl-zymosterol, 4-methyl-zymosterol, and zymosterol
were sterol intermediates upstream of fecosterol and
episterol. As shown in Table 2, ERG2 was overexpressed
in y0109R, possibly allowing for increased conversion of
fecosterol to episterol. Furthermore, in y0109R , the conversion of toxic episterol to ergosta-7,22-dienol was enhanced at the expense of ergosta-5,7,24(28)-trienol and,
ultimately, ergosterol. Still, FLC induced the increase in
lanosterol fraction at the expense of sterol intermediates
instead of ergosterol. Indeed, production of these precursor sterols has been demonstrated in erg3/erg3 mutant [32].
Likewise, our sterol analysis of the FLC-susceptible and
the FLC-resistant strains showed a similar sterol profile,
suggesting reduced activity of sterol C-5 desaturase
(ERG3) in the strain y0109R. The analysis of the ERG3
sequence from these strains identified one missense mutation which changed the aspartic acid in codon 19 to
glutamic acid in the FLC-resistant y0109R. These results
strongly imply a relationship between the presence of the
D19E mutation and the inactivation of Erg3 function.
In Saccharomyces cerevisiae, the induction of ergosterol biosynthesis genes encoding the enzymes that catalyzed late steps in ergosterol biosynthesis, such as ERG2,
could be mediated by either Ecm22 or Upc2 [33]. Recent
studies have shown that both Ecm22 levels and the amount
of Ecm22 bound to promoters decreased upon ergosterol
depletion, whereas Ecm22 is still able to increase the expression levels of its target genes in a Hap-dependent activator of ERG2 manner [34,35]. In the current study,
ZCF20, homologous to ScHAP1, was up-regulated 7.8fold, while the ECM22 was down-regulated. It was pos-

Downloaded from http://abbs.oxfordjournals.org/ by guest on August 4, 2016

responsive element in their promoters that belong to the

same Tac1 trans-acting regulation network [11,12,30]. In
the present study, we showed that the up-regulation of
CDR1, CDR2, and PDR17 suggested that the FLC-resistant strain y0109R had an activation of Tac1. Actually, TAC1
was observed to be up-regulated in y0109R, and was identified as having two missense mutations. Among them,
the known L47K mutation is not associated with azole
resistance [11]. The other mutation in TAC1 changed asparagine (N) to lysine (K) in codon 977. Indeed, the N977D
mutation has been confirmed its importance in conferring
hyperactivity to Tac1 [11]. Therefore, data in this study
strongly suggest that TAC1 can be hyperactive through
the N977K mutation and thereby up-regulates the expression of CDR1, CDR2, and PDR17 in y0109R .
In contrast to previous studies that have investigated
the changes in response to short-term azole drugs exposure in C. albicans [16,31], there were no genes involved
in the ergosterol biosynthesis pathway other than ERG2
and ERG9, which were found to be up-regulated in y0109R.
Accordingly, the current data further highlight the importance of differences in constitutive changes in gene expression that result in FLC resistance and transient changes
in the organism induced in order to respond to the presence of drug.
The sterol profile of the FLC-susceptible strain y0109S
was highest in enriched ergosterol and contained only minor amounts of intermediate ergosta-7,22-dienol. In
contrast, the sterol content in the resistant strain y0109R
suggested that resistance to FLC, AMB and nystatin was
the result of accumulation of sterol intermediates. The strain
y0109R contained lanosterol, 4,4-dimethyl-zymosterol, 4methyl-zymosterol and zymosterol, which were not de-

Microarray analysis of FLC resistance in a laboratory C. albicans strain

Conclusion
In the present study, a FLC-resistant C. albicans strain
was obtained by serial cultures of a FLC-susceptible strain
in inhibitory concentrations of FLC. Using cDNA

microarray to profile transcriptional expression of this

matched pair of C. albicans strains, we were able to identify genes whose expression changed during the acquisition of resistance to azoles. We showed that a point
mutation, the replacement of Asn by Lys at position 977
of the transcription factor gene TAC1, could modify the
activity of Tac1 into a hyperactive state, thereby resulting
in the up-regulation of CDR1 and CDR2. We also discovered that a single amino acid difference (D19E) in ERG3
could inactivate the Erg3 function, leading to decreased
ergosterol fraction content as the sterol intermediates accumulate in the resistant strain. This study also characterized the transcriptional alterations that accompany the development of resistant phenotype. Our results demonstrated that the laboratory FLC-resistant C. albicans strain
harbored known resistance mechanisms which were observed in the clinical isolates. Findings based on this model
could expand our understanding of potential new molecular mechanisms of FLC resistance in C. albicans.

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We suggested such alterations were collateral. We previously reported that y0109R had a great ability to form hyphal from yeast cells, especially relative to y0109S [38];
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Additionally, we identified several genes of unknown
and unclassified functions that were differentially expressed
between the pair of strains. These genes might be associated with FLC resistance in C. albicans, and hence, their
putative functions require further investigation.

Microarray analysis of FLC resistance in a laboratory C. albicans strain

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