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DOI: 10.1534/genetics.105.054767
A Mutation in Tac1p, a Transcription Factor Regulating CDR1 and CDR2,
Is Coupled With Loss of Heterozygosity at Chromosome 5 to Mediate
Antifungal Resistance in Candida albicans
Alix Coste,* Vincent Turner,* Francxoise Ischer,* Joachim Morschha¨user,
†
Anja Forche,
‡
Anna Selmecki,
‡
Judith Berman,
‡
Jacques Bille* and Dominique Sanglard*
,1
*Institute of Microbiology, University Hospital Lausanne, Lausanne CH-1011, Switzerland,
†
Institut fu
¨r Molekulare Infektionsbiologie,
Universita
¨tWu
¨rzburg, D-97070 Wu
¨rzburg, Germany and
‡
Departments of Genetics, Cell Biology and Development, and Microbiology,
University of Minnesota, Minneapolis, Minnesota 55455
Manuscript received December 21, 2005
Accepted for publication January 4, 2006
ABSTRACT
TAC1,aCandida albicans transcription factor situated near the mating-type locus on chromosome 5, is
necessary for the upregulation of the ABC-transporter genes CDR1 and CDR2, which mediate azole
resistance. We showed previously the existence of both wild-type and hyperactive TAC1 alleles. Wild-type
alleles mediate upregulation of CDR1 and CDR2 upon exposure to inducers such as fluphenazine, while
hyperactive alleles result in constitutive high expression of CDR1 and CDR2. Here we recovered TAC1
alleles from two pairs of matched azole-susceptible (DSY294; FH1: heterozygous at mating-type locus) and
azole-resistant isolates (DSY296; FH3: homozygous at mating-type locus). Two different TAC1 wild-type
alleles were recovered from DSY294 (TAC1-3 and TAC1-4) while a single hyperactive allele (TAC1-5) was
isolated from DSY296. A single amino acid (aa) difference between TAC1-4 and TAC1-5 (Asn977 to Asp or
N977D) was observed in a region corresponding to the predicted activation domain of Tac1p. Two TAC1
alleles were recovered from FH1 (TAC1-6 and TAC1-7) and a single hyperactive allele (TAC1-7) was
recovered from FH3. The N977D change was seen in TAC1-7 in addition to several other aa differences.
The importance of N977D in conferring hyperactivity to TAC1 was confirmed by site-directed
mutagenesis. Both hyperactive alleles TAC1-5 and TAC1-7 were codominant with wild-type alleles and
conferred hyperactive phenotypes only when homozygous. The mechanisms by which hyperactive alleles
become homozygous was addressed by comparative genome hybridization and single nucleotide
polymorphism arrays and indicated that loss of TAC1 heterozygosity can occur by recombination between
portions of chromosome 5 or by chromosome 5 duplication.
CANDIDA albicans is an opportunistic pathogen that
causes oral and systemic infections in immuno-
compromised patients as well as vaginal infections in im-
munocompetent women. To prevent and treat Candida
infections, immunocompromised patients are often
treated for a long time with antifungal agents among
which is the class of azoles. As azoles are fungistatic,
rather than fungicidal, C. albicans cells repetitively ex-
posed to these antifungals can adapt to the drug pres-
sure and eventually become resistant to azoles. The
most important mechanism of resistance to azoles is
the overexpression of multidrug transporters, encoded
by either the major facilitator efflux pump CaMDR1
(multidrug resistance 1) or the ABC transporters CDR1
(candida drug resistance) and CDR2. Upregulation of
CaMDR1 confers resistance to fluconazole, while up-
regulation of CDR1 and CDR2 confers resistance to mul-
tiple azoles (itraconazole, fluconazole, voriconazole).
Understanding the transcriptional control of these
genes, by both cis- and trans-acting effectors, is there-
fore important for determining how azole resistance
and transport mechanisms are regulated in C. albicans.
CaMDR1 expression is controlled by at least two
regulatory promoter cis-acting regions as reported re-
cently by Harry et al. (2005). Several elements of CDR
genes are important for the regulation of CDR1 and
CDR2.Abasal response element (BRE) is located be-
tween nt 860 and 810 in the CDR1 promoter, and a
drug response element (DRE) is present in the pro-
moters of both CDR1 and CDR2 (de Micheli et al. 2002).
The BRE regulates basal expression of CDR1 (de
Micheli et al. 2002), while the DRE sequence (59-
CGGAA/TATCGGATA-39) is crucial for the upregula-
tion of these genes in azole-resistant strains as well as for
the transient upregulation of both genes in the pres-
ence of different drugs such as oestradiol, progesterone,
or fluphenazine in azole-susceptible strains. In addition,
another BRE (located between 243 and 234) and a
1
Corresponding author: Institute of Microbiology, University Hospital
Lausanne, Rue du Bugnon 48, Lausanne CH-1011, Switzerland.
E-mail: dominique.sanglard@chuv.ch
Genetics 172: 2139–2156 (April 2006)
negative regulatory element (NRE) located within the
289 region have been reported in CDR1 (Puri et al.
1999; Gaur et al. 2004). Finally, in the same gene,
Karnani et al. (2004) identified SRE1 and SRE2 (steroid
response elements) between 696 and 521.
Trans-acting factors regulating CDR1 and CDR2 were
reported recently. C. G. Chen et al. (2004) described a
potential activator of CDR1 identified by screening of a
C. albicans genomic library expressed in a Saccharomyces
cerevisiae strain, which contained a CDR1 promoter/lacZ
fusion. This factor, CaNDT80, is a homolog to a meiosis-
specific transcription factor in S. cerevisiae (C. G. Chen
et al. 2004). Deletion of CaNDT80 in C. albicans con-
ferred hypersensitivity to azoles and decreased the
inducible expression of CDR1. Recently, our laboratory
discovered Tac1p (transcriptional activator of CDR), a
transcription factor belonging to the family of zinc-
finger proteins with a Zn
2
Cys
6
motif (Coste et al. 2004).
Tac1p binds to the DRE, which contains two CGG
triplets typical of the DNA-binding sites of Zn
2
Cys
6
tran-
scription factors. Tac1p is responsible for transient
upregulation of both CDR genes in azole-susceptible
strains in the presence of inducers. Interestingly, TAC1
is located close to (within 14 kb) the mating-type-like
(MTL) locus. Previous studies reported a strong corre-
lation between homozygosity at the mating-type locus
and azole resistance in a number of clinical isolates
(Rustad et al. 2002). In our previous study, we showed
that a clinical azole-resistant strain (DSY296) that is
homozygous at the mating-type locus contains a TAC1
allele that is sufficient to confer fluconazole resistance
to a laboratory strain lacking TAC1 (Coste et al. 2004).
This type of allele was defined as ‘‘hyperactive’’ because
it caused constitutive high expression of CDR1 and
CDR2 in a tac1D/Dmutant. In contrast, TAC1 alleles of
the matched azole-susceptible clinical strain (DSY294)
or of a laboratory strain (CAF2-1), which are strains
heterozygous at the mating-type locus, were not able to
confer azole resistance to a tac1D/Dmutant. These
alleles were defined as ‘‘wild-type’’ alleles. Using C.
albicans microarrays, we also showed that Tac1p regu-
lates the expression of at least three other genes: RTA3,
IFU5, and HSP12 (Coste et al. 2004; Karababa et al.
2004). Northern blot analysis showed that Tac1p also
regulates PDR16 (D. Sanglard, unpublished data), a
gene shown to be overexpressed in azole-resistant strains
upregulating CDR1 and CDR2 (DeDeken and Raymond
2004). Interestingly, all four of these Tac1p-regulated
genes contain a putative DRE in their promoters.
In S. cerevisiae, the functional homolog of CDR1 and
CDR2,PDR5, is known to be regulated by at least two
Zn
2
Cys
6
transcription factors (PDR1 and PDR3). These
transcription factors bind as homo- and heterodimers
to a cis-acting pleiotropic drug responsive element
containing two CGG triplets (Katzmann et al. 1994;
Carvajal et al. 1997). Point mutations in PDR1 and
PDR3 lead to increased PDR5 expression and drug
resistance (Carvajal et al. 1997; Nourani et al. 1997;
Anderson et al. 2003). Carvajal et al. (1997) found that
a F815S mutation (F815S) in the putative activation
domain of Pdr1p is responsible for strong constitutive
PDR5 expression. Point mutations in other regions of
PDR1 also affect the regulation of PDR5 expression, but
have a more moderate effect than the F815S mutation.
In this study, we analyzed TAC1 alleles of matched
azole-susceptible and azole-resistant clinical C. albicans
isolates. We identified a single point mutation (N977D)
in both hyperactive TAC1 alleles, which is sufficient to
confer hyperactivity as measured by upregulation of
CDR1 and CDR2 and levels of drug resistance. We also
show that hyperactive alleles carrying this mutation are
codominant with other wild-type alleles such that only
strains homozygous for hyperactive alleles show high
expression levels of CDR1 and CDR2. We also show that
homozygosity at MTL accompanies the acquisition of
TAC1 homozygosity via at least two mechanisms, but that
MTL homozygosity does not contribute to the azole
resistance phenotype.
MATERIALS AND METHODS
Strains and media: The C. albicans strains used in this study
are listed in Table 1. These strains were grown either in
complete medium YEPD (1% Bacto peptone, Difco Labora-
tories, Basel, Switzerland), 0.5% yeast extract (Difco), and 2%
glucose (Fluka, Buchs, Switzerland) or in minimal medium
yeast nitrogen base (Difco) and 2% glucose (Fluka). When
grown on solid media, 2% agar (Difco) was added to either of
the media. Escherichia coli DH5awas used as a host for plasmid
constructions and propagation. DH5awas grown in Luria–
Bertani broth (LB) or on LB plates, supplemented with
ampicillin (0.1 mg/ml) when required.
Yeast transformation: C. albicans cells from 0.2 ml stationary-
phase culture were resuspended in 0.1 ml of a solution
containing 200 mmlithium acetate (pH 7.5), 40% (w/v) PEG
8000, 15 mg/ml DTT, and 250 mg/ml denatured salmon sperm
DNA. Transforming DNA (1–5 mg) was added to the yeast
suspension, which was incubated for 60 min at 43.5°. Trans-
formation mixtures were plated directly onto selective plates.
For transformation involving the dominant marker SAT1,the
transformation mixture was incubated at room temperature
overnight in 1 ml YEPD and plated the day after on YEPD agar
plates containing 200 mg/ml of nourseothricin (Werner Bio-
agent, Jena, Germany).
Drug susceptibility testing: Drug susceptibility testing was
performed by spotting cells onto solid agar plates containing
the tested drugs. Yeast cultures were grown overnight in YEPD
and diluted to a density of 1.5 310
7
cells/ml and serial 10-fold
dilutions were performed to a final dilution step containing
1.5 310
3
cells/ml. Four microliters of each dilution were
spotted onto YEPD plates with or without drugs. Plates were
incubated for 48 hr at 35°.
Drug susceptibility testing was also performed in microtiter
plates with twofold serial dilutions of fluconazole (range is
from 128 to 0.06 mg/ml) or terbinafine (range is from 32 to
0.015 mg/ml). Yeast cultures were grown overnight in YEPD
and inoculated at a density of 10
4
cells/ml in a total volume of
200 ml containing the serial dilution of fluconazole or ter-
binafine. Microtiter plates were incubated at 35°during 48 hr
and optical densities read with a microtiter plate reader at a
2140 A. Coste et al.
TABLE 1
Strains used in this study
Strain Parental strain Genotype Reference
CAF2-1 SC5314 ura3DTimm434/URA3 Fonzi and Irwin (1993)
CAF4-2 CAF2-1 ura3DTimm434/ura3DTimm434 Fonzi and Irwin (1993)
DSY2875 CAF4-2 tac1-1DThisG/TAC1-2 Coste et al. (2004)
DSY2903 DSY2875 tac1-1DThisG/tac1-2DThisG-URA-hisG Coste et al. (2004)
DSY2906 DSY2903 tac1-1DThisG/tac1-2DThisG Coste et al. (2004)
DSY2937-35 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1 Coste et al. (2004)
DSY2925-47 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1-3 Coste et al. (2004)
DSY2925-18 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1-4 Coste et al. (2004)
DSY2984 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1-5 This study
VTY9 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1-5
D977N
This study
VTY21 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1-1
N977D
This study
VTY28 DSY2906 tac1-1DThisG/tac1-2DThisG, LEU2TTAC1-4
N977D
This study
DSY3010-80 DSY2906 tac1-1DThisG/tac1-2DThisG,LEU2TTAC1-6 This study
DSY3010-113 DSY2906 tac1-1DThisG/tac1-2DThisG,LEU2TTAC1-7-FH1 This study
DSY3013 DSY2906 tac1-1DThisG/tac1-2DThisG,LEU2TTAC1-7-FH3 This study
ACY11 DSY2906 tac1-1DThisG/tac1-2DThisG,LEU2TTAC1-6
N977D
This study
ACY12 DSY2906 tac1-1DThisG/tac1-2DThisG,LEU2TTAC1-7
D977N
This study
DSY294 Azole-susceptible clinical strain (MTLa/MTLa)Coste et al. (2004)
DSY3040 DSY294 ura3DTFRT/ura3DTFRT This study
DSY3058 DSY3040 TAC1-4/tac1-3DThisG-URA3-hisG This study
DSY3075 DSY3058 TAC1-4/tac1-3DThisG This study
DSY3082 DSY3075 tac1-3DThisG/tac1-4DThisG-URA3-hisG This study
DSY3089 DSY3082 tac1-3DThisG/tac1-4DThisG This study
DSY3102-2 DSY3089 tac1-3DThisG/tac1-4DThisG, LEU2TTAC1-5 This study
DSY3287-1 DSY3089 tac1-3DThisG/tac1-4DThisG, LEU2TTAC1-3 This study
DSY 3288-3 DSY3089 tac1-3DThisG/tac1-4DThisG, LEU2TTAC1-4 This study
DSY3053-1 DSY3040 TAC1-4/tac1-3DThisG-URA3-hisG This study
DSY3168-1 DSY3053-1 TAC1-4/tac1-3DThisG This study
DSY3219-2 DSY3168 TAC1-4/tac1-3DThisG, tac1TTAC1-5 This study
DSY3220-1 DSY3168 TAC1-4/tac1-3DThisG, tac1TTAC1-3 This study
DSY3221-3 DSY3168 TAC1-4/tac1-3DThisG, tac1TTAC1-4 This study
DSY3053-2 DSY3040 TAC1-3/tac1-4DThisG-URA3-hisG This study
DSY3168-2 DSY3053-2 TAC1-3/tac1-4DThisG This study
DSY3222-2 DSY3168-2 TAC1-3/tac1-4DThisG, tac1TTAC1-5 This study
DSY3223-1 DSY3168-2 TAC1-3/tac1-4DThisG, tac1TTAC1-3 This study
DSY3224-1 DSY3168-2 TAC1-3/tac1-4DThisG, tac1TTAC1-4 This study
DSY296 DSY294 Azole-resistant clinical strain (MTLa/MTLa)Coste et al. (2004)
DSY3041 DSY296 ura3DTFRT/ura3DTFRT This study
DSY3059 DSY3041 TAC1-5/tac1-5DThisG-URA3-hisG This study
DSY3076 DSY3059 TAC1-5/tac1-5DThisG This study
DSY3210-1 DSY3076 TAC1-5/tac1-5DThisG, tac1TTAC1-5 This study
DSY3211-4 DSY3076 TAC1-5/tac1-5DThisG, tac1TTAC1-3 This study
DSY3215-1 DSY3076 TAC1-5/tac1-5DThisG, tac1TTAC1-4 This study
DSY3083 DSY3076 tac1-5DThisG/tac1-5DThisG-URA3-hisG This study
DSY3090-8 DSY3083 tac1-5DThisG/tac1-5DThisG This study
DSY3284-1 DSY3090-8 tac1-5DThisG/tac1-5DThisG, LEU2TTAC1-5 This study
DSY3285-1 DSY3090-8 tac1-5DThisG/tac1-5DThisG, LEU2TTAC1-3 This study
DSY3286-2 DSY3090-8 tac1-5DThisG/tac1-5DThisG, LEU2TTAC1-4 This study
FH1 Azole-susceptible clinical strain (MTLa/MTLa)Marr et al. (1997)
FH3 FH1 Azole-resistant clinical strain (MTLa/MTLa)Marr et al. (1997)
DSY3132-14 FH1 TAC1-7/tac1-6DTFRT This study
DSY3132-11 FH1 TAC1-6/tac1-7DTFRT This study
DSY3133-15 FH3 TAC1-7/tac1-7DTFRT This study
DSY3157-2 FH1 TAC1-6/TAC1-7/TAC1-7 This study
DSY3301-4 DSY3157-2 TAC1-7/TAC1-7/TAC1-7 This study
Azole Resistance by TAC1 Mutation 2141
wavelength of 540 nm. The minimal inhibitory concentration
(MIC) was determined as the drug concentration required to
decrease the optical density of the drug-free culture by at least
50%.
Efflux of rhodamine 6G: To measure the drug efflux ca-
pacity of C. albicans strains with specific TAC1 alleles and
CDR1/CDR2 expression, rhodamine 6G (R6G) efflux was
measured by fluorescence assays with whole cells. C. albicans
cultures grown overnight in YEPD were diluted in 5 ml YEPD
and allowed to grow at 30°under constant agitation until a
density of 2 310
7
cells/ml was obtained. Cells were centri-
fuged, washed with 5 ml PBS (pH 7), and resuspended in 2 ml
PBS. The cells were incubated for 1 hr at 30°under constant
agitation in PBS to energy deprive cells. R6G was next added at
a concentration of 10 mg/ml and the incubation was contin-
ued for 1 hr, thus facilitating R6G accumulation. After this
incubation time, cells were sedimented by centrifugation,
washed with PBS at 4°, and resuspended in a final volume of
200 ml PBS. Fifty microliters of individual strains were diluted
in 150 ml PBS and aliquoted in a 96-well microtiter plate, which
was placed in a SpectraMax Gemini fluorimeter with temper-
ature control set at 30°. Baseline emission of fluorescence
(excitation wavelength: 344 nm; emission wavelength: 555 nm)
was recorded as relative fluorescence units (RFU) for 5 min
and glucose (1% final concentration) was next added to each
strain to initiate R6G efflux. As a negative control, no glucose
was added to separate aliquots of each strain. Data points were
recorded in duplicate for 60 min at 1-min intervals.
Immunoblots: C. albicans cell extracts for immunoblotting
were prepared by an alkaline extraction procedure from cells
grown to midlog phase. Briefly, cells (5 OD
540nm
) were
resuspended in an Eppendorf tube with 500 ml water and
150 ml of a solution containing 1.85 mNaOH and 7.5%
b-mercaptoethanol. This mixture was incubated on ice for
10 min. Proteins were next precipitated with 150 ml of a 50%
trichloroacidic acid solution and the suspension was left on ice
for another10 min. Precipitated proteins were sedimented by a
centrifugation step at maximal speed in a microfuge for 15 min.
The sediment was resuspended in 50 ml of loading buffer
(40 mmTris–HCl pH 6.8, 8 murea, 5% SDS, 0.1 mEDTA, 1%
b-mercaptoethanol, and 0.1 mg/ml bromophenol blue) and
incubated at 37°for 10 min. Nonsolubilized material was
cleared by a centrifugation step for 10 min. Ten microliters of
solubilized yeast proteins were separated by 10% SDS–PAGE
and transferred by Western blot on a nitrocellulose membrane.
The membrane was stained by Ponceau reagent (0.25%
Ponceau S in 40% methanol and 15% acetic acid) for 5 min
to verify that protein extracts were evenly transferred. Immu-
nodetection of Cdr1p and Cdr2p was performed with rabbit
polyclonal anti-Cdr1p and anti-Cdr2p antibodies as described
previously (de Micheli et al. 2002) by chemoluminescence with
an ECL kit according to the recommendations of the manu-
facturer (Amersham Biosciences, Otelfingen, Switzerland).
Construction of gene disruption cassettes: Four different
TAC1 disruption cassettes were designed in this study. Three
cassettes—C333, C357, and C343 in plasmids pDS1052,
pDS1142, and pDS1102—were designed using the ‘‘Ura’’-
blaster system. C333 and C357 bear the deletion of a small
portion of 271 bp between nt 11153 and 11424 with respect
to the first ATG codon of TAC1. C343 was designed to delete a
larger region of 1931 bp between nt 1502 and 12433. C358
carried by the plasmid pDS1196 integrates the SAT1-flipper
system (Reuss et al. 2004), in which a region of 1924 bp was
deleted between nt 1501 and 12425.
To construct these different deletion cassettes, the entire
TAC1 ORF was first amplified from genomic DNA using the
cloning primers CaZNC2–BamHI and CaZNC2–Xho (see sup-
plemental Table S3 at http://www.genetics.org/supplemental/).
For the construction of cassettes C333 and C343, TAC1 was
amplified from the genomic DNA of CAF2-1 but for the
construction of the cassette C357, TAC1 was amplified with
genomic DNA from DSY2875 to specifically amplify the
TAC1-2 allele. PCR fragments were cloned into pBluescript
KS1to yield pDS1048 and pDS1138 (supplemental Table
S2 at http://www.genetics.org/supplemental/). For disrup-
tion with cassettes C333 and C357, pDS1048 and pDS1138
were digested with PstI and BglII and the 3.7-kb PstI–BglII
fragment containing the ‘‘Ura’’-blaster cassette from pMB7
was cloned into compatible sites to yield pDS1052 and
pDS1142, respectively. For disruption with cassette C343, a
deletion was created from pDS1048 using primers Znc2–
BG2 and Znc2–PST (supplemental Table S3 at http://www.
genetics.org/supplemental/). The obtained PCR fragment
was digested with PstIandBglII and the 3.7-kb PstI–BglII frag-
ment from pMB7 was inserted to obtain pDS1102 (supple-
mental Table S2 at http://www.genetics.org/supplemental/).
For transformation in C. albicans, linear fragments were ob-
tained by digestion of the plasmids with NsiI and XmnI,
thus liberating cassettes C333 and C357, and by ApaIandSacI,
liberating C343. For the cassette C358, TAC1 was amplified from
CAF2-1 genomic DNA. The obtained PCR fragment was cloned
into the pMTL21 (Chambers et al. 1998) to yield pDS1141. A
deletion was created using pDS1141 as template with the
primers Znc2–Apa and Znc2–SacII. Next, the ApaI–SacII frag-
ment of pSFS2 comprising the SAT1-flipper cassette was cloned
into the previously ApaI- and SacII-digested PCR fragment to
obtain pDS1196. For the transformation in C. albicans,alinear
fragment was obtained by digestion of the plasmid with SacI
and SphI, thus liberating the C358 cassette. C. albicans strains
DSY3053-1 and DSY3053-2 were obtained using the C357 dis-
ruption cassette. DSY3058 and DSY3059 were obtained using
the C343 cassette. DSY3082, DSY3083, DSY2875, and DSY2903
were obtained using the C333 cassette. DSY3132-11, DSY3132-
14, and DSY3133-15 were obtained using the C358 cassette.
To obtain ura3 mutants of the clinical isolates DSY294 and
DSY296, the two URA3 alleles were deleted using the SAT1-
flipping strategy (Reuss et al. 2004). For this purpose, the
SAT1-flipper cassette was substituted for the MPA
R
-flipper
cassette in the previously described plasmid pSFIU4 (Strauss
et al. 2001) to result in pSFSU1, in which the SAT1 flipper is
flanked by URA3 upstream and downstream sequences. The
insert from this plasmid was then used to inactivate URA3 in
the clinical C. albicans isolates by two rounds of targeted in-
tegration and subsequent FLP-mediated excision of the SAT1-
flipper cassette, generating strains DSY3040 and DSY3041.
Transformationswere per formed by electroporation and selec-
tion of nourseothricin-resistant transformants was performed
as described (Reuss et al. 2004).
Construction of revertant strains: The revertant strains
from each homozygous mutant generated in this study were
obtained by transformation of C. albicans ura3 derivatives with
the pRC2312-derived plasmid pDS178 containing the URA3
and LEU2 markers as described previously (de Micheli et al.
2002). To generate revertants from tac1D/Dmutant strains,
TAC1 ORFs flanked by 500 bp were amplified from genomic
DNA of strains SC5314, DSY294, DSY296, and FH1 and FH3
with primers Znc2-5–BamB and Znc2-3–Xho (supplemental
Table S3 at http://www.genetics.org/supplemental) and in-
serted into pDS178 previously digested by BamHI and XhoIto
yield pDS1097 (containing the TAC1-1 allele), pDS1098-1 and
pDS1098-9 (containing the TAC1-4 and TAC1-3 alleles),
pDS1099 (containing the TAC1-5 allele), pDS1045 (contain-
ing the TAC1-6 allele), and pDS1048 (containing the TAC1-7
allele), respectively. For each amplified allele, TAC1 was
sequenced from several plasmids to rule out PCR artifacts.
These plasmids either were linearized by SalI and transformed
2142 A. Coste et al.
into C. albicans DSY2906, DSY3089, and DSY3090, allowing
integration into the genomic LEU2 locus, or were linearized
by BstBI, allowing integration in the TAC1 locus of strains
DSY3168-1, DSY3168-2, and DSY3076. For each reintegration
of TAC1 alleles, several independent transformants (approx-
imately four to five) were tested for phenotypes (susceptibility
assays, immunodetection of Cdr1p and Cdr2p) and correct
integration. The transformants containing identical TAC1
alleles generally had the same phenotypes, but only a single
revertant for individual alleles was selected and presented in
this study. Integration at the TAC1 locus for strains DSY2906,
DSY3089, and DSY3090 was also performed and yielded
phenotypes comparable to those obtained by integration at
the LEU2 locus (data not shown).
Southern blots: Southern blots were performed as de-
scribed previously (Sanglard et al. 1995). Radioactive signals
were revealed by exposure to Kodak BioMax MR films
(Amersham Biosciences, Otelfingen, Switzerland). Signals
obtained in blotted membranes were quantified by counting
of radioactivity with the help of an Instant Imager (Perkin-
Elmer, Rotkreuz, Switzerland).
Site-directed mutagenesis: For site-directed mutagenesis of
TAC1-1,TAC1-4, and TAC1-5, the previously cloned alleles
were amplified from pDS1097, pDS1098-1, and pDS1099,
respectively, using the forward primer Zn2-5BAMB (Table
S3) and with the reverse primers TAC1-Asn-Asp-977 (primer
for pDS1097 and pDS1098-1) and TAC1-Asp-Asn-977 (primer
for pDS1099). These primers introduce a modification of
codon 977 from Asn to Asp in TAC1-1 and TAC1-4 and from
Asp to Asn in TAC1-5. Amplified products were cloned in the
pDS178 backbone yielding pVT21, pVT28, and pVT9 contain-
ing the mutated TAC1-1
N977D
,TAC1-4
N977D
, and TAC1-5
D977N
alleles, respectively. These plasmids were linearized by SalI
and transformed into C. albicans DSY2906 as described above.
Single nucleotide polymorphism and comparative genomic
hybridization: Single nucleotide polymorphism (SNP) micro-
array hybridization and comparative genomic hybridization
(CGH) were performed as described previously (Forche et al.
2005; Selmecki et al. 2005). Additional SNP markers were
designed from specific regions of chromosome 5 using C.
albicans genome data deposited at http://candida.bri.nrc.ca/
candida/alignments/index.cfm?chr¼5. The search for SNP
markers not present on the microarrays was performed by
amplification of specific regions of chromosome 5 from geno-
mic DNA using different V5 and V3 primer pairs (see supple-
mental Table S3 at http://www.genetics.org/supplemental/)
followed by sequencing the PCR products using a AB Prism,
3130 genetic analyzer (AB Applied Biosystems). Sequences
were analyzed for polymorphisms using the Contig Express
software (InforMax).
RESULTS
Analysis of TAC1 alleles isolated from azole-suscep-
tible and azole-resistant C. albicans strains: To analyze
the different TAC1 alleles present in either azole-
susceptible or azole-resistant strains, two sets of clinical
strains were first chosen. A first set of matched strains
reported previously (Sanglard et al. 1995, 1998) and
consisting of the azole-susceptible strain DSY294 (also
known as C43) and the azole-resistant strain DSY296
(also known as C56) originated from an HIV-positive
patient with oropharyngeal candidiasis who was treated
with fluconazole. A second set of strains described by
Marr et al. (1998, 2001) consisted of azole-susceptible
strains FH1 and FH2 and of strains FH3–FH8, which
developed azole resistance. These strains were isolated
from a bone marrow transplant patient suffering from
invasive candidiasis and treated with fluconazole. FH1
and FH2 are heterozygous at the mating-type locus
(Rustad et al. 2002). FH3–FH8 are homozygous at the
mating-type locus (Rustad et al. 2002). TAC1 alleles of
strains DSY294, DSY296, and FH1–FH8 were cloned and
introduced in a tac1D/Dmutant (strain DSY2906) de-
rived from SC5314. The azole resistance phenotypes of
the transformants were analyzed by drug susceptibility
assays and immunoblotting detection of Cdr1p and
Cdr2p.
Sequencing of individual TAC1 alleles from DSY294
revealed two distinct alleles, TAC1-3 and TAC1-4 (Table 2).
Both of these alleles had the properties of wild-type
alleles when reintroduced in a tac1D/Dmutant. First,
they did not confer resistance to terbinafine or flucon-
azole (Figure 1A). Second, they did not result in con-
stitutive high levels of Cdr1p or Cdr2p expression
under normal growth conditions; rather, Cdr1p was
detected at basal levels and Cdr2p could not be detected
(Figure 1B, lanes 3 and 4). The Cdr1p and Cdr2p levels
were comparable to those found in CAF2-1 and in the
tac1D/Dmutant (Figure 1B, lanes 1–4). However, Cdrp1
and Cdr2p were still inducible as in CAF2-1, since ex-
posure of TAC1-3 or TAC1-4 revertant strains to flu-
phenazine led to high Cdr1p and Cdr2p levels (Figure
1B, lanes 1, 3, and 4). The identification of two distinct
TAC1 alleles in DSY294 is consistent with the heterozy-
gosity of DSY294 at the mating-type locus, given that
TAC1 is located at a distance of 14 kb from this locus.
In contrast, only a single TAC1 allele (TAC1-5, Table 2)
was recovered from the matched azole-resistant strain
DSY296. The isolation of a single TAC1 allele from
DSY296 is consistent with homozygosity of this strain at
the mating-type locus and suggests that a region .14 kb
underwent loss of heterozygosity (LOH) during the
acquisition of drug resistance.
The TAC1-5 allele was considered to be a hyperactive
allele. First, when reintroduced into a tac1D/Dmutant, it
conferred higher terbinafine resistance than that ob-
served in strains containing the wild-type TAC1-3 and
TAC1-4 alleles (Figure 1A). Second, it conferred consti-
tutively high levels of Cdr1p and Cdr2p (Figure 1B,
lane 5), which are comparable to levels seen in strains
carrying wild-type alleles and exposed to fluphenazine
(Figure 1B, lanes 1, 3, and 4).
Similar analyses of TAC1 alleles were performed in
FH1–FH8 strains. Two different TAC1 alleles were iso-
lated from FH1 and FH2 (Table 2). One allele, TAC1-6,
was wild type, and the other, TAC1-7, was hyperactive as
defined above for TAC1 alleles of DSY294 and DSY296.
TAC1-6 does not confer terbinafine resistance (Figure
1A) and does not express high levels of Cdr1p and
Cdr2p under normal growth conditions (Figure 1B,
Azole Resistance by TAC1 Mutation 2143
TABLE 2
TAC1 alleles present in the strains used in this study
Strain Name of allele Type of allele
Polymorphism of TAC1 alleles (position of the nonsynonymous codons)
47 104 131 170 189 199 206 207 377 396 558 772 776 829 869 904 935 937 941 944 977
CAF2-1 TAC1-1
orf19.3188
(contig10-10170)
Wild type TTA
(L)
TTT
(F)
CTA
(L)
ATG
(M)
TTT
(F)
AGT
(S)
CGT
(R)
GTT
(V)
GCT
(A)
AAC
(N)
ATT
(I)
AAT
(N)
GAC
(D)
GAA
(E)
CGA
(R)
GAG
(E)
TCA
(S)
TCG
(S)
CTG
(S)
AAT
(N)
AAT
(N)
TAC1-2
a
orf19.10700
(contig10-20170)
Wild type AAA
(K)
GTC
(V)
CTA
(L)
ATG
(M)
TTT
(F)
AAT
(N)
CAC
(H)
GCT
(A)
GCT
(A)
AGC
(S)
ATT
(I)
AAA
(K)
AAC
(N)
CAA
(Q)
CGA
(R)
GAG
(E)
TTA
(L)
TCG
(S)
CCG
(P)
AAT
(N)
AAT
(N)
DSY294 TAC1-3 Wild type AAA
(K)
TTT
(F)
ATA
(I)
ATG
(M)
TCT
(S)
AAT
(N)
CAC
(H)
GCT
(A)
GCT
(A)
AGC
(S)
GTT
(V)
AAA
(K)
AAC
(N)
GAA
(E)
CGA
(R)
GAG
(E)
TCA
(S)
TCG
(S)
CTG
(S)
AAT
(N)
AAT
(N)
TAC1-4 Wild type AAA
(K)
TTT
(F)
CTA
(L)
GTG
(V)
TTT
(F)
AAT
(N)
CAC
(H)
GCT
(A)
GTT
(V)
AGC
(S)
GTT
(V)
AAA
(K)
AAC
(N)
CAA
(Q)
CAA
(Q)
GAG
(E)
TCA
(S)
TCG
(S)
CTG
(S)
AAT
(N)
AAT
(N)
DSY296 TAC1-5 Hyperactive AAA
(K)
TTT
(F)
CTA
(L)
GTG
(V)
TTT
(F)
AAT
(N)
CAC
(H)
GCT
(A)
GTT
(V)
AGC
(S)
GTT
(V)
AAA
(K)
AAC
(N)
CAA
(Q)
CAA
(Q)
GAG
(E)
TCA
(S)
TCG
(S)
CTG
(S)
AAT
(N)
GAT
(D)
FH1 and FH2 TAC1-6 Wild type AAA
(K)
TTT
(F)
ATA
(I)
ATG
(M)
TCT
(S)
AAT
(N)
CAC
(H)
GCT
(A)
GCT
(A)
AGC
(S)
GTT
(V)
AAA
(K)
AAC
(N)
GAA
(E)
CGA
(R)
GGG
(G)
TCA
(S)
TTG
(L)
CTG
(S)
TAT
(Y)
AAT
(N)
TAC1-7 Hyperactive AAA
(K)
TTT
(F)
CTA
(L)
ATG
(M)
TTT
(F)
AAT
(N)
CAC
(H)
GCT
(A)
GCT
(A)
AGC
(S)
ATT
(I)
AAT
(N)
AAC
(N)
GAA
(E)
CGA
(R)
GAG
(E)
TCA
(S)
TCG
(S)
CCG
(P)
AAT
(N)
GAT
(D)
FH3 TAC1-7 Hyperactive AAA
(K)
TTT
(F)
CTA
(L)
ATG
(M)
TTT
(F)
AAT
(N)
CAC
(H)
GCT
(A)
GCT
(A)
AGC
(S)
ATT
(I)
AAT
(N)
AAC
(N)
GAA
(E)
CGA
(R)
GAG
(E)
TCA
(S)
TCG
(S)
CCG
(P)
AAT
(N)
GAT
(D)
a
This allele is not corrected in CGD and contains a stop codon in the ORF.
2144 A. Coste et al.
lane 6). In contrast, TAC1-7 conferred terbinafine resis-
tance (Figure 1A) and expressed constitutive high levels
of Cdr1p and Cdr2p (Figure 1B, lane 7). When strains
FH3–FH8 were screened for TAC1 alleles, all attempts
to recover alleles different from TAC1-7 were unsuccess-
ful. These results indicate that strains FH3–FH8 are
homozygous for the TAC1-7 allele. Consistent with this,
these strains were also homozygous at the MTL, suggest-
ing that they underwent an LOH event encompassing
.14 kb of chromosome 5.
Taken together, the analysis of TAC1 alleles from
clinical strains identified five new alleles in addition to
the TAC1-1 and TAC1-2 previously characterized from
strain CAF2-1. Nucleotide polymorphisms in all these
alleles are presented in Table 2. Because of the high
diversity of TAC1 alleles, their nomenclature was mod-
ified (Table 2) as compared to results of our previous
study (Coste et al. 2004). Among the five new alleles,
three were defined as wild type and two as hyperactive.
Furthermore, the two hyperactive alleles were the only
alleles that were homozygous in two independent
clinical, azole-resistant strains, a feature consistent with
the linked homozygosity at the mating-type locus.
TAC1 alleles can be hyperactive through a mutation in
the C-terminal domain: TAC1 sequences were aligned to
identify differences between wild-type and hyperactive
alleles. Wild-type TAC1-4 and hyperactive TAC1-5 alleles
differed by only one base in codon 977 (Table 2). This
nonsynonymous point mutation changes asparagine
(N) in TAC1-4 to aspartatic acid (D) in TAC1-5. Fur-
thermore, none of the wild-type alleles contain this
point mutation. Importantly, the TAC1-7 hyperactive
allele contains the same nucleotide change that yields a
N977D mutation in Tac1p. These results strongly sug-
gest a relationship between the presence of the N977D
mutation and the hyperactivity of TAC1-5 and TAC1-7.
Nineteen other codons with nonsynonymous polymor-
phisms were detected among the seven allele sequences
compared in this study (Table 2). Since these were
present in both wild-type and hyperactive alleles, they
were not associated with azole resistance.
To test the role of the N977D mutation in TAC1 hyper-
activity, Asp
977
was introduced by site-directed mutagen-
esis into wild-type alleles TAC1-1,TAC1-4, and TAC1-6.
The complementary experiment, replacing Asp
977
with
Asn
977
in the hyperactive alleles TAC1-5 and TAC1-7, was
Figure 1.—Analysis of drug resistance properties dependent on TAC1 alleles. (A) Drug susceptibility testing of C. albicans tac1D/D
mutant and TAC1 revertant strains with different TAC1 alleles. Drug susceptibility assays were carried out by plating serial dilutions
of overnight cultures onto YEPD agar plates containing different drugs as indicated. Plates were incubated for 48 hr at 35°. MIC
assays were performed as described in materials and methods. (B) Immunodetection of Cdr1p and Cdr2p in tac1D/Dmutant
and TAC1 revertant strains with different TAC1 alleles. Protein extracts of each strain were separated on SDS-10% polyacrylamide
gels and immunoblotted with rabbit polyclonal anti-Cdr1p and anti-Cdr2p as described previously (de Micheli et al. 2002). C.
albicans strains were grown in liquid YEPD to midlog phase and exposed (1) or not () to fluphenazine (10 mg/ml) for 20 min.
The following Ura
1
strains correspond to the following genotypes: CAF2-1, TAC1-1/TAC1-2; DSY2903, tac1-1D/tac1-2D; DSY2925-
47, tac1D/D1TAC1-3; DSY2925-18, tac1D/D1TAC1-4; DSY2984, tac1D/D1TAC1-5; DSY3010-80, tac1D/D1TAC1-6; DSY3010-
113, tac1D/D1TAC1-7-FH1; DSY3013, tac1D/D1TAC1-7-FH3. For phenotypes and genotypes of the different strains of this study
refer to supplemental Table S1 at http://www.genetics.org/supplemental/.
Azole Resistance by TAC1 Mutation 2145
also performed. The new alleles TAC1-1
N977D
,TAC1-
4
N977D
,TAC1-6
N977D
,TAC1-5
D977N
, and TAC1-7
D977N
were
then introduced into a tac1D/Dmutant strain and the
drug resistance phenotypes, as well as the Cdr1p and
Cdr2p levels, were measured in cells with or without ex-
posure to fluphenazine. The presence of Asp
977
in the
modified alleles TAC1-1
N977D
,TAC1-4
N977D
, and TAC1-
6
N977D
increased resistance to fluconazole and terbina-
fine relativeto the correspondingwild-type alleles (Figure
2A). Moreover, the presence of Asp
977
in the modified
alleles resulted in constitutive high levels of Cdr1p and
Cdr2p (Figure 2B, lanes 2, 4, and 8). In contrast, ex-
pression of the corresponding wild-type alleles resulted in
basal levels of Cdr1p. High levels of Cdr1p and Cdr2p
expression were seen in strains carrying the wild-type
alleles only upon fluphenazine exposure (Figure 2B,
lanes 1, 3, and 7). When Asp
977
was replaced by Asn
977
in
the hyperactive alleles (modified alleles TAC1-5
D977N
and
TAC1-7
D977N
), the ability to grow on plates containing anti-
fungal agents and the constitutive high levels of Cdr1p
and Cdr2p were not maintained (Figure 2). Modified
alleles TAC1-5
D977N
and TAC1-7
D977N
(Figure 2B, lanes 6 and
10) did not mediate constitutive high levels of Cdr1p
and Cdr2p as compared to the nonmodified alleles TAC1-
5and TAC1-7 (Figure 2B, lanes 5 and 9). The phenotypes
obtained by the strains carrying these modified hyperac-
tive alleles were similar to those obtained with strains con-
taining the wild-type alleles TAC1 -1 ,TAC1-4,andTAC1-6
(Figure 2B, lanes 1, 3, and 7). Taken together, these anal-
yses demonstrate that a single point mutation, the replace-
ment of Asn by Asp at position 977 of Tac1p, is sufficient
to modify the activity of the transcription factor into a
hyperactive state.
Role of TAC1 in drug susceptibility and Cdr1p/Cdr2p
levels in clinical strains: As mentioned above, strains
DSY294 and DSY296 are matched azole-susceptible and
azole-resistant isolates. To demonstrate that azole re-
sistance was coupled with the presence of hyperactive
alleles, TAC1 was inactivated in the background of these
clinical strains. First, the ura3 auxotrophic marker
was introduced into these strains using the dominant
marker caSAT1 (Reuss et al. 2004). The deletion of TAC1
in DSY294 had a moderate effect on fluconazole MIC
in both the heterozygous and the homozygous mutants
(Figure 3A, top). The fluconazole MIC values decreased
from 1 mg/ml for DSY294 to 0.25 and 0.5 mg/ml in both
mutant strains. Furthermore, in the tac1D/Dmutant,
fluphenazine exposure did not increase Cdr1p and
Cdr2p levels (Figure 3B, lane 3). This result is similar
to observations of CAF2-1 and its tac1D/Dderivative
strain (Coste et al. 2004). These results highlight the
crucial role of TAC1 in both basal and induced
Figure 2.—Effect of the mutation N977D on the properties of TAC1. (A) Drug susceptibility testing of C. albicans tac1D/Dmu-
tant and TAC1 revertant strains with different TAC1 alleles containing the N977D substitution. Drug susceptibility assays were
carried out as described in Figure 1. MIC assays were performed as described in materials and methods. (B) Immunodetection
of Cdr1p and Cdr2p in C. albicans tac1D/Dmutant and TAC1 revertant strains containing different TAC1 alleles with the N977D
substitution. See legend of Figure 1B for other details. The following strains correspond to the following genotypes: DSY2937-35,
tac1D/D1TAC1-1; VTY21, tac1D/D1TAC1-1
N977D
; VTY28, tac1D/D1TAC1- 4
N977D
; ACY11, tac1D/D1TAC 1-6
N977D
; ACY12, tac1D/D1
TAC1-7
D977N
; VTY9, tac1D/D1TAC1-5
D977N
. See legend of Figure 1 for other strains and genotype designations.
2146 A. Coste et al.
expression of CDR1 and CDR2 in this clinical strain. The
deletion of one copy of TAC1-5 from DSY296 resulted in
a slight decrease of fluconazole resistance (MIC varying
from 128 to 64 mg/ml), while deletion of both TAC
copies in DSY296 resulted in a significant reduction in
fluconazole resistance (MIC varying from 128 to 4 mg/
ml) (Figure 3A, bottom). A slight decrease in Cdr1p and
Cdr2p levels was observed in TAC1-5/tac1-5Das com-
pared to the wild-type strain (Figure 3B, right, lane 8),
thus indicating that the TAC1-5 hyperactive allele copy
number has an impact on expression levels of these
proteins. In the tac1-5D/Dhomozygous mutant, Cdr1p/
Cdr2p levels were almost undetectable both with and
without fluphenazine exposure, demonstrating a direct
relationship between the presence of TAC1 hyperactive
alleles and azole resistance in these clinical strains
(Figure 3B, lane 9). Interestingly, the MIC of flucona-
zole, but not of terbinafine, was higher in the tac1D/D
DSY296-derived strain (4 mg/ml) than in the tac1D/D
DSY294-derived strain (0.5 mg/ml). This discrepancy
may be explained by the presence of a mutation in both
ERG11 alleles in DSY296 (G464S) that alters binding of
azoles to Erg11p and therefore contributes to flucona-
zole resistance (Sanglard et al. 1998).
To characterize the properties of TAC1 alleles from
DSY294 and DSY296, the activity of TAC1-3,TAC1-4, and
TAC1-5 was first assessed in the DSY294 and DSY296
backgrounds, which are mating type heterozygous and
homozygous, respectively. Each type of TAC1 allele was
reintroduced into the tac1D/Dmutant strains derived
from DSY294 and DSY296. TAC1 activity profiles for
these alleles were similar tothose observed in theCAF2-1
background. TAC1-3 and TAC1-4 did not confer resis-
tance to fluconazole or terbinafine (Figure 3A) in either
strain backgrounds. Consistently, they exhibited basal
levels of Cdr1p and no Cdr2p and did mediate high
Cdr1p/Cdr2p levels in the presence of fluphenazine
(Figure 3B, lanes 5, 6, 11, and 12). TAC1-5 restored resis-
tance to fluconazole and terbinafine in either strain
backgrounds (Figure 3A) and constitutive high Cdr1p
and Cdr2p levels (Figure 3B, lanes 4 and 10).
To correlate Cdr1p and Cdr2p levels with the capacity
to efflux an ABC-transporter substrate, rhodamine 6G
efflux was monitored in clinical strains DSY294 and
Figure 3.—Analysis of TAC1 alleles from the clinical strains DSY294 and DSY296. (A) Drug susceptibility testing of C. albicans
tac1D/Dmutant and TAC1 revertant clinical strains containing specific TAC1 alleles. Drug susceptibility assays were carried out
onto YEPD medium containing 2.5 mg/ml of fluconazole and 1 mg/ml cyclosporin A for the DSY294-derived strains and 5 mg/ml
of fluconazole and 1 mg/ml cyclosporin A for the DSY296-derived strains. Cyclosporin A alone had no effect on the growth of these
strains. All strains were spotted onto agar medium containing 20 mg/ml of terbinafine. Plates were incubated for 48 hr at 35°. MIC
assays were performed as described in materials and methods. (B) Immunodetection of Cdr1p and Cdr2p in C. albicans tac1D/D
mutant and TAC1 revertant clinical strains. See legend of Figure 1 for other details. The following strains correspond to the fol-
lowing genotypes: DSY294, TAC1-3/TAC1-4; DSY296, TAC1-5/TAC1-5; DSY3058, TAC1-4/tac1-3D; DSY3082; tac1-3D/tac1-4D;
DSY3287-1, tac1-3D/tac1-4D1TAC1-3; DSY3288-3, tac1-3D/tac1-4D1TAC1-4; DSY3102-2, tac1-3D/tac1-4D1TAC1-5; DSY3059,
TAC1-5/tac1-5D; DSY3083, tac1-5D/D; DSY3285-1, tac1-5D/D1TAC1-3; DSY3286-2, tac1-5D/D1TAC1-4; DSY33284-1, tac1-5D/D1
TAC1-5.
Azole Resistance by TAC1 Mutation 2147
DSY296 as well as in the tac1D/Dderivatives (supple-
mental Figure S1, A and B, at http://www.genetics.org/
supplemental/). The DSY296 strain exhibited higher
rhodamine 6G efflux rates (average V
max
: 5.7 RFU/sec)
than the DSY294 strain (average V
max
: 1.3 RFU/sec),
which is consistent with the differences in Cdr1p and
Cdr2p levels between the two strains. In the tac1D/D
mutant strains from both isolates, rhodamine 6G efflux
rates were similar to the rates observed in DSY294. The
presence of TAC1-5 in the background of DSY294 tac1D/
Dmutants resulted in higher rhodamine 6G efflux rates
(V
max
values between 3.8 and 4.1 RFU/sec) than those
observed in DSY294, in tac1D/Dmutants, and in TAC1-3
or TAC1-4 revertant strains (supplemental Figure S1A
at http://www.genetics.org/supplemental/). Thus, the
TAC1-5 hyperactive allele is necessary for increased
Cdr1p/Cdr2p levels, which is also correlated to en-
hanced efflux rates and is independent of strain back-
ground. Importantly, since the MTL locus in DSY294 is
heterozygous, these studies also show that MTL homo-
zygosity does not have a detectable effect on azole
resistance.
Codominance of TAC1 alleles with the N977D sub-
stitution: Homozygosity at the mating-type locus was
reported to correlate with the occurrence of azole resis-
tance (Rustad et al. 2002). Hence, azole-resistant strains
used in this study are homozygous at the mating-type
locus and consequently contain a single TAC1 allele. It is
possible that the development of the drug resistance
phenotype necessitates hyperactive TAC1 alleles in a
homozygous state, suggesting that these alleles are
recessive to wild-type alleles. This rationale is supported
by the profiles of TAC1 alleles in strain FH1: although
this strain contains both a wild-type and a hyperactive
TAC1 allele, this strain remains fluconazole susceptible.
TAC1 status in strains DSY294 and DSY296: The
above-described analyses were performed in tac1D/Dho-
mozygous mutant strains and therefore the dominance/
recessivity relationships of TAC1 alleles could not be
determined. To address this question, TAC1 alleles
(TAC1-3–TAC1 -5 )werereintroducedinDSY294and
DSY296 TAC1/tac1Dheterozygotes, thus generating
heterozygous strains, each carrying two different TAC1
alleles.
When the TAC1 -5 allele was introduced at the genomic
TAC1 locus in the heterozygous mutant strain TAC1-5/
tac1-5D, the obtained revertant (TAC1-5/tac 1-5D1TAC1-
5) exhibited phenotypes similar to those observed in
DSY296. The fluconazole and terbinafine MICs were
similar in both strains: while the fluconazole MICs were
128 mg/ml for bothstrains, the terbinafine MICs were 16
and 8 mg/ml for DSY296 and the revertant, respec-
tively (Figure 4A). Constitutive high levels of Cdr1p
and Cdr2p were also similar between these strains
(Figure 4B, lanes 2 and 5). When wild-type allele
TAC1-3 or TAC1-4 was introduced into the TAC1-5/
tac1-5Dstrain, the transformants had decreased resis-
tance phenotypes compared to DSY296. Fluconazole
MIC decreased from 128 mg/ml to 32 and 16 mg/ml
for the TAC1-3 and TAC1-4 alleles, respectively; ter-
binafine MICs decreased from 16 to 4 and 2 mg/ml,
respectively (Figure 4A). Moreover, constitutive high
Cdr1p and Cdr2p levels were reduced in these hetero-
zygotes compared to DSY296 (Figure 4B, lanes 2, 6,
Figure 4.—Codominance of the
TAC1-5 hyperactive allele. (A) Drug sus-
ceptibility testing of the C. albicansclinical
isolates DSY294 and DSY296, hetero-
zygous (TAC1-5/tac1-5D) and homozy-
gous (tac1-5D/D) mutants derived from
DSY296, and transformants of the hetero-
zygous mutant in which a TAC1-3,TA C1 -4,
or TAC1-5 allele was reintroduced. See
legend of Figure 1 for other details. (B)
Immunodetection of Cdr1p and Cdr2p
in the strains listed above. See legend of
Figure 1 for other details. The following
strains correspond to the following geno-
types: DSY3211-4, TAC1- 5/ ta c1-5D1TAC1-
3; DSY3215-1, TAC1-5/tac1 -5 D1TAC 1- 4;
DSY3210-1, TAC 1- 5/ ta c1-5D1TAC1-5.See
the legend of Figure 3 for other strain
and genotype designations.
2148 A. Coste et al.
and 7). Cdr1p levels in these TAC1-5/tac1-5D1TAC1-3
and TAC1-5/tac1-5D1TAC1-4 strains were slightly
higher than those observed in the azole-susceptible
strain DSY294 (Figure 4B, lanes 1, 6, and 7). Cdr2p
levels, although barely detectable in the heterozygote
with TAC1-3, were not detectable in the heterozygote
with TAC1-4 (Figure 4B, lanes 6 and 7) as in DSY294
(Figure 4B, lane 1). Induction of Cdr1p and Cdr2p
expression by fluphenazine did occur in these hetero-
zygotes. Thus these two heterozygous strains have
phenotypes intermediate between that of the azole-
susceptible strain DSY294 and the azole-resistant strain
DSY296. For example, the heterozygotes with TAC1-5
and TAC1-3 alleles or TAC1-5 and TAC1-4 alleles have
fluconazole MICs of 32 and 16 mg/ml, respectively, vs.
1mg/ml for DSY294 (TAC1-3/TAC1-4) and 128 mg/ml
for DSY296 (TAC1-5/TAC1-5). Similarly, DSY294-derived
heterozygous strains TAC1-3/tac1-4Dand tac1-3D/TAC1-
4transformed with the TAC1-5 allele had an intermedi-
ate phenotype in terms of drug resistance and Cdr1p
and Cdr2p levels (data not shown). Taken together, our
results suggest that the hyperactivity of the TAC1-5 allele
is decreased in the presence of a wild-type allele. This
implies that the hyperactive TAC1-5 allele is codominant
with wild-type alleles TAC1-3 and TAC1-4. Moreover,
these results confirm the codominance hypothesis of
the azole resistance phenotype that was proposed from
fusion experiments between DSY296 and an azole-
susceptible strain with opposite mating type (Coste
et al. 2004).
TAC1 status in strains FH1 and FH3: The develop-
ment of drug resistance in strain FH1 is of interest
because this strain contains both a wild-type allele
(TAC1-6) and a hyperactive allele (TAC1-7). Consistent
with this, strain FH1 exhibited intermediate levels of
drug resistance and Cdr1p/Cdr2p levels. FH1 has MICs
of 8 and 32 mg/ml for fluconazole and terbinafine,
respectively (Figure 5A). Cdr1p and Cdr2p levels in
normal growth conditions are higher in FH1 than those
in CAF2-1 and DSY294 and are still inducible after
fluphenazine exposure (Figure 5B, lane 1).
To analyze the phenotypes of individual alleles, each
TAC1 allele in strain FH1 was deleted. Whereas the FH1
strain possesses fluconazole and terbinafine MICs of 8
and 32 mg/ml, respectively (Figure 5A), the drug resis-
tance of the TAC1-6/tac1-7Dmutant decreased (flucon-
azole and terbinafine MICs of 1 and 8 mg/ml; Figure
5A). Both Cdr1p and Cdr2p levels decreased as well
(Figure 5B, lanes 1 and 4). In contrast, when comparedto
FH1, the fluconazole resistance of the tac1-6D/TAC1-7
mutant increased from 8 to 16 mg/ml and Cdr1p and
Cdr2p levels increased as well (Figure 5B, lanes 1 and 3).
These phenotypes resemble those of FH3 and are
almost identical to those observed in the FH3-derived
TAC1-7/tac1-7Dstrain (Figure 5, A and B, lane 2).
In summary, the hyperactive TAC1-7 allele possesses
codominant properties similar to those described for
TAC1-5. The presence of TAC1-7 together with a wild-
type allele results in intermediate drug resistance
properties. Strains carrying two hyperactive alleles (i.e.,
DSY296 and FH3) exhibit the strongest phenotypes in
terms of drug susceptibility and Cdr1p/Cdr2p levels.
Therefore, development of high resistance to azoles in
aTAC1 heterozygous strain carrying one hyperactive
Figure 5.—Analysis of the
TAC1 alleles from clinical strains
FH1 and FH3. (A) Drug suscepti-
bility testing of C. albicans FH1
and FH3 derivatives lacking one
specific TAC1 allele. See legend
of Figure 1 for other details. (B)
Immunodetection of Cdr1p and
Cdr2p in C. albicans FH1 and
FH3 derivatives lacking one spe-
cific TAC1 allele. See legend of
Figure1 for other details. The fol-
lowing strains correspond to the
following genotypes: DSY3132-
11, TAC1-6/tac1-7D; DSY3132-14,
TAC1-7/tac1-6D; DSY3133-15,
TAC1-7/tac1-7D; FH1, TAC1-6/
TAC1-7; FH3, TAC1-7/TAC1-7.
Azole Resistance by TAC1 Mutation 2149
allele is due to LOH involving removal of the TAC1 wild-
type copy and maintenance of the hyperactive allele.
Since TAC1 is only 14 kb from the MTL, it appears that
the mechanisms that result in LOH at TAC1 are often
accompanied by LOH at MTL.
Stages in the development of azole resistance: The
mechanisms by which hyperactive alleles can become
homozygous in azole-resistant strains remain unknown.
One possibility is that, during azole exposure of a strain
heterozygous at the TAC1 locus, the chromosome
carrying a TAC1 hyperactive allele can be duplicated.
This would then be followed by loss of one chromosome
5 copy with a wild-type TAC1 allele, thus resulting in
homozygosity of the remaining chromosome 5 copies.
Alternatively, the chromosome 5 copy with the wild-type
TAC1 allele could be lost first and then the remaining
copy could be duplicated. A third possibility is that the
region containing TAC1 (and MTL) undergoes a local
recombination event that results in gene conversion of a
long region of the chromosome.
Development of in vitro fluconazole resistance: To ask how
LOH and fluconazole resistance occurs, we followed the
in vitro acquisition of fluconazole resistance in strain
FH1 and analyzed the newly resistant strains for their
chromosome 5 and TAC1 status. FH1 was spotted onto a
YEPD plate supplemented with 10 mg/ml of flucon-
azole. The plate was incubated at 30°until resistant
colonies appeared. The medium contained cyclospor-
ine A to suppress residual growth of C. albicans in the
presence of fluconazole as described (Marchetti et al.
2000). One resistant colony (DSY3157-2) was plated
onto YEPD with 10 mg/ml fluconazole and fast-growing
colonies were obtained. One of these, DSY3301-4, as well
as strains FH1, FH3, and DSY3157-2, were analyzed for
azole susceptibility and Cdr1p/Cdr2p protein levels.
Strains FH1, DSY3157-2, and DSY3301-4 showed in-
creasing fluconazole resistance (MIC of 8, 16, and
32 mg/ml, respectively, Figure 6A). These strains also
exhibited increasing Cdr1p and Cdr2p levels as com-
pared to FH1 under normal growth conditions (Figure
6B, lanes 1, 3 and 4). Furthermore, DSY3301-4 and FH3
had identical fluconazole MICs (32 mg/ml) and ex-
hibited similar Cdr1p and Cdr2p levels in both the
absence and the presence of fluphenazine (Figure 6B,
lanes 2 and 4).
Analysis of TAC1 and chromosome 5 status: The step-
wise increase of fluconazole resistance from FH1 to
DSY3157-2 and DSY3301-4 could have occurred if TAC1-
7was duplicated in DSY3157-2 while TAC1-6 was main-
tained. In DSY3301-4, TAC1-6 could have been lost,
leaving two TAC1-7 copies. To test these step hypotheses,
we determined the status of the MTL loci. Both FH1 and
DSY3157-2 were heterozygous (MTLa/MTLa) while
both FH3 and DSY3301-4 were mating type homozygous
(MTLa/MTLa) (Figure 6C). Southern blot analysis of
the TAC1 loci of strains FH1, FH3, DSY3157-2, and
DSY3301-4 confirmed that FH1 and DSY3157-2 con-
tained two distinct alleles (1.8 and 3.2 kb), while FH3
contained only the 3.2-kb signal corresponding to TAC1-
7. Interestingly, the signal ratio for TAC1-7:TAC1-6 was
0.98:1 in FH1 and 2.1:1 in DSY3157-2. This analysis is
consistent with the idea that, in strain DSY3157-2, the
genotype is TAC1-7/TAC1-7/TAC1-6. Comparative ge-
nome hybridization array analysis (Figure 7) supports
the first part of this step hypothesis: strains FH1 and FH3
are disomic for all genes on chromosome 5, while chro-
mosome 5 is trisomic in strain DSY3157-2. In addition,
chromosome 5 is trisomic in DSY3301-4, suggesting that
this strain carries three copies of TAC1-7,MTLa, and all
of the other genes on chromosome 5. Thus, either the
copy of chromosome 5 carrying MTLa and TAC1-6
was lost and another copy of a remaining chromosome
5 was duplicated or, alternatively, a region of chromo-
some 5 containing MTLa and TAC1-6 underwent gene
conversion.
Analysis of chromosome 5 alterations: To distinguish
between the above mechanisms of LOH in strain
DSY3301-4, we conducted SNP microarray hybridization
for 13 SNP loci evenly distributed across chromosome
5(Forche et al. 2005). This analysis showed that 12
of these loci were homozygous in all four strains. One
SNP locus (marker 104), located within the SNF1 ORF
(orf19.1936), was heterozygous in strains FH1, FH3, and
DSY3157-2. This locus became homozygous in strain
DSY3301-4 (Figure 8, top).
To obtain additional information, 10 more genes,
not present on the SNP arrays (CRH12,orf19.4251,
orf19.1926, orf19.4288, TRX1,TRR1,ZNC3, orf19.4225,
orf19.2646, and orf19.6680, solid triangles in Figure 8),
were sequenced to determine the allelic status in each
of the four strains. Of these 10 genes, 6 were uninforma-
tive because they were homozygous for all four strains.
The remaining 4 genes (CRH12, orf19.4251, orf19.1926,
orf19.4288; see Table 3) were heterozygous in strains
FH1, FH3, and DSY3157-2 and homozygous in strain
DSY3301-4 (markers B, E, F, and J of Figure 8, top). Since
the SNP loci that became homozygous in DSY3301-4 are
distributed all along chromosome 5, this result is consis-
tent with the idea that all of chromosome 5 became
homozygous in DSY3301-4. It is likely that in DSY3301-4
a homolog carrying the TAC1-7 allele replaced the chro-
mosome carrying TAC1-6.
Interestingly, the mechanism by which FH3 became
homozygous for TAC1-7 appears to be different from
that of DSY3301-4; this strain shows LOH only between
the MTL and TAC1 loci. Rather, it appears that a mitotic
recombination event occurred between the two chro-
mosome 5 copies of strain FH3, thus leading to gene
conversion of the TAC1-6 and MTLa to TAC1-7 and
MTLa(see Figure 9, top, for schematic).
TAC1 and chromosome 5 status in clinical strains DSY294
and DSY296: To ask about the mechanism by which
strain DSY296 became azole resistant, we analyzed the
organization of chromosome 5 homologs in this strain
2150 A. Coste et al.
as well. Comparative genome hybridization array anal-
ysis indicated that both strains DSY294 and DSY296
carried two copies of chromosome 5 (data not shown).
SNP microarray hybridization revealed that 10 of the 13
SNP markers on the array were homozygous in both
strains. Markers 110 and 111 were homozygous only in
DSY296 and marker 109 was heterozygous in both
strains (Figure 8, bottom). Seven other genes (CRH12,
orf19.4251, orf19.1926, TRX1,TRR1, orf19.4225 and
orf19.6680) were amplified and sequenced along chro-
mosome 5 as described above. DSY294 and DSY296 were
homozygous for TRX1, orf19.1926, and orf19.6680 and
heterozygous for orf19.4225, orf19.4251, TRR1, and
CRH12 (Figure 8, bottom). Since strain DSY296 is het-
erozygous for five polymorphisms tested here (marker
109, orf19.4225, orf19.4251, TRR1 and CRH12), we
conclude that LOH at TAC1 and MTL was not caused by
duplication of all of chromosome 5. Rather, it appears
that mitotic recombination between two copies of
chromosome 5 likely resulted in LOH at these loci
(see Figure 9, bottom, for schematic). We propose that
an intermediate strain, carrying one TAC1-3 and one
TAC1-5 allele (derived from TAC1-4), underwent mi-
totic recombination and gene conversion leading to
homozygosis of the TAC1-5 alleles in strain DSY296.
Taken together, our results show that increased anti-
fungal drug resistance due to constitutively high expres-
sion of the ABC transporters Cdr1p and Cdr2p can be
achieved when TAC1 alleles carry the N977D muta-
tion and become homozygous. Homozygosis can occur
Figure 6.—Analysis of FH1-derived strains after in vitro fluconazole exposure. (A) Drug susceptibility testing of C. albicans FH3-,
FH1-, and FH1-derived strains selected for their fluconazole resistance. DSY3157-2 was derived from a single colony of FH1 that
arose after spotting onto YEPD medium with 10 mg/ml fluconazole. DSY3301-4 was derived from DSY3157-2 as a fast-growing
colony onto medium with 10 mg/ml fluconazole. Drug susceptibility assays were carried out as described in Figure 1 onto YEPD
medium containing 10 mg/ml fluconazole and 1 mg/ml cyclosporin A. Cyclosporin A alone had no effect on the growth of these
strains. Plates were incubated for 48 hr at 35°. MIC assays were performed as described in materials and methods. (B) Immu-
nodetection of Cdr1p and Cdr2p in C. albicans strains FH1, FH3, DSY3157-2, and DSY3301-4. See legend of Figure 1 for other
details. (C) PCR analysis of the mating-type locus. PCR was performed as described (Rustad et al. 2002). ‘‘a’’ and ‘‘a’’ denote
analysis performed to detect MTLa and MTLaloci, respectively. (D) Southern blot analysis of the TAC1 alleles in C. albicans
FH-derivative strains. Genomic DNA of each strain was digested by EcoRI and Southern blot was performed as described in
materials and methods. Radioactivity of signals was quantified as discussed in materials and methods. Restriction maps of both
TAC1-6 and TAC1-7 alleles indicate restriction site polymorphism for EcoRI. The solid bar indicates the position of the labeled probe,
which corresponded to the region located between the first TAC1 initiation codon and the PstI restriction site. Digestion of genomic
DNA with EcoRI is expected to yield positive signals at 1.8 and 3.2 kb for TAC1-6 and TAC1-7,respectively.
Azole Resistance by TAC1 Mutation 2151
either by mitotic recombination between chromosome
5 copies that results in gene conversion or by the
presence of extra copies of chromosome 5 carrying
the hyperactive TAC1 allele accompanied by loss of
chromosome 5 carrying the wild-type TAC1 allele.
DISCUSSION
A mutation in TAC1 is involved in azole resistance: In
this work, we established for the first time a link between
a point mutation in a transcription factor and the
constitutive high expression of the multidrug trans-
porters Cdr1p and Cdr2p responsible for antifungal
drug resistance. It was previously shown that azole
resistance can be due to point mutations in ERG11,
which encodes the enzyme target of azoles. It is believed
that these mutations can alter the affinity of azoles for
their target and therefore can participate in the de-
velopment of resistance (Sanglard et al. 1998; Perea
et al. 2001). Among azole-resistant strains with altered
ERG11 alleles, Marichal et al. (1999) observed three
‘‘hot spots’’ localized in the region 105–165, 266–287,
and 405–488 of Erg11p. Resistance to other antifungal
drugs such as 5-fluorocytosine (5-FC) and caspofungin
was also shown to be due to point mutations in specific
Figure 8.—SNP analysis
of chromosome 5. The
map of chromosome 5 was
obtained by assembly of the
different contig sequences
shown by arrows. An asterisk
(*) indicates SNPs of chro-
mosome 5 as measured on
the SNP microarray: 102,
1855/2172; 103, HST3; 104,
SNF1; 109, 1899/2008; 110,
1445/2395; 111, 1922/
2344; 112, PDE1; 113,
1969/2162; 114, DPH5; 115,
HEX1; 116, 2093/2390; 117,
1817/2082; 118, 1341/
2493; 119, F16n1; 120,
2340/2493. ‘‘:’’ i n d i c a t e s
additional markers of chro-
mosome 5: A, orf19.1976
(TRX1); B, orf19.1926; C,
ZNC3;D,orf19.4225;E,
orf19.4251; F, orf19.4288;
G, orf19.1942 (TRR1); H,
orf19.2646; I, orf19.6680; J,
CRH12. Color codes indicate modifications in SNPs for individual strains. For the strains DSY294 and DSY296, sequences of the C,
F, and H markers were not available. The hatched regions on chromosome 5 delimitate the maximal region of a recombination.
MRS, major repeat sequence.
Figure 7.—CGH of FH-derived strains. The ge-
nomes of the tested strains were hybridized
against the SC5314 genome according to the pro-
tocol published by Selmecki et al. (2005). Each
gene on chromosome 5 is represented by its rel-
ative intensity as compared to signals obtained in
SC5314.
2152 A. Coste et al.
genes (Dodgson et al. 2004; Park et al. 2005). Resistance
to 5-FC is restricted to clade I and due to a single point
mutation, C301T, in the FUR1 gene, encoding a phos-
phoribosyltransferase. This nonsynonymous mutation
changes arginine to cysteine at position 101 of Fur1p
(Dodgson et al. 2004). Recently, Park et al. (2005)
showed that the modification of the serine 645 of
CaFks1p, a subunit of the 1,3-b-d-glucan synthase, is
sufficient to confer reduced susceptibility to echinocan-
dins in C. albicans.
The single nucleotide mutation in codon 977 (A to G
at nt 2929) in TAC1 corresponds to a nonsynonymous
modification from Asn to Asp in Tac1p. This mutation is
located within a putative C-terminal activation domain
of the transcription factor. The importance of this
region for transcriptional activity was confirmed by
preliminary experiments that deleted the C-terminal
region from aa 801 to the C-terminal end of Tac1p. This
deletion resulted in a truncated Tac1p unable to activate
the expression of at least CDR2 in presence of fluphen-
azine (D. Sanglard, unpublished results). Further
experiments will be needed to elucidate how the
N977D point mutation transforms Tac1p into a hyper-
active state. Several mutations in the C-terminal activa-
tion domain region of the S. cerevisiae transcription
factors Pdr1p or Pdr3p are also responsible for antifun-
gal drug resistance and for upregulation of PDR5. These
include pdr1-3 (F815S; Carvajal et al. 1997), pdr1-8
(L1036W; Carvajal et al. 1997), pdr1-12 (L1044Q;
Wendler et al. 1997), PDR1-101 (T879M; Reid et al.
1997), PDR1 (R821H; Tuttle et al. 2003), pdr3-17
(G834D), pdr3-18 (G834S), pdr3-19 (L837S), and pdr3-
20 (G957N; Carvajal et al. 1997). Gao et al. (2004)
showed that a strain carrying the hyperactive pdr1-3
allele expressed PDR5 in a drug-independent manner.
This can be associated with enhanced promoter occu-
pancy of coactivator complexes, including SAGA, Me-
diator, chromatin-remodeling SWI/SNF complex, and
TATA-binding protein. Using chromatin immunopre-
cipitation, loss of contacts between histones and DNA
was demonstrated at PDR5 promoter and coding se-
quences (Gao et al. 2004). Other mechanisms to activate
zinc-finger regulators have also been described, including
nuclear-cytoplasmic shuffling (Gorner et al. 1998; Santos
and de Larrinoa 2005), dimerization (Rottensteiner
et al. 1997), DNA binding, phosphorylation (Sadowski
et al. 1996; Kren et al. 2003), and unmasking of the
activation domain (Sadowski et al. 1996). Auto-induction
also can be envisaged as in the case of PDR3 (Delahodde
et al. 1995). Tac1p is present in the nucleus of cells
in normal growth conditions (Coste et al. 2004). Thus,
it is likely that Tac1p hyperactivity involves constitu-
tive binding to the DRE in the promoter of CDR1 and
CDR2, constitutive phosphorylation of the protein,
and/or a change in the conformation of the protein,
which can lead to the constant unmasking of the ac-
tivation domain.
TABLE 3
Nucleotide polymorphisms in chromosome 5 in FH1–FH3 and derivative strains
Strains
Genes and position of SNPs with respect to ATG
orf19.4251 orf19.1926 orf19.4288: CRH12
576 899 945 684 708 720 741 870 876 893 135 404 510
FH1, FH3, DSY3157-2 C or T A or G C or T A or G A or G A or G C or T A or G C or T C or G C or T C or T C or T
DSY3301-4 T A T A G G T A T C C T C
Azole Resistance by TAC1 Mutation 2153
TAC1 hyperactive alleles are codominant with wild-
type alleles: Much like PDR1 F815S and other PDR1
gain-of-function alleles, hyperactive TAC1 N977D alleles
are codominant with wild-type alleles. This suggests
that high levels of antifungal drug resistance cannot be
achieved in the presence of wild-type alleles. Rather,
homozygosis of the hyperactive alleles is necessary for
the development of high levels of azole resistance. Codo-
minance of these alleles probably reflects the fact that
Tac1p is a transcription factor belonging to the Zn
2
–
Cys
6
family, which often dimerize to bind a cis-acting
element (Mamnun et al. 2002). When two different
TAC1 alleles are expressed in the same cell, heterodimer
formation is likely. A corollary to this hypothesis, which
remains to be tested, is that heterodimers containing
one hyperactive and one wild-type Tac1p cannot direct
high levels of basal Cdr1p and Cdr2p expression.
Codominance is a feature shared with other alleles of
genes involved in antifungal resistance such as ERG11 or
FUR1.White (1997) showed that the R467K mutation
in ERG11 alone is not sufficient to confer azole re-
sistance. Loss of allelic variation in ERG11 is required to
confer strong azole resistance (White 1997). This ob-
servation was also made independently for other ERG11
alleles in our laboratory (Sanglard et al. 1998). Dodgson
et al. (2004) found a semidominant relationship between
the hyperactive and wild-type alleles of FUR1 for the
development of 5-FC resistance.
Duplication of and recombination between chromo-
some 5 homologs as mechanisms resulting in TAC1
homozygosity: To observe strong drug resistance, the
codominance of TAC1 N977D alleles requires of loss of
allelic variation. This property of TAC1 may explain the
link between MTL homozygosity and antifungal drug
resistance. In this study, we reconstituted the loss of
allelic variation at the TAC1 locus in vitro. Drug resis-
tance increased in two steps: the first step corresponded
to a duplication of the TAC1-7 hyperactive allele along
with all other chromosome 5-linked genes; the second
step involved the replacement of the remaining wild-
type TAC1-6 allele by the hyperactive TAC1-7 allele while
the strain remained trisomic for chromosome 5. SNP
analysis suggests that the mechanism by which this
occurred was loss of the MTLa chromosome 5 homolog
and gain of a third copy of the MTLahomolog. The
order of these events is not clear, but appears to have
involved all of chromosome 5.
We also observed other mechanisms of LOH: duplica-
tion and exchanges of parts of chromosome 5. Clinical
strain FH3 did not undergo LOH for all investigated
SNP markers and for 10 other genes situated on
chromosome 5 (see Figure 8). In FH3, the LOH that
led to homozygosity of TAC1 and of MTL includes
flanking regions that compose a 250-kb region delimi-
tated by heterozygous loci orf19.1926 and orf19.4251
(Figure 9, hatched region of chromosome 5). Similarly,
SNP analysis of strains DSY294 and DSY296 revealed that
a mitotic recombination event in DSY296 comprising an
300-kb fragment bordered by heterozygous loci 1899/
2008 and orf19.4225 resulted in LOH at TAC1 and MTL.
This homozygosity was not restricted to the MTLa/a
genes but extended into the PAP genes within MTL
Figure 9.—Schematic of
chromosome 5 alterations
in matchedazole-susceptible
and azole-resistant strains
obtained from patients or
developed in vitro.(Top)
Chromosome 5 alterations
in FH1-derived strains both
in vitro and in vivo. Chromo-
some 5 containing TAC1-7
and MTLawas duplicated
after in vitro fluconazole ex-
posure in DSY3157-2; a chro-
mosome 5 copy with TAC1-6
and MTLa was lost in
DSY3301-4 and replaced by
another chromosome 5 copy
containing TAC1-7 and
MTLaafter a second flucon-
azole exposure; in FH3, a
portion of chromosome 5
underwent mitotic recombi-
nation between markers B
and E, resulting in TAC1-7
and MTLahomozygosity.
(Bottom) Chromosome 5 al-
terations in DSY294 and DSY296in vivo. In DSY296, a portion of chromosome 5 underwent mitotic recombination betweenmarkers 109
and D, resulting in TAC1-5 and MTLahomozygosity. An intermediate strain between DSY294 and DSY296 in which a single nucleotide
change in TAC1-4 yielded TAC1-5 resulting in Asn
977
to Asp could have existed. TAC1 -3,-4,and-6are defined as wild-typealleles; TAC1-5
and -7are defined as hyperactive alleles. ‘‘a’’ and ‘‘a’’designate MTL types. Position of markers 109, B, D, and E are indicated in Figure 8.
2154 A. Coste et al.
as well (data not shown). This observation is not in
agreement with the findings of Goldman et al. (2004),
which indicated that recombination occurring within
the MTL locus of azole-resistant clinical isolates leads to
homozygosity for the a/agenes but not to other genes
of the MTL locus.
The experiments undertaken with strains DSY3157-2
and DSY3301-4 also revealed that chromosome 5 can
become trisomic prior to the loss of one chromosome 5
copy. These results are partially consistent with those
presented by Wuet al. (2005), who showed that MTL
homozygosity can occur by loss of one copy of chromo-
some 5 followed by duplication of the remaining copy.
Our work suggests that several mechanisms contribute
to LOH, which is consistent with the recognized elas-
ticity of the C. albicans genome (Iwaguchi et al. 2001; X.
Chen et al. 2004; Iwaguchi et al. 2004). Although the
number of isolates investigated here was limited, LOH
at chromosome 5 was obtained by mitotic recombina-
tion in the investigated clinical isolates, whereas it was
obtained by chromosome 5 loss and duplications in
laboratory conditions. This difference might reflect a
fitness cost for such events under the conditions en-
countered in the host. Therefore chromosome 5 mitotic
recombinations rather might be selected in vivo. Addi-
tional azole-resistant clinical isolates should be investi-
gated to address this question.
Association among azole resistance, TAC1, and the
mating-type locus: The short distance between TAC1
and MTL has interesting consequences. Under selective
drug pressure, codominance of TAC1 favors LOH at
this locus and indirectly contributes to the appearance
of MTL homozygous yeast strains. Importantly, our
work with different TAC1 alleles in different strain back-
grounds that were heterozygous or homozygous for
MTL did not detect any contribution of MTL in drug
resistance. Nonetheless, selective pressure for LOH at
the TAC1 locus appears to facilitate the exchange of
genetic material within a yeast population. For example,
mating-competent strains that emerge from LOH in this
region have the capacity to mate with cells of the op-
posite mating type. Even though mating in a C. albicans
population under in vivo conditions has been reported,
evidence for genetic rearrangement and exchange after
mating is still sparse. However, the link between TAC1
and mating-type locus enables us to test the effect of
drug pressure on the ability to propagate a drug resis-
tance genotype.
Genetic transfer of drug resistance in bacteria is a
common feature; however, genetic transfer in fungi and
especially in C. albicans has not yet been reported.
Animal models will be used in future studies to test this
hypothesis. Interestingly, in some clinical isolates, azole
resistance mediated by upregulation of CDR1 and CDR2
is not linked to homozygosity at the mating-type locus
(D. Sanglard, unpublished results). The TAC1 alleles
of these isolates are probably dominant and may carry
mutation(s) different from the N977D mutation de-
scribed here. A catalog of existing mutations in TAC1
alleles in the population of azole-resistant isolates is
necessary to identify and characterize other mutations
responsible for Tac1p hyperactivity as well as their dis-
tribution in the population of clinical strains. First, this
catalog will help to establish critical domains necessary
for TAC1 hyperactivity and, second, it will assist the
design of molecular diagnostic tools for the detection of
such alleles.
This work was supported by a grant (no. 3200B0-100747/1) from the
Swiss Research Foundation to D.S. Sequence data for C. albicans were
obtained from the Stanford Genome Technology Center website at
http://www-sequence.stanford.edu/group/candida. Sequencing of
C. albicans was accomplished with the support of the National Institute
of Dental Research and the Burroughs Wellcome Fund. Joachim
Morschha
¨user was supported by the European Community project
QLK2-CT-2001-02377. Anna Selmecki was supported by an Integrative
Fellowship from the Center for Microbial and Plant Genomics,
University of Minnesota. Anja Forche and Judith Berman were
supported by grant AI62427 from the National Institutes of Health.
LITERATURE CITED
Anderson, J. B., C. Sirjusingh,A.B.Parsons,C.Boone,C.Wickens
et al., 2003 Mode of selection and experimental evolution of an-
tifungal drug resistance in Saccharomyces cerevisiae. Genetics 163:
1287–1298.
Carvajal, E., H. B. van den Hazel,A.Cybularz-Kolaczkowska,
E. Balzi and A. Goffeau, 1997 Molecular and phenotypic
characterization of yeast PDR1 mutants that show hyperactive
transcription of various ABC multidrug transporter genes. Mol.
Gen. Genet. 256: 406–415.
Chambers, S. P., S. E. Prior,D.A.Barstow and N. P. Minton,
1988 The pMTL nic- cloning vectors. I. Improved pUC poly-
linker regions to facilitate the use of sonicated DNA for nucleo-
tide sequencing. Gene 68: 139–149.
Chen, C. G., Y. L. Yang,H.I.Shih,C.L.Suand H. J. Lo, 2004 CaNdt80
is involved in drug resistance in Candida albicans by regulating
CDR1. Antimicrob. Agents Chemother. 48: 4505–4512.
Chen, X., B. B. Magee,D.Dawson,P.T.Magee and C. A. Kumamoto,
2004 Chromosome 1 trisomy compromises the virulence of
Candida albicans. Mol. Microbiol. 51: 551–565.
Coste, A. T., M. Karababa,F.Ischer,J.Bille and D. Sanglard,
2004 TAC1, transcriptional activator of CDR genes, is a new
transcription factor involved in the regulation of Candida albicans
ABC transporters CDR1 and CDR2. Eukaryot. Cell 3: 1639–
1652.
DeDeken, X., and M. Raymond, 2004 Constitutive activation of
the PDR16 promoter in a Candida albicans azole-resistant clinical
isolate overexpressing CDR1 and CDR2. Antimicrob. Agents
Chemother. 48: 2700–2703.
Delahodde, A., T. Delaveau and C. Jacq, 1995 Positive autoregu-
lation of the yeast transcription factor Pdr3p, which is involved in
control of drug resistance. Mol. Cell. Biol. 15: 4043–4051.
de Micheli, M., J. Bille,C.Schueller and D. Sanglard, 2002 A
common drug-responsive element mediates the upregulation of
the Candida albicans ABC transporters CDR1 and CDR2, two genes
involved in antifungal drug resistance. Mol. Microbiol. 43: 1197–
1214.
Dodgson, A. R., K. J. Dodgson,C.Pujol,M.A.Pfaller and D. R.
Soll, 2004 Clade-specific flucytosine resistance is due to a
single nucleotide change in the FUR1 gene of Candida albicans.
Antimicrob. Agents Chemother. 48: 2223–2227.
Fonzi, W. A., and M. Y. Irwin, 1993 Isogenic strain construction and
gene mapping in Candida albicans. Genetics 134: 717–728.
Forche, A., G. May and P. T. Magee, 2005 Demonstration of loss of
heterozygosity by single-nucleotide polymorphism microarray
Azole Resistance by TAC1 Mutation 2155
analysis and alterations in strain morphology in Candida albicans
strains during infection. Eukaryot. Cell 4: 156–165.
Gao, C., L. Wang,E.Milgrom and W. C. Shen, 2004 On the mech-
anism of constitutive Pdr1 activator-mediated PDR5 transcription
in Saccharomyces cerevisiae: evidence for enhanced recruitment of
coactivators and altered nucleosome structures. J. Biol. Chem.
279: 42677–42686.
Gaur, N. A., N. Puri,N.Karnani,G.Mukhopadhyay,S.K.Goswami
et al., 2004 Identification of a negative regulatory element
which regulates basal transcription of a multidrug resistance
gene CDR1 of Candida albicans. FEMS Yeast Res. 4: 389–399.
Goldman, G. H., M. E. da Silva Ferreira,E.dos Reis Marques,
M. Savoldi,D.Perlin et al., 2004 Evaluation of fluconazole re-
sistance mechanisms in Candida albicans clinical isolates from
HIV-infected patients in Brazil. Diagn. Microbiol. Infect. Dis.
50: 25–32.
Gorner, W., E. Durchschlag,M.T.Martinez-Pastor,F.Estruch,
G. Ammerer et al., 1998 Nuclear localization of the C2H2 zinc
finger protein Msn2p is regulated by stress and protein kinase
A activity. Genes Dev. 12: 586–597.
Harry, J. B., B. G. Oliver,J.L.Song,P.M.Silver,J.T.Little et al.,
2005 Drug-induced regulation of the MDR1 promoter in Can-
dida albicans. Antimicrob. Agents Chemother. 49: 2785–2792.
Iwaguchi, S. I., M. Sato,B.B.Magee,P.T.Magee,K.Makimura
et al., 2001 Extensive chromosome translocation in a clinical
isolate showing the distinctive carbohydrate assimilation profile
from a candidiasis patient. Yeast 18: 1035–1046.
Iwaguchi, S., M. Suzuki,N.Sakai,Y.Nakagawa,P.T.Magee et al.,
2004 Chromosome translocation induced by the insertion of
the URA blaster into the major repeat sequence (MRS) in Can-
dida albicans. Yeast 21: 619–634.
Karababa, M., A. T. Coste,B.Rognon,J.Bille and D. Sanglard,
2004 Comparison of gene expression profiles of Candida albi-
cans azole-resistant clinical isolates and laboratory strains ex-
posed to drugs inducing multidrug transporters. Antimicrob.
Agents Chemother. 48: 3064–3079.
Karnani, N., N. A. Gaur,S.Jha,N.Puri,S.Krishnamurthy et al.,
2004 SRE1 and SRE2 are two specific steroid-responsive mod-
ules of Candida drug resistance gene 1 (CDR1) promoter. Yeast
21: 219–239.
Katzmann, D. J., P. E. Burnett,J.Golin,Y.Mahe and W. S. Moye-
Rowley, 1994 Transcriptional control of the yeast PDR5 gene
by the PDR3 gene product. Mol. Cell. Biol. 14: 4653–4661.
Kren, A., Y. M. Mamnun,B.E.Bauer,C.Schuller,H.Wolfger
et al., 2003 War1p, a novel transcription factor controlling weak
acid stress response in yeast. Mol. Cell. Biol. 23: 1775–1785.
Mamnun, Y. M., R. Pandjaitan,Y.Mahe,A.Delahodde and
K. Kuchler, 2002 The yeast zinc finger regulators Pdr1p and
Pdr3p control pleiotropic drug resistance (PDR) as homo-
and heterodimers in vivo. Mol. Microbiol. 46: 1429–1440.
Marchetti, O., J. M. Entenza,D.Sanglard,J.Bille,M.P.Glauser
et al., 2000 Fluconazole plus cyclosporine: a fungicidal combi-
nation effective against experimental endocarditis due to Can-
dida albicans. Antimicrob. Agents Chemother. 44: 2932–2938.
Marichal, P., L. Koymans,S.Willemsens,D.Bellens,P.Verhasselt
et al., 1999 Contribution of mutations in the cytochrome P450
14a-demethylase (Erg11p, Cyp51p) to azole resistance in Candida
albicans. Microbiology 145: 2701–2713.
Marr, K. A., T. C. White,J.A.van Burik and R. A. Bowden,
1997 Development of fluconazole resistance in Candida albicans
causing disseminated infection in a patient undergoing marrow
transplantation. Clin. Infect Dis. 25: 908–910.
Marr, K. A., C. N. Lyons,T.R.Rustad,R.A.Bowden and T. C.
White, 1998 Rapid, transient fluconazole resistance in Candida
albicans is associated with increased mRNA levels of CDR. Antimi-
crob. Agents Chemother. 42: 2584–2589.
Marr, K. A., C. N. Lyons,K.Ha,T.R.Rustad and T. C. White,
2001 Inducible azole resistance associated with a heteroge-
neous phenotype in Candida albicans. Antimicrob. Agents Che-
mother. 45: 52–59.
Nourani, A., D. Papajova,A.Delahodde,C.Jacq and J. Subik,
1997 Clustered amino acid substitutions in the yeast transcrip-
tion regulator Pdr3p increase pleiotropic drug resistance and
identify a new central regulatory domain. Mol. Gen. Genet. 256:
397–405.
Park, S., R. Kelly,J.N.Kahn,J.Robles,M.J.Hsu et al.,
2005 Specific substitutions in the echinocandin target Fks1p ac-
count for reduced susceptibility of rare laboratory and clinical
Candida sp. isolates. Antimicrob. Agents Chemother. 49: 3264–
3273.
Perea, S., J. L. Lopez-Ribot,W.R.Kirkpatrick,R.K.McAtee,R.A.
Santillan et al., 2001 Prevalence of molecular mechanisms of
resistance to azole antifungal agents in Candida albicans strains
displaying high-level fluconazole resistance isolated from human
immunodeficiency virus-infected patients. Antimicrob. Agents
Chemother. 45: 2676–2684.
Puri, N., S. Krishnamurthy,S.Habib,S.E.Hasnain,S.K.Goswami
et al., 1999 CDR1, a multidrug resistance gene from Candida al-
bicans, contains multiple regulatory domains in its promoter and
the distal AP-1 element mediates its induction by miconazole.
FEMS Microbiol. Lett. 180: 213–219.
Reid, R. J., E. A. Kauh and M. A. Bjornsti, 1997 Camptothecin sen-
sitivity is mediated by the pleiotropic drug resistance network in
yeast. J. Biol. Chem. 272: 12091–12099.
Reuss, O., A. Vik,R.Kolter and J. Morschhauser, 2004 The SAT1
flipper, an optimized tool for gene disruption in Candida albicans.
Gene 341: 119–127.
Rottensteiner, H., A. J. Kal,B.Hamilton,H.Ruis and H. F. Tabak,
1997 A heterodimer of the Zn2Cys6 transcription factors Pip2p
and Oaf1p controls induction of genes encoding peroxisomal
proteins in Saccharomyces cerevisiae. Eur. J. Biochem. 247: 776–783.
Rustad, T. R., D. A. Stevens,M.A.Pfaller and T. C. White,
2002 Homozygosity at the Candida albicans MTL locus associ-
ated with azole resistance. Microbiology 148: 1061–1072.
Sadowski, I., C. Costa and R. Dhanawansa, 1996 Phosphorylation
of Ga14p at a single C-terminal residue is necessary for galactose-
inducible transcription. Mol. Cell. Biol. 16: 4879–4887.
Sanglard, D., K. Kuchler,F.Ischer,J.L.Pagani,M.Monod et al.,
1995 Mechanisms of resistance to azole antifungal agents in
Candida albicans isolates from AIDS patients involve specific mul-
tidrug transporters. Antimicrob. Agents Chemother. 39: 2378–
2386.
Sanglard, D., F. Ischer,L.Koymans and J. Bille, 1998 Amino acid
substitutions in the cytochrome P450 lanosterol 14a-demethylase
(CYP51A1) from azole-resistant Candida albicans clinical isolates
contributing to the resistance to azole antifungal agents. Antimi-
crob. Agents Chemother. 42: 241–253.
Santos, M., and I. F. de Larrinoa, 2005 Functional characteriza-
tion of the Candida albicans CRZ1 gene encoding a calcineurin-
regulated transcription factor. Curr. Genet. 48: 88–100.
Selmecki, A., S. Bergmann and J. Berman, 2005 Comparative
genome hybridization reveals widespread aneuploidy in Candida
albicans laboratory strains. Mol. Microbiol. 55: 1553–1565.
Strauss, A., S. Michel and J. Morschhauser, 2001 Analysis of
phase-specific gene expression at the single-cell level in the
white-opaque switching system of Candida albicans. J. Bacteriol.
183: 3761–3769.
Tuttle, M. S., D. Radisky,L.Liand J. Kaplan, 2003 A dominant
allele of PDR1 alters transition metal resistance in yeast. J. Biol.
Chem. 278: 1273–1280.
Wendler, F., H. Bergler,K.Prutej,H.Jungwirth,G.Zisser et al.,
1997 Diazaborine resistance in the yeast Saccharomyces cerevisiae
reveals a link between YAP1 and the pleiotropic drug resistance
genes PDR1 and PDR3. J. Biol. Chem. 272: 27091–27098.
White, T. C., 1997 The presence of an R467K amino acid substitu-
tion and loss of allelic variation correlate with an azole-resistant
lanosterol 14-alpha-demethylase in Candida albicans. Antimicrob.
Agents Chemother. 41: 1488–1494.
Wu, W., C. Pujol,S.R.Lockhart and D. R. Soll, 2005 Chro-
mosome loss followed by duplication is the major mechanism
of mating-type locus homozygosis in Candida albicans. Genetics
169: 1311–1327.
Communicating editor: A. P. Mitchell
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