OUP user menu

A yeast cell-based system for screening Candida glabrata multidrug resistance reversal agents and selection of loss-of-function pdr1 mutants

Eduard Goffa, Alexandra Bialkova, Monika Batova, Vladimira Dzugasova, Julius Subik
DOI: http://dx.doi.org/10.1111/j.1567-1364.2010.00702.x 155-159 First published online: 1 March 2011


In the pathogenic yeast Candida glabrata, multidrug resistance is associated with the overexpression of drug efflux pumps caused by gain-of-function mutations in the CgPDR1 gene. CgPdr1p transcription factor, which activates the expression of several drug efflux transporter genes, is considered to be a promising target for compounds sensitizing the multidrug-resistant yeast cells. Here, we describe a cell-based screening system for detecting the inhibitory activity of compounds interfering with the CgPdr1p function in a heterologous genetic background of the hypersensitive Saccharomyces cerevisiae mutant strain. The screening is based on the ability to abrogate the growth defect of cells suffering from the galactose-induced and CgPdr1p-driven overexpression of a dominant lethal pma1(D378N) allele placed under the control of the ScPDR5 promoter. The system allows rapid identification of multidrug resistance reversal agents inhibiting the CgPdr1p activity or loss-of-function Cgpdr1 mutations, and is amenable to high-throughput screening on solid or liquid media.

  • CgPDR1
  • CgPdr1p inhibitors
  • Cgpdr1 mutants
  • transcription factor
  • Saccharomyces cerevisiae
  • Candida glabrata


The yeast Candida glabrata is an opportunistic human fungal pathogen. After C. albicans, it is the second most frequent cause of candidemia (Pfaller, 1999; Diekema, 2002) and vaginal tract as well as respiratory tract fungal infections (Sobel & Chaim, 1997; Sojakova, 2004; Tortorano, 2004). Contrary to the evolutionarily closely related Saccharomyces cerevisiae (Dujon, 2004), it possesses a lower susceptibility to azole antifungals (Kaur, 2005) and can aerobically take up cholesterol from serum, thus overcoming the inhibition of ergosterol biosynthesis by azole antifungals (Nakayama, 2007).

The most important mechanism of azole resistance in C. glabrata is associated with drug efflux that is mediated by overproduced plasma membrane transporters (Sanglard, 1999; Berila, 2009; Ferrari, 2009). This yeast species possesses homologues of almost all of the genes involved in multidrug resistance in S. cerevisiae (Dujon, 2004; Bialkova & Subik, 2006; Drobna, 2008). The CgPDR1 gene (Vermitsky & Edlind, 2004; Tsai, 2006) is a single homologue of the ScPDR1 and ScPDR3 genes, which encode the two main multidrug resistance transcriptional activators in S. cerevisiae (Balzi, 1987; Delaveau, 1994). Its expression is enhanced by its own gene product (Tsai, 2006), dysfunctional mitochondria (Sanglard, 1999; Tsai, 2006), anionic phospholipid deficiency (Batova, 2008), ketoconazole (Thakur, 2008) and gain-of-function mutations. Many such mutations in CgPDR1 have proven to be responsible for its overexpression, leading to upregulation of the main drug efflux transporter genes CgCDR1, CgCDR2 and CgSNQ2, and establishment of azole resistance in C. glabrata clinical isolates recovered from different patients worldwide (Tsai, 2006; Berila, 2009; Ferrari, 2009).

There are several strategies to combat multidrug resistance in fungal and cancer cells (Cannon, 2009). Some of them are based on the inhibition of drug efflux transporter activities (Conseil, 2000; Monk, 2005) or suppression of gene expression preventing their biosynthesis (Gottesman, 2002; Sidorova, 2007; Stepanov, 2008). Recently, we have described a screening system for identification of compounds interfering with the function of the ScPdr3p multidrug resistance transcription factor in S. cerevisiae (Kozovska & Subik, 2003; Kozovska, 2004). In this study, we report the development of a heterologous screening system for multidrug resistance reversal agents specifically inhibiting the CgPdr1p function that is essential for the development of multidrug resistance in pathogenic C. glabrata.

Materials and methods

Strains and culture conditions

The S. cerevisiae strains used in this study were as follows: FY1679-28C (MATa ura3-52 trp1-63 leu2-1 his3-200), FY1679-28C/TDEC (MATa ura3-52 trp1-63 leu2-1 his3-200 pdr1∷TRP1 pdr3∷HIS3) (Delaveau, 1994; Carvajal, 1997), EC61 (MATa pdr1-3 pdr3Δ∷HIS3 ura3-52 trp1-Δ63 leu2-Δ1 his3Δ200 GAL2+) and EC62 (MATa pdr1-6 pdr3∷HIS3 ura3-52 trp1-Δ63 leu2-Δ1 his3Δ200 GAL2+) (Carvajal, 1997). Cells were grown on glucose-rich (YPD) medium (2% glucose, 1% yeast extract, 2% Bacto peptone) or on minimal (YNB) medium containing 0.67% yeast nitrogen base without amino acids, 2% glucose or 2% galactose and appropriate nutritional requirements. The media were solidified with 2% Bacto agar. The Escherichia coli XL1-Blue strain was used as the host for transformation, plasmid amplification and preparation. The bacterial strains were grown at 37 °C in Luria–Bertani medium (1% tryptone, 1% NaCl, 0.6% yeast extract, pH 7.5) supplemented with 100 mg L−1 ampicillin for selection of transformants.

Drug susceptibility assay

Drug susceptibility was determined by spot assay. Five microliters of suspension of three independent clones grown overnight at 30 °C were spotted in serial dilutions onto minimal medium supplemented with various concentrations of cycloheximide and fluconazole. Growth was scored after 5 days of incubation at 30 °C. Alternatively, cells grown on minimal medium containing galactose were replica plated onto minimal medium containing galactose supplemented with various concentrations of cycloheximide (0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5 and 2.0 mg L−1).

Recombinant DNA techniques and plasmids

Standard protocols were used for generating recombinant DNAs, restriction enzyme analysis, gel electrophoresis and hybridization as described by Sambrook (1989). Plasmid DNA from E. coli was prepared by the alkaline lysis method. Plasmid DNA from yeast cells was extracted according to Ward (1990). Yeast transformation was carried out using the modified lithium acetate protocol (Nourani, 1997) or by electroporation (Thompson, 1997). Plasmids pYES2-PGAL1-PDR3 (2 μm URA3) and pYCp-PPDR5-pma1(D378N) have been described previously (Kozovska, 2004). Plasmid pYES2 (2 μm URA3) was used as an empty vector to introduce a plasmid-borne URA3 gene into host strains and to clone the CgPDR1 (Cg4672) gain-of-function allele expressed from the PGAL1 promoter. The latter was PCR amplified from a pCgACU-P2F5 plasmid carrying the CgPDR1 gene from the clinical isolate Cg8R (Cg4672) (Tsai, 2006). PCR was carried out with a high-fidelity KOD Hot Star DNA polymerase (Merck, Germany) with primers containing sequences recognized by BamHI and EcoRI (underlined): forward BamHICgPDR1-1 5′-GATATATGGATCCCGTTATTGAGAGAATATGC-3′; reverse EcoRICgPDR1_STOP 5′-GATATATGAATTCTCATTCAGAATCGAAGGG-3′.

DNA sequence analysis

The DNA sequence of the Cgpdr1 allele was determined by an ABI Prism 3100 DNA sequencer (Applied Biosystems, Foster City, CA) using double-stranded plasmid DNA purified using Qiagen Plasmid kits (Qiagen, Hilden, Germany) and a set of synthetic 19mer oligonucleotide primers corresponding to the CgPDR1 coding sequence distributed at intervals of about 300–350 bp. Sequence data were analyzed using programs based on the blast algorithm by computing performed at the NCBI.

Results and discussion

A previously developed screening system for multidrug resistance reversal agents acting at the level of gene expression in S. cerevisiae was based on the restoration of growth on media containing galactose of the ZK11-1 test strain harboring two fusion genes on plasmids: PGAL1-PDR3 and PPDR5-pma1 (Kozovska & Subik, 2003). To screen for inhibitors of CgPdr1p, the C. glabrata multidrug resistance transcriptional activator, the PDR3 gene in the pYES2-PGAL1-PDR3 plasmid, was replaced by the gain-of-function allele of the CgPDR1 gene derived from drug-resistant cells of this human pathogen. This allele was PCR amplified from a plasmid-borne CgPDR1 sequence of Cg4672 (Tsai, 2006), digested with BamHI plus EcoRI and ligated into a truncated pYES2-PGAL1-PDR3 plasmid (Kozovska, 2004) from which the BamHI–EcoRI DNA fragment containing the entire PDR3 gene had been removed. The structure of the resulting pYES2-PGAL1-CgPDR1 plasmid was verified by restriction analysis and the CgPDR1 function was assessed by determination of the sensitivity to cycloheximide and fluconazole of the corresponding S. cerevisiae FY1679-28C/TDEC transformants. As shown in Table 1, the galactose-induced expression of CgPDR1 from the PGAL1 promoter rendered yeast cells resistant to cycloheximide [minimum inhibitory concentrations (MIC)=1.5 μg mL−1] and fluconazole (MIC=8 μg mL−1), thus indicating their functional expression in the heterologous genetic background. After growth for 12 generations under nonselective conditions in a glucose-rich medium, 26% of the cells retained the plasmid-borne PGAL1-CgPDR1 gene, indicating the mitotic stability of this newly constructed plasmid was comparable to that of PGAL1-PDR3 (Kozovska, 2004).

View this table:

Susceptibility to cycloheximide and fluconazole of the Saccharomyces cerevisiae FY1679-28C/TDEC transformants bearing indicated plasmids

PlasmidMIC (μg mL−1)

When transformants harboring the PGAL1-CgPDR1 gene were subsequently transformed by a YCp-PPDR5-pma1(D378N) centromeric plasmid (Kozovska & Subik, 2003) containing a dominant lethal pma1 allele expressed from the PPDR5 promoter and known to be recognized by CgPdr1p (Tsai, 2006), double transformants were obtained. They were prototrophic, and, like the ZK11-1 strain (Kozovska & Subik, 2003), failed to grow on minimal medium containing galactose as the carbon and energy source (Fig. 1). One double transformant, named SE41, was selected as the test strain suitable for the identification of drugs interfering with the CgPdr1p function. Any drug inhibiting the CgPdr1p function would be expected to downregulate the expression of pma1 and restore the ability of SE41 cells to grow in the presence of galactose and, hence, may serve as the multidrug resistance reversal agent in the pathogenic C. glabrata. The zone of yeast growth around the reversal agent applied on a cellulose disk to the lawn of the SE41 cells on solid minimal medium containing galactose can indicate the presence of the CgPdr1p inhibitor. Such a zone of growth can be clearly distinguished from loss-of-function Cgpdr1 mutants, which appear spontaneously as galactose-positive clones with a frequency of 0.66 × 10−6. Using the SE41 test strain, small compound libraries can be screened for multidrug resistance reversal agents that can be subsequently analyzed in more detail using wild-type strains and multidrug-resistant C. glabrata clinical isolates. Unfortunately, the lead compounds that bind to multidrug resistance transcription factor and inhibit its function have not yet been identified.


Galactose-induced growth defect of 14 independent double transformants of the Saccharomyces cerevisiae strain FY1679-28C/TDEC bearing the PGAL1-CgPDR1 and PPDR5-pma1(D378N) fusion genes. Five microliters of suspensions (107 and 106 cells mL−1, respectively) of each individual transformant were spotted onto media and growth was scored after 5 days.

The screening system described above is also suitable for a positive selection of mutants harboring loss-of-function alleles of the CgPDR1 gene. As shown in Fig. 1, potential loss-of-function Cgpdr1 mutants appeared in independent double transformants as the galactose-positive clones on the background of cells failing to grow in the presence of galactose that induced the expression of the pma1 dominant lethal allele. Phenotypic and molecular analyses of one galactose-positive clone selected from the SE41 test strain revealed that its cells, in the presence of galactose, were hypersensitive to cycloheximide (MIC=0.1 μg mL−1). This was due to a mutation that replaced the serine codon with a termination stop codon (Ser947Ter) in the plasmid-borne CgPDR1 gene. This mutation resulted in a truncated CgPdr1p devoid of a C-terminal activation domain required for the transactivation function of this transcriptional activator.

Despite the loss of its normal biological function, the loss-of-function Cgpdr1(Ser947Ter) mutant allele was able to enhance the sensitivity to cycloheximide of the S. cerevisiae EC61 and EC62 mutant strains containing gain-of-function pdr1-3 and pdr1-6 mutations along with a disrupted PDR3 gene. MICs of cycloheximide for the EC61 and EC62 mutant strains corresponded to 1.5 μg mL−1. On the other hand, growth of their transformants was significantly reduced already at a cycloheximide concentration of 0.4 μg mL−1, compared with that on the control medium without cycloheximide (Fig. 2). This indicates that a truncated CgPdr1p either attenuates the function of a hyperactive ScPdr1p by heterodimer formation (Mamnun, 2002) or inhibits the transcription of their target genes by competing for the promoters or interacting subunit of the mediator complex (Thakur, 2008). No drug-sensitizing effect was, however, observed in the FY1679-28C wild-type strain containing functional PDR1 and PDR3 genes. At present, we do not have a plausible explanation for this observation.


Suppression of cycloheximide resistance in gain-of-function pdr1-3 and pdr1-6 mutant strains expressing loss-of-function Cgpdr1(Ser947Ter) allele from the PGAL1 promoter. Cells grown on minimal medium containing galactose were replica plated on media containing indicated concentrations of cycloheximide. Growth was scored after 6 days.

Based on the genetic background of S. cerevisiae, which is generally regarded as safe, we have developed a test strain that is suitable for the identification of inhibitors of C. glabrata CgPdr1p transcriptional activator as well as for positive selection of loss-of-function Cgpdr1 alleles. The screen can be carried out either on solid or in liquid media containing galactose, allowing a high-throughput screening of compound libraries. The identified CgPdr1p inhibitors enhancing activities of antimycotics may be useful for the development of novel strategies to combat fungal infections caused by the multidrug-resistant C. glabrata strains. Moreover, the loss-of-function Cgpdr1 alleles may help identify amino acids essential for the transactivation function of the main transcriptional activator involved in the control of multidrug resistance in this opportunistic human fungal pathogen.


The authors thank H.F. Tsai and E. Carvajal for plasmid and strains, and D. Hanson for careful reading of the manuscript. This work was supported by grants from the Slovak Research and Developmental Agency (LPP-0022-06, LPP-0011-07, VVCE-0064-07) and the Slovak Grant Agency of Science (VEGA 1/0001/09).


  • Editor: André Goffeau


View Abstract