Cph1p negatively regulates MDR1 involved in drug resistance in
Candida albicans
Hsiu-Jung Lo a,b, Kuo-Yun Tseng a,1, Yeong-Yi Kao c, Ming-Yang Tsao a, Han-Lun
Lo c, Yun-Liang Yang c,d,*
a
National Institute of Infectious Diseases and Vaccinology, National Health
Research Institutes, Miaoli, Taiwan
b
School of Dentistry, China Medical University, Taichung, Taiwan
c
Department of Biological Science and Technology, National Chiao Tung
University, Hsinchu, Taiwan
d
Institute of Molecular Medicine and Bioengineering, National Chiao Tung
University, Hsinchu, Taiwan
ARTICLE INFO
Article history:
Received 4 July 2014
Accepted 22 January 2015
Keywords:
Candida albicans
Drug resistance
Efflux pump
Virulence factor
Regulation
* Corresponding author. Present address: Department of Biological Science and
Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC. Tel.: +886 3
571 2121 x56920; fax: +886 3 572 9288.
E-mail address: yyang@mail.nctu.edu.tw (Y.L. Yang).
1
Present address: Institute of Cellular and Systems Medicine, National Health
Research Institutes, Miaoli, Taiwan.
ABSTRACT
The cph1/cph1 efg1/efg1 double mutant in Candida albicans is defective in
filamentous growth and is avirulent in a mouse model. We previously reported that
Efg1p but not Cph1p is involved in drug resistance by negatively regulating ERG3
in C. albicans. In the current study, we have found that overexpression of CPH1 in
Saccharomyces cerevisiae increases susceptibility to the antifungal drug
fluconazole. Furthermore, in C. albicans, null mutation of CPH1 increased the
expression of MDR1 as well as decreased susceptibility to fluconazole and
voriconazole but not to amphotericin B. These findings indicate that although Efg1p
and Cph1p may have the same effects on virulence, they have opposite effects on
drug resistance in C. albicans.
1. Introduction
In the past two decades, yeast infections have increased significantly. Among
them, Candida albicans is the most frequently isolated fungal pathogen in humans
and causes morbidity in immunocompromised hosts [1,2]. Coincident with the
increased use of antifungal agents, the incidence of drug resistance has increased
[3,4]. However, the mechanisms of drug resistance are not well characterised.
Overexpression of efflux pumps and the drug target are major mechanisms
contributing to drug resistance in clinical isolates of C. albicans. At least two active
efflux pumps are involved in drug resistance: CDR1, belonging to the ATP-binding
cassette (ABC) family; and MDR1, belonging to the major facilitator superfamily
(MFS). The CDR1 gene was identified by complementing the hypersensitive
phenotype to azoles, cycloheximide and chloramphenicol in the PDR5 null mutant
in Saccharomyces cerevisiae [5]. Overexpression of CDR1 in S. cerevisiae causes
resistance to azole-based antifungal drugs. Recently, two transcription factors,
CaNdt80p and CaTac1p, have been identified as positive regulators of CDR1.
Consistently, mutations in NDT80 and TAC1 increase the susceptibility to more
than one azole type drug [6–9].
The MDR1 gene was primarily identified by its ability to confer both benomyl and
methotrexate resistance when transformed into S. cerevisiae [10]. Its expression is
induced by drugs such as benomyl, methotrexate, 4-nitroquinoline N-oxide (4NQO), o-phenanthroline and sulfometuron methyl [11]. Overexpression of MDR1
contributes to fluconazole resistance in C. albicans [12]. Mrr1p has been reported
as a positive regulator [13], and both Cap1p and Rep1p are negative regulators of
MDR1 [14,15].
The predominant target of the azole-based drugs is lanosterol demethylase, the
product of ERG11. ERG3, a gene upstream of ERG11, that encodes the sterol
5,6-desaturase, produces a toxic product, 14-methylergosta-8,24-dien-3,6-diol,
following azole treatment [16]. Furthermore, it has been reported that null mutation
of ERG3 causes fluconazole resistance [17].
STE12 in S. cerevisiae, a transcription factor that is the target of the pheromone
response mitogen-activated protein kinase cascade, is involved in mating and
filamentous growth. CPH1 in C. albicans was identified by complementing both
defects in mating and filamentous growth of ste12 mutants [18]. Cph1p and Ste12p
have 74% identical residues among the region containing DNA-binding domains.
Furthermore, they use identical residues for pheromone-induced transcription
activation. Thus, like Ste12p in S. cerevisiae, Cph1p in C. albicans also binds to
pheromone response elements (PREs) and is involved in mating and filamentous
growth [18,19]. However, a null mutation of CPH1 does not affect filamentous
growth in medium containing serum or the virulence in a mouse systemic infection
model [20] owing to other Cph1p-independent pathways. It has been reported that
the cph1/cph1 efg1/efg1 double mutant fails to form filaments in vitro and does not
cause lethal infections in a mouse model [20,21]. The observation that Efg1p, but
not Cph1p, regulates ERG3 and that the null mutations of EFG1 increase the
susceptibility of the cells to antifungal agents demonstrates that a transcription
factor can be responsible both for drug resistance and virulence [22]. Later, more
transcription factors, such as Ndt80p, were reported to be involved in different
pathways by regulating different sets of targets [9,23].
Recently, a 35-bp MDR1 promoter element (MDRE) containing the Mcm1p-binding
site has been identified. Interestingly, a sequence similar to the PRE overlaps the
boundary of the MDRE [24]. In the current study, we found that Cph1p negatively
regulates the expression of MDR1 involved in drug resistance in C. albicans.
2. Materials and methods
2.1. Strains and media
The S. cerevisiae strain used in this study was Sigma 10560-2B MATa his3::hisG
leu2::hisG ura3-52 [6]. A high-copy-number vector pRS426 alone or HLB126
(pRS426-CPH1) was transformed into S. cerevisiae 10560-2B to investigate the
effects on drug susceptibility of overexpressing CPH1. The C. albicans strains used
in this study were SC5314 (CPH1/CPH1), the wild-type strain [25]; JKC19, the
cph1/cph1 null mutant strain, ura3::imm434/ ura3::imm434
cph1::hisG/cph1::hisG -URA3-hisG [18]; and YLO323 (cph1/cph1::CPH1), the
cph1/cph1 null mutant with a copy of the wild-type CPH1 allele rescued strain,
ura3::imm434/ ura3::imm434 cph1::hisG/cph1::hisG::CPH1-URA3. YLO323
was constructed in this study because we failed to detect the CPH1 mRNA of
JKC28 strain (ura3::imm434/ ura3::imm434 cph1::hisG/cph1::hisG::CPH1URA3). Yeast–peptone–dextrose (YPD) (1% yeast extract, 2% peptone and 2%
dextrose) and synthetic dextrose (SD) (0.67% yeast nitrogen base without amino
acid and 2% dextrose) were prepared as described previously [26]. The
compounds added to media were from Difco (Henry St, Detroit, MI, USA) unless
otherwise stated.
2.2. Antifungal susceptibility testing
Etest was used to determine the susceptibility to antifungal agents as described
previously [22]. Homogenised isolated colonies grown on SD agar medium
overnight were transferred in 0.85% NaCl to achieve a density of 5  106 cells/mL.
A sterile swab was dipped into inoculum suspension and was used to swab the
entire agar surface of SD agar medium evenly. Fluconazole Etest strips (0.016–256
mg/L; AB BIODISK, Solna, Sweden) were applied on the plate when the excess
moisture was absorbed completely. The agar dilution method was also used to
determine the susceptibility to antifungal agents for C. albicans. First, cells were
diluted to an optical density at 600 nm (OD600) of 2 (ca. 2  107 CFU/mL) and were
spotted (ca. 0.5 L per spot) onto different drug plates with 10-fold serial dilutions.
Stock solutions of antifungal drugs were prepared by dissolving drugs in dimethyl
sulfoxide (DMSO). The final concentrations of fluconazole and voriconazole were
25 mg/L and 2 mg/L, respectively, in medium containing 1% DMSO. The broth
microdilution method was performed according to the guidelines published by the
Clinical and Laboratory Standards Institute (CLSI) [27]. The highest concentrations
of amphotericin B, fluconazole and voriconazole were 0.5, 16 and 1 mg/L,
respectively, and a two-fold serial dilution was applied. The final growth of each
isolate was measured using a Biotrak II Visible Plate Reader (Amersham
Biosciences, Piscataway, NJ, USA) after 2 days of incubation at 30 C. Growth of
cells in the absence of drug was defined as 100 and the relative growth of cells in
the presence of different concentrations of drug was normalised accordingly.
2.3. RNA isolation
Candida albicans cells were harvested after being grown in 20 mL of YPD liquid
medium in the absence or presence of 100 g/mL 4-NQO at 30 C for 1 h (OD600 =
0.7~1.0). Total RNA was isolated by the method of acid hot phenol followed by
RQ1 RNase-Free DNase (Promega, Madison, WI, USA) treatment to digest the
possible contaminating DNA.
2.4. Northern blot analysis
RNA samples (20 g each) were separated by electrophoresis on 1%
formaldehyde-agarose gel and were then transferred to Nylon membranes with a
positive charge (Roche, Indianapolis, IN, USA) in 20 SSC (3 M NaCl, 0.3 M
sodium citrate). The membrane was hybridised to a digoxigenin (DIG)-labelled
probe prepared using a PCR DIG Probe Synthesis Kit (Roche) according to the
manufacturer’s instructions. The level of ACT1 mRNA was used as the loading
control.
2.5. Quantitative analysis of mRNA level by real-time PCR
Real-time PCR was performed in a Rotor-Gene 3000 instrument (Corbett
Research, Sydney, Australia) with a TITANIUM Taq PCR Kit (BD Clontech,
Mountain View, CA,USA) and SYBR Green I Nucleic Acid Stain (Cambrex,
Rockland, ME, USA) to determine the level of mRNA as described previously [8].
The sample set-up was processed automatically using a CAS-1200TM (Corbett
Research). Real-time PCR was performed according to the manufacturer’s
instructions and the expression of ACT1 (for miconazole treatment) or CaSNF3 (for
4-NQO treatment) in each strain was used as the loading control. The relative
quantitation was based on two standard curves for comparison and the results
were given as a ratio [28]. The level of mRNA isolated from wild-type cells in the
absence of drug was defined as 1. The relative level of mRNA isolated from
different strains in the absence or presence of drugs was normalised accordingly.
3. Results and discussion
3.1. Overexpression of CPH1 increased susceptibility to fluconazole in
Saccharomyces cerevisiae
We determined whether Cph1p, like Efg1p, is involved in drug resistance as well as
virulence. First, the involvement of CPH1 in fluconazole susceptibility in S.
cerevisiae was investigated. Interestingly, cells overexpressing CPH1 were more
susceptible to fluconazole than control cells (24 mg/L vs. 64 mg/L) (Fig. 1),
suggesting that Cph1p may be a negative regulator of drug resistance.
3.2. The null mutation of CPH1 increased the expression of MDR1 in Candida
albicans
To elucidate the regulatory network of Cph1p in C. albicans, we determined
whether Cph1p regulates the expression of known genes involved in drug
resistance. The results are summarised in Table 1. Expression of MDR1 in wildtype CPH1/CPH1 cells was induced significantly by 4-NQO (1 vs. 97.9), which is
consistent with a previous report [29]. In the presence of 4-NQO, the null mutation
of CPH1 further increased the expression of MDR1 by ca. 2.8-fold (97.9 vs. 269.8).
The level of MDR1 mRNA in the cph1/cph1 mutant cells containing a wild-type
copy of CPH1 was reduced to that of the CPH1/CPH1 wild-type cells (104.8 vs.
97.9), suggesting that the increased level of MDR1 mRNA results from the null
mutation of CPH1. In the absence of 4-NQO, the level of MDR1 mRNA in the
cph1/cph1 mutant cells was similar to that in the CPH1/CPH1 wild-type cells,
suggesting that Cph1p is not involved in the basal expression of MDR1.
To further assess whether Cph1p regulates the expression of MDR1, levels of
MDR1 mRNA of different C. albicans strains were determined by northern blot
assay. In the absence of 4-NQO, the level of MDR1 mRNA was low and was barely
detectable (Fig. 2, lane 1). As expected, expression of MDR1 in the CPH1/CPH1
wild-type cells was highly induced by the addition of 4-NQO (Fig. 2, comparing
lanes 1 and 2). Expression of MDR1 was further induced by the null mutation of
CPH1 (Fig. 2, comparing lanes 2 and 4), suggesting that Cph1p is a repressor of
MDR1. That the level of MDR1 mRNA in the cph1/cph1 mutant cells containing a
wild-type copy of CPH1 was reduced to that of the wild-type CPH1/CPH1 cells
demonstrated that the increased level of MDR1 mRNA in the cph1/cph1 cells is
indeed caused by the null mutation of CPH1.
In general, inhibition of one efflux pump may result in overexpression of another
different efflux pump as a compensatory mechanism. Thus, we also determined
whether the null mutation of CPH1 affects the expression of CDR1 in the presence
of 4-NQO. First, unlike MDR1, the expression of CDR1 was not induced
significantly by 4-NQO. Expression of CDR1 was similar both in the CPH1/CPH1
wild-type cells and in the cph1/cph1 null mutant cells in the absence (1.0 vs. 1.0)
and presence (1. 8 vs. 2.2) of 4-NQO.
Expression of MDR1 is barely detectable in the presence of miconazole, consistent
with previous reports that the expression of MDR1 is not induced by miconazole. In
contrast, expression of CDR1, CDR2, ERG3 and ERG11 were induced by
miconazole by ca. 3.2-, 6.9-, 3.2- and 3.4-fold, respectively. These findings indicate
that Cph1p acts as a transcription repressor of MDR1 specifically in C. albicans
even though the expression of CDR1 (0.8; 3.2 vs. 2.4), CDR2 (0.7; 6.9 vs. 4.9),
ERG3 (0.9; 3.2 vs. 2.9) and ERG11 (0.7; 3.2 vs. 2.5) in the presence of miconazole
was slightly reduced by the null mutation of CPH1.
Recently, Mcm1p has been identified as a positive regulator of MDR1. The
promoter of MDR1 from SC5314, the wild-type laboratory strain, does not contain
the PRE sequence (TGAAACA), a conserved binding site of Ste12p. Interestingly,
sequence TGACACA, similar to a PRE, was identified near the binding site of
Mcm1p [24]. Furthermore, in some clinical isolates, such as B792, a PRE sequence
can be found at the same location [10]. Here we proposed a working model for how
Cph1p regulates the transcription of MDR1. Cph1p alone or in the Cph1p–Mcm1p
complex binds the MDR1 promoter and represses the expression of MDR1. Under
inducing conditions, Cph1p will be released from the promoter of MDR1 either by
modification of Cph1p or the involvement of another regulator. Alternatively, the
effect of the complex of Cph1p–Mcm1p will be reversed by the involvement of
another regulator. Whether Cph1p regulates the expression of MDR1 through
directly binding to its promoter is under investigation.
3.3. Null mutation in CPH1 decreased susceptibility to antifungal drugs in Candida
albicans
Since the null mutation of CPH1 increased the expression of MDR1, it is a
possibility that cph1/cph1 mutant cells are less susceptible to antifungal drugs than
wild-type cells. First, the agar dilution method was used to determine the
susceptibility to antifungal drugs of different strains as described previously [6].
Cells of different strains grew well on medium containing DMSO in the absence of
drug (Fig. 3, left panel), suggesting that the null mutation of CPH1 does not affect
the growth rate of C. albicans as previously reported [14]. Interestingly, more
cph1/cph1 mutant cells grew on medium containing fluconazole (Fig. 3, middle
panel) and voriconazole (Fig. 3, right panel) than the wild-type strain. The drug
susceptibilities of the cph1/cph1 mutant cells were restored by a wild-type copy of
CPH1 (cph1/cph1::CPH1), suggesting that the decreased drug susceptibilities in
the cph1/cph1 cells results from the null mutation in CPH1. Thus, null mutations on
CPH1 decreased the susceptibilities to both fluconazole and voriconazole.
To confirm the effect of Cph1p on drug susceptibility, we also quantified the drug
susceptibility of different strains by a broth microdilution method. Again, the
cph1/cph1 mutant cells were more resistant to fluconazole (Fig. 4B) and
voriconazole (Fig. 4C) than the CPH1/CPH1 wild-type cells and the cph1/cph1 with
a copy of CPH1 wild-type allele (cph1/cph1::CPH1) cells, consistent with the results
of the agar dilution assay. To investigate whether Cph1p is also involved in drug
resistance of a different type of drug, susceptibility to amphotericin B was also
determined. In contrast, the null mutation of CPH1 had no effect on susceptibility to
amphotericin B (Fig. 4A).
These results showed that Cph1p negatively regulates the expression of MDR1 but
not other tested genes known to be involved in drug resistance, including CDR1,
CDR2, ERG3 and ERG11. Interestingly, mutations in CPH1 decreased
susceptibility to other azole drugs in addition to fluconazole, although MDR1 was
reported to be the efflux pump contributing to fluconazole resistance specifically [4].
In the absence of stimulus, cph1/cph1 mutant cells did not lose their fitness in vitro
(Fig. 3A) or in vivo [20], since the null mutation of CPH1 does not affect the basal
expression of MDR1. The observation that cph1/cph1 mutant cells were more
resistant to azole drugs may result from higher expression of MDR1 and/or other
novel genes. Hence, a genome-wide approach to identify additional targets of
Cph1p involved in azole drug resistance is needed. Furthermore, it would be
interesting to investigate whether there are residues of Cph1p specifically
responsible for regulating MDR1.
Genes regulating both drug resistance and virulence have been suggested
previously. Recently, we reported that Efg1p is involved in drug resistance by
negatively regulating ERG3 [22]. A null mutation of EFG1 increased the
susceptibility to drugs. In current study, we showed that Cph1p, a protein involved
in virulence, is also involved in negatively regulating the expression of MDR1,
which is responsible for drug resistance in C. albicans. And in this case, a null
mutation of CPH1 decreases the susceptibility to drugs. These findings indicate
that although Efg1p and Cph1p can have the same effects on virulence, they
regulate different targets resulting in opposite effects on drug resistance in C.
albicans. It is important to investigate how a transcription factor regulates both
virulence and drug resistance. There are multiple interconnected pathways in C.
albicans responsible for different sets of environmental conditions [30]. Hence, how
Cap1p, Cph1p, Mrr1p and Rep1p co-regulate drug resistance pathways,
particularly through MDR1, in C. albicans requires further investigation.
Acknowledgments: The authors would like to thank Ms Y.H. Lin, C.L. Su and C.G.
Chen (National Health Research Institutes, Miaoli, Taiwan) for their technical
assistance. The authors would also like to thank Dr G.R. Fink for Saccharomyces
cerevisiae strain 10560-2B.
Funding: This work was supported in part by grants from the National Science
Council [102-2320-B-009-001 to Y-LY and 102-2311-B-400-001 to H-JL] and the
National Health Research Institutes [IV-103-PP-08 to H-JL].
Competing interests: None declared.
Ethical approval: Not required.
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Fig. 1. Susceptibility to fluconazole of Saccharomyces cerevisiae strain 10560-2B
containing (A) pRS426 vector alone or (B) HLB126 overexpressing CPH1 as
determined by Etest. The results were photographed after 2 days of growth on
synthetic dextrose agar medium at 30 C.
Fig. 2. Levels of MDR1 mRNA of different Candida albicans strains. The upper
panels represent MDR1 mRNA and the lower panels represent ACT1 mRNA used
as the loading control. Cells were grown in the absence (lanes 1, 3 and 5) or
presence (lanes 2, 4 and 6) of 4-nitroquinoline N-oxide (4-NQO). Lanes 1 and 2,
CPH1/CPH1 wild-type cells (SC5314); lanes 3 and 4, chp1/cph1 mutant cells
(JKC19); and lanes 5 and 6, cph1/cph1::CPH1 cells (YLO323).
Fig. 3. Susceptibilities to antifungal drugs of three Candida albicans strains
[CPH1/CPH1 wild-type cells (SC5314), chp1/cph1 mutant cells (JKC19) and
cph1/cph1::CPH1 cells (YLO323)] as determined by the agar dilution assay. (Left
panel) Yeast–peptone–dextrose (YPD) medium containing an equal amount of
dimethyl sulfoxide (DMSO) in the absence of drug was used as the growth control.
Fluconazole (middle panel) and voriconazole (right panel) were prepared to final
concentrations of 25 mg/L and 2 mg/L, respectively, in DMSO. The results were
photographed after 2 days of growth at 30 C.
Fig. 4. Susceptibilities of three Candida albicans strains [CPH1/CPH1 wild-type
cells (SC5314), chp1/cph1 mutant cells (JKC19) and cph1/cph1::CPH1 cells
(YLO323)] to (A) amphotericin B, (B) fluconazole and (C) voriconazole as
determined by the broth microdilution assay. Growth of cells in the absence of drug
was defined as 100.