The present study examines the molecular mechanism underlying in vitro-induced resistance to FLC (fluconazole), KTC (ketaonazole), MCZ (miconazole) and CHX (cycloheximide) in AS (azole-susceptible) strains of Candida albicans when exposed to CaCDR1/CaCDR2 inducers like FPZ (fluphenazine) and steroids [PRG (progesterone) and β-EST (β-oestradiol)]. By employing spot and checkerboard titre assays, we provide evidence of an in vitro-induced antagonism between tested drugs and inducers, which was accompanied with a concomitant increase in CaCDR1 and CaCDR2 transcript levels. Notably, unlike AS isolates, parental WT (wild-type) and Δcdr2 null strains, Δcdr1 as well as Δcdr1cdr2 nulls, when challenged with the inducers could not display antagonism. Our results validated by Northern blotting, reporter gene transcription and TRO (transcription run on) assays show that in vitro-induced antagonism between tested drugs and inducer in AS isolates was mainly due to a transient and reversible transcriptional activation of CaCDR1. Notwithstanding our earlier observation that consistent high transcript levels of CaCDR1 in clinical AR (azole-resistant) isolates were maintained due to the combination of its transcriptional activation and enhanced mRNA stability via elongated poly(A) tails, this study shows that transient and reversible transcriptional activation of CaCDR1 was the major determinant of induced antagonism in AS isolates. The distinct strategies between sustained (in AR isolates) and transiently induced resistance mechanisms (in AS isolates) adopted by Candida should become useful in improving therapeutic approaches.

INTRODUCTION

Azoles, which target the ergosterol biosynthesis pathway by inhibiting the P450 cytochrome 14α-lanosterol demethylase (CaERG11), are frontline antifungals most commonly used to treat Candida albicans infections [1]. However, repeated and prolong usage of azoles leads to the development of AR (azole-resistant) strains. Biochemical and molecular analyses of AR strains have shown that factors contributing to the development of clinical resistance are numerous and include the extent of immuno-suppression of host, level of exposure to azoles and intrinsic resistance of the fungus to antifungal drugs [2]. The molecular mechanisms of clinical azole resistance include an increased cellular content or alteration of accessibility to drug target, decreased permeability of the cell membrane and active drug extrusion by membrane transporters [3]. The enhanced drug extrusion is one of the most commonly observed mechanisms encountered in AR isolates, which is assisted by the stable up-regulation of MDR (multi-drug resistance) genes such as CaCDR1/CaCDR2 and CaMDR1 encoding ABC transporter (ATP-binding-cassette transporter) and MFS (Major Facilitator Superfamily) multidrug transporters respectively [36].

Regulation at transcription level is generally accepted to be the principal mechanism for controlling the MDR gene expression and so is the case with CaCDR1 for its transient and consistent up-regulation in clinical AS (azole-susceptible) and AR isolates respectively [710]. The identification of various consensus and specific cis-regulatory elements as well as trans-acting factors matches well with the transcriptional regulation of CaCDR1 [917]. It has been shown earlier that CaCDR1 in its 5′ flanking region harbours various consensus [Sp1 (specificity protein 1), AP-1 (activator protein 1) and Y-box] sequences as well as specific BRE (basal response element), NRE (negative response element) and DRE (drug response element)/SRE (steroid response element) [912]. Various trans-acting factors regulating CaCDR1 expression have also been identified. For example, Non DiTyrosine 80 [CaNDT80, a homologue of a meiosis-specific TF (transcription factor)] in Saccharomyces cerevisiae has been identified as a potential activator of CaCDR1 [13]. Coste et al. [14] identified a TF TAC1 (Transcriptional Activator of CDR genes 1) that binds to the DRE in CaCDR1/CaCDR2 promoters. In contrast, TF belonging to the zinc cluster family, FCR1 (Fluconazole Resistance 1) [15], as well as the global repressor, Tup1 (Thymidine uptake 1), acts as a negative regulator of CaCDR1 expression [16]. Recent genome-wide location profiling [ChIP (chromatin immunoprecipitation)-chip] shows that another TF of the zinc cluster family, Upc2 (Uptake control 2), regulating ERG genes, also targets CaCDR1 [17]. However, our recent studies have shown that along with the transcriptional control, post-transcriptional event involving mRNA stability governed by hyperadenylation of its 3′-UTR (3′-untranslated region) also regulates CaCDR1 levels in clinical AR isolates [8,18]. A number of studies indicate that hMDR1 (human CaCDR1 homologue) encoding P-gp (P-glycoprotein) can be rapidly and transiently induced in vitro in cultured cell lines on exposure to a variety of chemotherapeutic agents by either its transcriptional activation or by enhanced mRNA stability [1923]. However, the clinical relevance of in vitro-induced CaCDR1 expression of C. albicans is not well understood. The present study provides evidence of an in vitro-induced antagonism between tested drugs [FLC (fluconazole), KTC (ketaonazole), MCZ (miconazole) and CHX (cycloheximide)] and CaCDR1/CaCDR2 inducers [FPZ (fluphenazine), PRG (progesterone) and β-EST (β-oestradiol)], which occur mainly by transiently and reversibly induced expression of CaCDR1 in clinical AS. We further show that this in vitro rapid, transient and reversible induced CaCDR1 overexpression in AS isolates is solely contributed by its transcriptional activation with no change in its mRNA stability or 3′-UTR polyadenylation status.

MATERIALS AND METHODS

Materials

Medium and other chemicals were obtained from Hi-Media (Mumbai, India). Luria–Bertani broth and agar media was purchased from Difco, BD Biosciences. FPZ, PRG, β-EST, KTC, MCZ, CHX and R6G (rhodamine6G) were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Oligonucleotides used were commercially synthesized by Sigma–Aldrich (Table 1). All molecular biology grade chemicals used in the present study were obtained from Sigma Chemical Co.

Table 1
Oligonucleotides and plasmids used in this study
NameDescriptionSequences* or source (reference)
Oligonucleotides   
 KM1 Forward primer for amplification of CaCDR1 N-terminal region 5′-CTTTTCCACTGGTAACTACT-3′ 
 KM2 Reverse primer for amplification of CaCDR1 N-terminal region 5′-ACTGTATCCTACGAAGTTACCATTGACCC-3′ 
 CDR2-F Forward primer for amplification of CaCDR2 ORF 5′-GGTCCTTATACCGAAGCTGC-3′ 
 CDR2-R Reverse primer for amplification of CaCDR2 ORF 5′-TTTTTTCATCTTCTTTTCTCT-3′ 
 ACT1-F-RM Forward primer for amplification of ACT1 ORF 5′-GTTAGAAAAGAATTATACGG-3′ 
 ACT1-R-RM Reverse primer for amplification of ACT1 ORF 5′-GAAACATTTGTGGTGAACAATGG-3′ 
 CT-CDR1-F-RM Forward primer for RT–PCR amplification of CaCDR1 5′-ATTTGGTACCATACATTAAATTTGCTGGTGGG-3′ 
 CT-CDR1-R-RM Reverse primer for RT–PCR amplification of CaCDR1 5′-GTTTCTCGAGTTTCTTATTTTTTT TCTCTCTGTTACCC-3′ 
 Oligo(dC9T6)AP-RM 5′ anchored anti-sense primer for PAT assay of CDR1 mRNA 5′-GATCAAACTCGAG-CCGCGG-TCTAGACCCCCCCCCTTTTTT-3′ 
 AP-RM Reverse-anchored primer for RT–PCR amplification of CaCDR1 5′-GATCAAA-CTCGAG-CCGCGG-TCTAGA-3′ 
 Gu4/DSY294-UTR-F-RM Forward primer for CaCDR1 3′-UTR amplification and cloning 5′-GATCTTAATTAAATTAAACAGTTTGTTTTTTGACATGG-3′ 
 T7 promoter primer Primer for sequencing of T/A cloned PCR product 5′-TAATACGACTCACTATAGGG-3′ 
 SP6 promoter primer Primer for sequencing of T/A cloned PCR product 5′-ATTTAGGTGACACTATAG-3′ 
Plasmids   
 pGEM®T-Easy vector system II Plasmid backbone used for T/A DNA cloning purpose Promega 
 pGu4-poly(A)-RM Plasmid harbouring poly(A) tailed RT-PCR product of CaCDR1 of AS clinical isolate, Gu4 [18
 pGu4-FPZ-poly(A)-RM Plasmid harbouring FPZ induced ‘poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, Gu4 This study 
 pGu4-PRG-poly(A)-RM Plasmid harbouring PRG induced poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, Gu4 This study 
 pGu4-β-EST-poly(A)-RM Plasmid harbouring β-EST induced ‘poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, Gu4 This study 
 pGu5-poly(A)-RM Plasmid harbouring poly(A) tailed RT–PCR product CaCDR1 of AR clinical isolate, Gu5 [18
 pDSY294-poly(A)-RM Plasmid harbouring poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 [18
 pDSY294-FPZ-poly(A)-RM Plasmid harbouring FPZ induced ‘poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 This study 
 pDSY294-PRG-poly(A)-RM Plasmid harbouring PRG induced poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 This study 
 pDSY294-β-EST-poly(A)-RM Plasmid harbouring β-EST induced poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 This study 
 pDSY296-poly(A)-RM Plasmid harbouring poly(A) tailed RT–PCR product of CaCDR1 of AR clinical isolate, DSY296 [18
NameDescriptionSequences* or source (reference)
Oligonucleotides   
 KM1 Forward primer for amplification of CaCDR1 N-terminal region 5′-CTTTTCCACTGGTAACTACT-3′ 
 KM2 Reverse primer for amplification of CaCDR1 N-terminal region 5′-ACTGTATCCTACGAAGTTACCATTGACCC-3′ 
 CDR2-F Forward primer for amplification of CaCDR2 ORF 5′-GGTCCTTATACCGAAGCTGC-3′ 
 CDR2-R Reverse primer for amplification of CaCDR2 ORF 5′-TTTTTTCATCTTCTTTTCTCT-3′ 
 ACT1-F-RM Forward primer for amplification of ACT1 ORF 5′-GTTAGAAAAGAATTATACGG-3′ 
 ACT1-R-RM Reverse primer for amplification of ACT1 ORF 5′-GAAACATTTGTGGTGAACAATGG-3′ 
 CT-CDR1-F-RM Forward primer for RT–PCR amplification of CaCDR1 5′-ATTTGGTACCATACATTAAATTTGCTGGTGGG-3′ 
 CT-CDR1-R-RM Reverse primer for RT–PCR amplification of CaCDR1 5′-GTTTCTCGAGTTTCTTATTTTTTT TCTCTCTGTTACCC-3′ 
 Oligo(dC9T6)AP-RM 5′ anchored anti-sense primer for PAT assay of CDR1 mRNA 5′-GATCAAACTCGAG-CCGCGG-TCTAGACCCCCCCCCTTTTTT-3′ 
 AP-RM Reverse-anchored primer for RT–PCR amplification of CaCDR1 5′-GATCAAA-CTCGAG-CCGCGG-TCTAGA-3′ 
 Gu4/DSY294-UTR-F-RM Forward primer for CaCDR1 3′-UTR amplification and cloning 5′-GATCTTAATTAAATTAAACAGTTTGTTTTTTGACATGG-3′ 
 T7 promoter primer Primer for sequencing of T/A cloned PCR product 5′-TAATACGACTCACTATAGGG-3′ 
 SP6 promoter primer Primer for sequencing of T/A cloned PCR product 5′-ATTTAGGTGACACTATAG-3′ 
Plasmids   
 pGEM®T-Easy vector system II Plasmid backbone used for T/A DNA cloning purpose Promega 
 pGu4-poly(A)-RM Plasmid harbouring poly(A) tailed RT-PCR product of CaCDR1 of AS clinical isolate, Gu4 [18
 pGu4-FPZ-poly(A)-RM Plasmid harbouring FPZ induced ‘poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, Gu4 This study 
 pGu4-PRG-poly(A)-RM Plasmid harbouring PRG induced poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, Gu4 This study 
 pGu4-β-EST-poly(A)-RM Plasmid harbouring β-EST induced ‘poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, Gu4 This study 
 pGu5-poly(A)-RM Plasmid harbouring poly(A) tailed RT–PCR product CaCDR1 of AR clinical isolate, Gu5 [18
 pDSY294-poly(A)-RM Plasmid harbouring poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 [18
 pDSY294-FPZ-poly(A)-RM Plasmid harbouring FPZ induced ‘poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 This study 
 pDSY294-PRG-poly(A)-RM Plasmid harbouring PRG induced poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 This study 
 pDSY294-β-EST-poly(A)-RM Plasmid harbouring β-EST induced poly(A) tailed RT–PCR product of CaCDR1 of AS clinical isolate, DSY294 This study 
 pDSY296-poly(A)-RM Plasmid harbouring poly(A) tailed RT–PCR product of CaCDR1 of AR clinical isolate, DSY296 [18
*

The restriction sites introduced into the primers are underlined, while the flanking bases are in italics.

Bacterial and yeast strains and growth media

Plasmids and C. albicans strains used are listed in Tables 1 and 2 respectively. For induction experiments, C. albicans cells at mid-exponential phase were pretreated with FPZ (10 μg/ml), PRG (1 mM) and β-EST (1 mM) for 30 min unless otherwise indicated. The final concentrations used are given in parentheses.

Table 2
Strains used in this study
StrainsDescription/genotypeReference
CAF2-1 Δura3::imm434/URA3 [44
DSY449 Δcdr1::hisG/Δcdr1::hisG [45
DSY653 Δcdr2::hisG-URA3-hisG/Δcdr2::hisG [5
DSY654 Δcdrl::hisG/Δcdrl::hisG [5
 Δcdr2::hisG-URA3-hisG/Δcdr2::hisG  
Gu4 Fluconazole-susceptible clinical isolate (MTL-a/MTL-α) [46
Gu4L2 PCDR1-lacZ integrated at CaCDR1 locus [8
Gu5 Fluconazole-resistant clinical isolate (MTL-α/MTL-α) [46
Gu5L2 PCDR1-lacZ integrated at CaCDR1 locus [8
DSY294 Fluconazole-susceptible clinical isolate (MTL-a/MTL-α) [47
DSY294L2 PCDR1-lacZ integrated at CaCDR1 locus [8
DSY296 Fluconazole-resistant clinical isolate (MTL-α/MTL-α) [47
DSY296L2 PCDR1-lacZ integrated at CaCDR1 locus [8
StrainsDescription/genotypeReference
CAF2-1 Δura3::imm434/URA3 [44
DSY449 Δcdr1::hisG/Δcdr1::hisG [45
DSY653 Δcdr2::hisG-URA3-hisG/Δcdr2::hisG [5
DSY654 Δcdrl::hisG/Δcdrl::hisG [5
 Δcdr2::hisG-URA3-hisG/Δcdr2::hisG  
Gu4 Fluconazole-susceptible clinical isolate (MTL-a/MTL-α) [46
Gu4L2 PCDR1-lacZ integrated at CaCDR1 locus [8
Gu5 Fluconazole-resistant clinical isolate (MTL-α/MTL-α) [46
Gu5L2 PCDR1-lacZ integrated at CaCDR1 locus [8
DSY294 Fluconazole-susceptible clinical isolate (MTL-a/MTL-α) [47
DSY294L2 PCDR1-lacZ integrated at CaCDR1 locus [8
DSY296 Fluconazole-resistant clinical isolate (MTL-α/MTL-α) [47
DSY296L2 PCDR1-lacZ integrated at CaCDR1 locus [8

Drug susceptibility testing

Susceptibilities to different drugs (see the Figure 1a legend for drug concentrations used) were tested by spotting cells on to solid agar plates either alone or in the presence of FPZ, PRG and β-EST as described previously [24]. Yeast cultures were grown overnight in YEPD (yeast extract peptone dextrose) medium and diluted to a density of 1.5×107 cells/ml and 10-fold serial dilutions were performed to a final dilution step containing 1.5×103 cells/ml. Then, 5 μl of each dilution was spotted on to YEPD plates with or without drugs. Plates were photographed after 48 h of incubation at 30°C.

Checkerboard titre assay

The interaction between the tested drugs namely FLC, KTC, MCZ, CHX and CaCDR1/CaCDR2 inducers namely FPZ, PRG and β-EST was evaluated by the checkerboard method as recommended by the NCCLS. In brief, serial double dilutions of tested anticandidal compounds and inducers were prepared (in μg/ml) ranging from 0.25 to 128 for FLC, 0.019 to 10 for KTC, 0.019 to 10 for MCZ, 0.039 to 20 for CHX and 0.097 to 50 for FPZ, 0.017 to 10 for both PRG and β-EST respectively. After making dilutions, a 100 μl suspension of Candida strains adjusted to 5×105 CFU (colony forming units)/ml was added to each well and cultured at 30°C for 48 h in RPMI 1640 medium. Then visual reading of MICs (minimal inhibitory concentrations) was performed, and the D492 (attenuance at 492 nm) values were measured. The background D492 was subtracted from the D492 of each well. Each isolate was tested in triplicate on different days. Each checkerboard test generates many different combinations and by convention the FIC (fractional inhibitory concentration) value of the most effective combination is used in calculating the FICI (FIC index).

FICI was calculated by adding both FICs:

 
formula

where MICAalone and MICBalone are the MICs of drug/inducer A and B when acting alone and CAcomb and CBcomb are concentrations of drugs/inducers A and B at the isoeffective combinations respectively.

Off-scale MICs were converted to the next highest or next lowest doubling concentration. The interpretation of the FICI was as follows: an FICI value of ≤0.5 represented synergy, a value of 1–4 represented indifference and a value of >4 represented antagonism [2527].

Drug-resistant inducibility of clinical AS isolates

Figure 1
Drug-resistant inducibility of clinical AS isolates

(a) Drug susceptibility testing. Drug susceptibility in the presence of inducers (FPZ, PRG and β-EST) was measured by spot assays either alone or along with the indicated drugs as described under the Material and methods section. The final drug concentrations used (the solvent used is given in parentheses) for clinical AS isolates were: FLC 2 μg/ml (water), KTC 0.05 μg/ml (methanol), MCZ 0.1 μg/ml (methanol) and CHX 300 μg/ml (water). For WT parental (CAF2-1), Δcdr1, Δcdr2 and Δcdr1cdr2 null strains, the final FLC concentration used was 0.5 μg/ml. (b) R6G efflux assay. The results are shown as relative fold induction of R6G efflux from induced AS isolates as compared with its uninduced cells (considered as ‘1’). The quantities reported are means±S.D. (indicated by error bars) for three independent experiments. (c) Northern-blot analysis. Transcript levels of CaCDR1 and CaCDR2 in uninduced and induced AS isolates were detected by Northernblot analysis. The membranes were subsequently de-probed and rehybridized with the ACT1 probe to monitor equal RNA loading and transfer. Hybridization signal intensities of CaCDR1 and CaCDR2 transcript levels were quantified using densitometry scanning of phosphoimages. Relative intensity (RI) of CaCDR1 and CaCDR2 transcript levels with respect to corresponding ACT1 mRNA of each AS isolate is plotted. Un, uninduced.

Figure 1
Drug-resistant inducibility of clinical AS isolates

(a) Drug susceptibility testing. Drug susceptibility in the presence of inducers (FPZ, PRG and β-EST) was measured by spot assays either alone or along with the indicated drugs as described under the Material and methods section. The final drug concentrations used (the solvent used is given in parentheses) for clinical AS isolates were: FLC 2 μg/ml (water), KTC 0.05 μg/ml (methanol), MCZ 0.1 μg/ml (methanol) and CHX 300 μg/ml (water). For WT parental (CAF2-1), Δcdr1, Δcdr2 and Δcdr1cdr2 null strains, the final FLC concentration used was 0.5 μg/ml. (b) R6G efflux assay. The results are shown as relative fold induction of R6G efflux from induced AS isolates as compared with its uninduced cells (considered as ‘1’). The quantities reported are means±S.D. (indicated by error bars) for three independent experiments. (c) Northern-blot analysis. Transcript levels of CaCDR1 and CaCDR2 in uninduced and induced AS isolates were detected by Northernblot analysis. The membranes were subsequently de-probed and rehybridized with the ACT1 probe to monitor equal RNA loading and transfer. Hybridization signal intensities of CaCDR1 and CaCDR2 transcript levels were quantified using densitometry scanning of phosphoimages. Relative intensity (RI) of CaCDR1 and CaCDR2 transcript levels with respect to corresponding ACT1 mRNA of each AS isolate is plotted. Un, uninduced.

R6G efflux assay

Glucose-mediated efflux of R6G was carried out as described earlier [28]. Briefly, approx. 1.0×107 cells from a culture grown overnight were inoculated in 50 ml of YEPD and grown for 3 h at 30°C. Approx. 30 min before harvesting, cells were pretreated with FPZ (10 μg/ml), PRG (1 mM) and β-EST (1 mM). The cells were pelleted, washed twice with PBS without glucose and subsequently resuspended as 2% cell suspension in PBS without glucose. The cells were then de-energized for 45 min in DOG (deoxy-glucose; 5 mM) and DNP (2,4-dinitrophenyl; 5 mM) in PBS without glucose. The de-energized cells were pelleted, washed and resuspended as 2% (w/v) cell suspension in PBS without glucose to which R6G was added at a final concentration of 10 μM and incubated for 40 min at 30°C. The equilibrated cells with R6G were then washed and resuspended as 2% (w/v) cell suspension in PBS with 2% (w/v) glucose. An aliquot of 1 ml was taken after the indicated time and centrifuged at 9000 g for 2 min. The absorption of the supernatant was measured at 527 nm.

RNA isolation and Northern-blot hybridization

RNA isolation and Northern-blot analyses were carried out essentially by standard protocols as described earlier [29]. Equal loading of RNA was assessed by rRNA bands. RNA was electrophoresed on denaturing formaldehyde gel and blotted and UV cross-linked on to nylon membranes (Hybond-N+; Amersham Pharmacia Biotech). Membrane-bound RNA was stained with Methylene Blue before hybridization to check rRNA bands for equal loading and transfer. The relative intensities of CaCDR1 and ACT1 mRNA signals in Northern hybridizations were quantified by exposure of the hybridized membrane in an FLA5000 Fuji PhosphoImager. The list of primers used for making the probes is given in Table 1.

β-Galactosidase reporter assay

The β-galactosidase reporter assay was performed using duplicate samples of cells from three independent experiments as described previously [8]. β-Galactosidase activity was determined by the standard equation and is expressed in Miller units per mg of protein. Miller units are arbitrary units (a.u.).

 
formula

where t is the time of reaction expressed in minutes and v is the culture volume assayed expressed in millilitres.

TRO (transcription run on) assay

TRO experiments were performed as described previously [8] with the following modifications. Cells were grown at 30°C in YEPD with agitation until the culture reached an D600 of 1.0 and pretreated with FPZ (10 μg/ml), PRG (1 mM) or β-EST (1 mM) for 30 min. An aliquot of yeast cells (6×108 ml−1) was used to perform TRO. The cells were centrifuged for 5 min at 4000 g and resuspended in 5 ml of cold TMN (10 mM Tris, 100 mM NaCl and 5 mM MgCl2, pH 7.4). The cells were again centrifuged for 5 min at 4000 g and the cell pellet was resuspended in 900 μl of sterile cold DEPC (diethyl pyrocarbonate)-treated water. Then the cell suspension was transferred to a fresh microcentrifuge tube containing 50 μl of 10% N-dodecyl sarcosine sodium sulfate (sarkosyl) and was incubated for 20 min on ice. After the permeabilization step, cells were recovered by low-speed centrifugation at 2415 g for 2 min at 4°C and the supernatant was removed. In vivo transcription was reinitiated by resuspending the permeabilized cell fraction in 120 μl of 2.5×transcription buffer (50 mM Tris/HCl, pH 7.7, 500 mM KCl and 80 mM MgCl2), 16 μl of AGC mix (10 mM each of ATP, GTP and CTP), 6 μl of dithiothreitol (0.1 M), 1 unit of RNase inhibitor/μl, 10 mM phosphocreatine, 1.2 μg of creatine kinase/μl and 15 μl of [α-32P]UTP (3000 Ci/mmol and 10 μCi/μl). Cells were maintained on ice at all times. The final volume was adjusted to 300 μl with DEPC-treated water and the mix was incubated for 15 min at 30°C to allow transcription elongation. The reaction was stopped by adding 1 ml of ice-cold DEPC-treated water to the mix. Cells were recovered by centrifugation to remove non-incorporated radioactive nucleotides. Total RNA was isolated using TRIzol® reagent (Sigma) as per the manufacturer’s specifications except that 200 μl of ice-cold acid-washed 0.4–0.6 mm diameter glass beads (Sigma Chemical Co.) were also used for efficient and complete lysis of permeabilized cells. Isolated total labelled RNA was again precipitated by adding 2.5 M ammonium acetate and an equal volume of propan-2-ol. The mixture was stored overnight at –20°C. To pellet the RNA, tubes were centrifuged at 13148 g for 15 min in the micro-centrifuge. The propan-2-ol was removed and the labelled RNA pellet was washed twice with 70% ethanol, dried and resuspended in 100 μl of DEPC-treated water. This double precipitation of RNA was used to minimize DNA contamination. Total extracted RNA was spectrophotometrically quantified. An aliquot was used for specific radioactivity determination in a Tri-CARB 2900 TR liquid scintillation analyser (Packard Instrument Co.). All of the in vivo labelled RNA of each isolate (~2–2.5×106 c.p.m.) was subsequently used for reverse Northern hybridization with a dot-blotted nylon membrane (Hybond-N+; Amersham Pharmacia Biotech) containing PCR amplified gene-specific N-terminal CaCDR1 sequences (nt –242 to +256 from the TSP), ACT1 (positive control) and pBlueScript-KS(+) (negative control) as probes, as per the manufacturer’s recommendations. Northern blots were scanned with a phosphoimager scanner (FLA-5000, Fuji PhosphoImager). Signal intensities of hybridized nuclear RNA were quantified using densitometry scanning.

Thiolutin chase assay

A potent in vivo transcriptional inhibitor, thiolutin was used to measure the CaCDR1 mRNA t½ (half-life) as described previously [8,18]. Briefly, 100 ml of cells was grown to a D600 of 1.0 at 30°C and pretreated with FPZ (10 μg/ml), PRG (1 mM) or β-EST (1 mM) for 30 min. Transcription was subsequently stopped by the addition of an optimized concentration (40 μg/ml) of thiolutin. Aliquots of cells were taken at the indicated times after transcriptional shut-off by thiolutin. Total RNA was isolated using Ambion’s RiboPure™-Yeast RNA isolation kit (catalogue no. 1926) as per the manufacturer’s instructions. Equal RNA loading was assessed by staining the agarose gel with ethidium bromide prior to blotting. For Northern blots, approx. 25 μg of total RNA from the above samples was hybridized with a single probe derived from CaCDR1-specific primers (primer pairs: KM1 and KM2; Table 1), which has been used throughout the present study. Hybridization signal intensity was quantified with a phosphoimager scanner (FLA-5000, FLA5000 Fuji PhosphorImager) and normalized to the band intensity at time T0 and plotted as a line graph.

PAT (polyadenylation test)

An improved variation of 3′-RACE (3′-rapid amplification of cDNA ends) PAT involving G-tailing of mRNA was employed to determine poly(A) tail length as described previously [8,18].

Guanylation of mRNA

Total RNA isolated from mid-exponential phase grown untreated or 30 min pretreated [FPZ (10 μg/ml), PRG (1 mM) and β-EST (1 mM)] AS isolates using TRIzol® reagent (as per the manufacturer’s specifications) were enriched with poly(A)+ (polyadenylated) mRNA using the Oligotex mRNA Mini kit protocol (Qiagen). The purified poly(A)+-enriched mRNA was used directly for polyguanylation using yeast PAP [poly(A) polymerase] (catalogue no. E74225Y; Amersham Pharmacia Biotech/US Biochemicals) as per the manufacturer’s recommendations. To abolish the higher-ordered secondary structure at the 3′-end of mRNA, samples (0.1 μg) were heated at 65°C for 5 min and immediately placed on ice. They were incubated for 1 h at 37°C with 600 units of PAP and 0.5 mM GTP in a 25 μl reaction mixture containing 20 mM Tris/HCl (pH 7.0), 50 mM KCl, 0.7 mM MnCl2, 0.2 mM EDTA, 100 μg/ml BSA and 10% glycerol. An additional 300 units of PAP were added and incubation was continued for an additional 1 h. The reaction was terminated by heat treatment at 65°C for 10 min, chilled on ice and kept at –80°C until further use.

RT–PCR (reverse transcription–PCR)

Polyguanylated mRNA (as described above) was applied directly for RT–PCR analysis using hot start conditions (65°C, 30 s). The first strand cDNA synthesis (RT, 42°C for 1 h) was primed by oligo(dC9T6)-anchor primer (1 μM). The RT reaction was stopped by denaturation at 70°C for 10 min; the synthesized cDNA product (1:4 dilution) was used for PCR amplification with 1 μM of each CaCDR1 3′-UTR specific forward primers, Gu4/DSY294-UTR-F-RM and reverse anchor primer (AP-RM) as mentioned in Table 1 (PCR parameters: initial denaturation of 95°C for 5 min followed by 35 cycles denaturation at 95°C for 30 s, annealing at 55°C for 30 s, elongation at 72°C for 1 min and final extension at 72°C for 10 min). As a positive control, CaCDR1 ORF (open reading frame)-specific forward and reverse primers, CT-CDR1-F-RM and CT-CDR1-R-RM (corresponding to position 4271–4293 and 4475–4503 in the CaCDR1 genomic sequence) were also used (results not shown). The negative control (without RT) established that the PCR products generated in the RT–PCR were not due to genomic DNA contamination (results not shown). The RT–PCR product of each isolate was electrophoresed on a 1.2% agarose gel in 1×TAE (Tris/acetate/EDTA). The gel-purified 3′-RACE product (Qiagen PCR clean up kit) of each isolate was cloned directly in pGEM®T-Easy vector using T/A cloning kit (Promega) as per the manufacturer’s recommendations. All the cloned RT–PCR amplified products were confirmed by appropriate restriction digestion analysis and sequenced further to determine the exact and precise length of CaCDR1 transcript poly(A) tail.

Custom service nucleotide sequencing

Multiple CaCDR1 3′-UTR specific clones harbouring poly(A) tail length of each isolate (as described above) were sequenced directly by extension from both sense and anti-sense strands using T7 promoter/T7 promoter primer and SP6 promoter/SP6 promoter primer (Table 1) by exploiting Big Dye terminator chemistry and automated DNA sequencing (ABI Prism 3100). Approx. 8–10 random clones from at least three independent RT–PCR reactions of each isolate were sequenced. Reproducibility of the sequencing was confirmed by processing all samples at least twice.

RESULTS

In vitro-induced drug resistance in clinical AS isolates display up-regulation of CaCDR1 and CaCDR2

To check the inducible drug-resistance potential, AS isolates (Gu4 and DSY294) were spotted on YEPD plates containing drugs such as FLC, KTC, MCZ and CHX in the presence of well-known CaCDR1 inducers such as FPZ, PRG and β-EST [10]. Gu4 and DSY294 strains in the presence of the tested inducers showed marked increase in MDR (Figure 1a). Notably, the presence of inducers in Δcdr1 (DSY449) as well as in Δcdr1cdr2 (DSY654) null cells did not cause any significant change in susceptibilities to tested drugs (Figure 1a). However, exposure of inducers to Δcdr2 null cells (DSY653) and its corresponding parental WT (wild-type) cells exhibited relatively enhanced resistance to tested drugs (Figure 1a). The results of the spot assays were further confirmed by checkerboard assays. Table 3 summarizes the FICI values of AS isolates (Gu4 and DSY294), Δcdr2 null cells (DSY653) and its corresponding WT strain (CAF2-1) in the presence of the inducers (FPZ, PRG and β-EST) and tested drugs (FLC, KTC, MCZ and CHX). The FICI values were higher than 4 in each case, thus confirming antagonism observed in spot assays. Notably, an FICI value above 4 confirms antagonism between the tested drugs and the inducers [25]. In the case of Δcdr1 (DSY449), Δcdr1cdr2 (DSY654) nulls and AR isolates (Gu5 and DSY296), there was no significant difference in the MIC80 (minimal inhibitory concentration for 80% of input cells) values of FLC, KTC, MCZ and CHX in the presence of inducers (results not shown). The enhanced efflux of R6G, a well-known substrate of CaCdr1p in induced AS isolates (1.8-, 2.3- and 1.8-fold for Gu4 and 2.0-, 2.3- and 2.2-fold for DSY294, in the presence of FPZ, PRG and β-EST respectively; Figure 1b), further supports the observed antagonism. Northern-blot analysis in induced and non-induced cells scraped from the plates mimicking the conditions of spot assays, displayed relatively higher CaCDR1 and CaCDR2 transcripts levels (Figure 1c). Considering the fact that CaCDR1 up-regulation elicits enhanced resistance to tested drugs as compared with CaCDR2 (Figure 1a), we focused on detailed analysis of CaCDR1. The possible involvement of other genes, however, in induction of MDR phenotype in AS isolates cannot be ruled out. Notably, no difference in drug-resistance profiling, R6G efflux and CaCDR1 transcript levels for both uninduced and induced AR isolates was observed (Supplementary Figure S1 at http://www.biosci.org/bsr/031/bsr0310031add.htm).

Table 3
*FICI values of the representative strains
FLCKTCMCZCHX
Gu4     
 +FPZ 
 +PRG 
 +β-EST 10 
DSY294     
 +FPZ 
 +PRG 10 
 +β-EST 10 
CAF2-1     
 +FPZ 
 +PRG 
 +β-EST 
DSY653     
 +FPZ 
 +PRG 
 +β-EST 10 
FLCKTCMCZCHX
Gu4     
 +FPZ 
 +PRG 
 +β-EST 10 
DSY294     
 +FPZ 
 +PRG 10 
 +β-EST 10 
CAF2-1     
 +FPZ 
 +PRG 
 +β-EST 
DSY653     
 +FPZ 
 +PRG 
 +β-EST 10 
*

FICI=FICA+FICB=CAcomb/MICAalone+CBcomb/MICBalone, where MICAalone and MICBalone are the MICs of drug/inducer A and B when acting alone and CAcomb and CBcomb are concentrations of drugs/inducers A and B at the isoeffective combinations respectively [2527].

Induced up-regulation of CaCDR1 is mediated by its transcriptional activation

We evaluated if observed induced CaCDR1 up-regulation was due to its transcriptional activation. For this, we used two different approaches. First, we performed the β-galactosidase reporter assay in which CaCDR1 promoter was placed upstream of the lacZ reporter [8]. Interestingly, β-galactosidase activity observed in both the induced AS isolates was relatively higher than their corresponding untreated counterparts (Figure 2a; 4.5-, 4.6- and 5.4-fold for Gu4 and 3.3-, 7.6- and 6.6-fold for DSY294 in the presence of FPZ, PRG and β-EST respectively). To validate this further, we examined the CaCDR1 promoter activity in its native chromosomal context by TRO experiments. TRO analyses of newly transcribed RNA were done on permeabilized cell fraction harbouring the intact nuclei isolated from uninduced and induced AS strains, as described previously [8]. As depicted in Figure 2(b), induced AS isolates exhibited an increased CaCDR1 transcriptional rate as compared with its untreated counterparts (10-, 12.3- and 12.2-fold for Gu4 and 24.2-, 23.9- and 20.1-fold for DSY294 in the presence of FPZ, PRG and β-EST respectively).

Transcriptional analysis of CaCDR1 in induced AS isolates

Figure 2
Transcriptional analysis of CaCDR1 in induced AS isolates

(a) β-Galactosidase reporter assay. Induced β-galactosidase reporter activity driven by CaCDR1 promoter was normalized to that of corresponding untreated cells (considered as ‘1’), and represented as fold induction. Results are expressed as the means±S.D. (indicated by error bars) for three independent experiments. (b) TRO analysis. Approx. 2 μg (each) of CaCDR1, ACT1 and empty vector pBlueScript-KS(+) DNA was blotted and immobilized on charged nylon membranes using a dot-blot assembly apparatus. The blots were probed with total labelled nuclear run-on RNA. DNA from a non-recombinant pBS-KS(+) plasmid was used as a negative control to determine the background and non-specific binding of labelled nuclear RNA to a random DNA fragment. Hybridization signal intensities of nuclear RNA were quantified using densitometry scanning of phosphoimages. Un, uninduced.

Figure 2
Transcriptional analysis of CaCDR1 in induced AS isolates

(a) β-Galactosidase reporter assay. Induced β-galactosidase reporter activity driven by CaCDR1 promoter was normalized to that of corresponding untreated cells (considered as ‘1’), and represented as fold induction. Results are expressed as the means±S.D. (indicated by error bars) for three independent experiments. (b) TRO analysis. Approx. 2 μg (each) of CaCDR1, ACT1 and empty vector pBlueScript-KS(+) DNA was blotted and immobilized on charged nylon membranes using a dot-blot assembly apparatus. The blots were probed with total labelled nuclear run-on RNA. DNA from a non-recombinant pBS-KS(+) plasmid was used as a negative control to determine the background and non-specific binding of labelled nuclear RNA to a random DNA fragment. Hybridization signal intensities of nuclear RNA were quantified using densitometry scanning of phosphoimages. Un, uninduced.

Rapid inducible CaCDR1 transcription activation is transient and reversible

The clinical AS isolates were subjected to Northern-blot analysis up to 2 h of challenge with the inducers. As depicted in Figure 3(a), CaCDR1 mRNA levels in both the induced AS isolates declined with time. The noted gradual reduction in CaCDR1 promoter-driven β-galactosidase reporter activity substantiated the Northern-blot results (Figure 3b). Coinciding with these results, there was also a decline in the magnitude of CaCDR1 run-on transcriptional rate with time which paralleled the levels of the endogenous mRNA (Figure 3c). Taken together, an overall kinetics analysis by Northern, reporter and TRO assays (Figures 3a–3c) confirmed that in vitro-induced CaCDR1 expression in AS isolates is transient and reverts to basal level within 2 h of treatment (Figures 2 and 3).

Kinetics of CaCDR1 expression in induced AS isolates

Figure 3
Kinetics of CaCDR1 expression in induced AS isolates

(a) Northern-blot analysis. AS isolates were exposed to inducers over a time interval of 0–120 min. Equal RNA loading and transfer were monitored by subsequently de-probing and rehybridization of membranes with ACT1 probe. (b) β-Galactosidase reporter assay. CaCDR1 promoter-driven β-galactosidase reporter analysis of AS isolates after exposure to inducers over a time interval of 0–120 min was measured. Data represent the fold induction of CaCDR1 promoter-driven reporter activity as compared with untreated cells (considered as ‘1’) from three independent experiments. (c) TRO analysis. TRO assays were done on permeabilized cell fractions harbouring the intact nuclei from AS isolates at the indicated times after addition of inducers. The labelled nuclear run-on RNA for each indicated time point was hybridized to ACT1 and CaCDR1 specific PCR amplicon, which were immobilized by dot-blotting on to a nylon membrane. Relative positions of the individual dot-blots on the nylon membrane are shown in the scheme (middle-top). Un, uninduced.

Figure 3
Kinetics of CaCDR1 expression in induced AS isolates

(a) Northern-blot analysis. AS isolates were exposed to inducers over a time interval of 0–120 min. Equal RNA loading and transfer were monitored by subsequently de-probing and rehybridization of membranes with ACT1 probe. (b) β-Galactosidase reporter assay. CaCDR1 promoter-driven β-galactosidase reporter analysis of AS isolates after exposure to inducers over a time interval of 0–120 min was measured. Data represent the fold induction of CaCDR1 promoter-driven reporter activity as compared with untreated cells (considered as ‘1’) from three independent experiments. (c) TRO analysis. TRO assays were done on permeabilized cell fractions harbouring the intact nuclei from AS isolates at the indicated times after addition of inducers. The labelled nuclear run-on RNA for each indicated time point was hybridized to ACT1 and CaCDR1 specific PCR amplicon, which were immobilized by dot-blotting on to a nylon membrane. Relative positions of the individual dot-blots on the nylon membrane are shown in the scheme (middle-top). Un, uninduced.

CaCDR1 transcript stability does not change in induced AS isolates

Because the increase in steady-state CaCDR1 mRNA was due to de novo mRNA synthesis, we explored if changes in its mRNA decay rates, could also contribute to its transiently induced levels. For determining the CaCDR1 mRNA t½ in uninduced and induced AS strains, we treated cells with thiolutin to inhibit in vivo transcription [8]. Total RNA was isolated at different time points after blocking the transcription and was analysed by RNA gel blots (Figure 4a). After probing the blots with a CaCDR1-specific probe, CaCDR1 mRNA could be detected in both the induced AS isolates at time T0 and the signal intensity diminished progressively with time at a rate (mRNA t½ was ~60 min) similar to that of uninduced AS isolates (Figure 4b). Neither of the induced AS isolates showed any significant change in poly(A) tail length of CaCDR1 3′-UTR (Table 4; a maximum variation of ±2 bases was observed between different clones). A minimum of ten random clones were subjected to sequencing to determine the precise poly(A) tail length.

CaCDR1 mRNA decay assay on exposure to inducers

Figure 4
CaCDR1 mRNA decay assay on exposure to inducers

Exponentially grown cultures of C. albicans were treated with inducers and subsequently incubated with the optimized thiolutin concentration (40 μg/ml) to inhibit ongoing in vivo transcription. Total RNA was isolated at the times indicated thereafter and fractionated on a 1% (w/v) agarose/2.2 M formaldehyde denaturing gel. (a) The gel was stained with ethidium bromide before blotting to monitor equal loading of the RNA and subsequently blotted on to a charged nylon membrane. The blot was hybridized with [α-32P]dATP-labelled CaCDR1 specific probe. Time points in minutes are indicated below the equal loaded RNA gels. (b) The hybridization signals were quantified using densitometry scanning in a phosphoimager scanner. The signal intensity at each time point was normalized to that of time T0 (expressed as a percentage) and plotted as a line graph. Un, uninduced.

Figure 4
CaCDR1 mRNA decay assay on exposure to inducers

Exponentially grown cultures of C. albicans were treated with inducers and subsequently incubated with the optimized thiolutin concentration (40 μg/ml) to inhibit ongoing in vivo transcription. Total RNA was isolated at the times indicated thereafter and fractionated on a 1% (w/v) agarose/2.2 M formaldehyde denaturing gel. (a) The gel was stained with ethidium bromide before blotting to monitor equal loading of the RNA and subsequently blotted on to a charged nylon membrane. The blot was hybridized with [α-32P]dATP-labelled CaCDR1 specific probe. Time points in minutes are indicated below the equal loaded RNA gels. (b) The hybridization signals were quantified using densitometry scanning in a phosphoimager scanner. The signal intensity at each time point was normalized to that of time T0 (expressed as a percentage) and plotted as a line graph. Un, uninduced.

Table 4
Representative CaCDR1 transcript t½ and its corresponding poly(A) tail length

CaCDR1 transcript t½* and its poly(A) tail length† in both the mentioned AS and AR isolates have been reported earlier [8,18].

StrainsCaCDR1 transcript t½ (min)CaCDR1 transcript poly(A) tail length
Gu4   
 Un-induced ~ 60* [826±2† [18
Gu4-induced   
 +FPZ ~ 60 25±2 
 +PRG ~ 60 25±2 
 +β-EST ~ 60 25±2 
Gu5 > 180* [835±2† [18
DSY294   
 Un-induced ~ 60 24±2† [18
DSY294-induced   
 +FPZ ~ 60 22±2 
 +PRG ~ 60 23±2 
 +β-EST ~ 60 24±2 
DSY296 > 180* [836±2† [18
StrainsCaCDR1 transcript t½ (min)CaCDR1 transcript poly(A) tail length
Gu4   
 Un-induced ~ 60* [826±2† [18
Gu4-induced   
 +FPZ ~ 60 25±2 
 +PRG ~ 60 25±2 
 +β-EST ~ 60 25±2 
Gu5 > 180* [835±2† [18
DSY294   
 Un-induced ~ 60 24±2† [18
DSY294-induced   
 +FPZ ~ 60 22±2 
 +PRG ~ 60 23±2 
 +β-EST ~ 60 24±2 
DSY296 > 180* [836±2† [18

DISCUSSION

Our present work addresses and differentiates the induced drug-resistance mechanism(s) from acquired resistance of C. albicans. We show by spot and checkerboard titre assays that antagonism between inducers (FPZ, PRG and β-EST) of efflux pump encoding genes, CaCDR1/CaCDR2 and tested drugs (FLC, KTC, MCZ and CHX) reduces the susceptibility of AS isolates mainly via up-regulation of CaCDR1 (Figures 1a–1c and Table 3). The spot assays of Δcdr1 showed that despite induced CaCDR2 transcript levels, there was no significant increase in drug resistance (Figure 1a), indicating that, in contrast to CaCDR1, the contribution of CaCDR2 in the development of in vitro-induced drug resistance is limited. These results are in agreement with earlier reports where in contrast to CaCDR2 expression, a relatively higher transient inducible response of CaCDR1 transcript is observed in FLC-susceptible isolates [10].

Although in a previous study by Coste et al. [14], it has been reported that CaCDR1/CaCDR2 can be up-regulated in AS isolates by exposing the cells transiently to drug (FPZ) and steroid (β-EST). Accordingly, a 22-bp DRE containing two 6-bp repetitive elements with 5′-CGG-3′ triplets and corresponding TF, TAC1 has been shown to be required for the transient induction of CaCDR1/CaCDR2 [14]. However, the functionality and molecular mechanisms of the transiently induced CaCDR1 in regulating the induced MDR phenotype of AS isolates remains to be examined. Thus, considering that CaCDR1 plays a major role in induced MDR in C. albicans, this study is focused to dissect the molecular mechanism underlying the transiently induced up-regulation of its transcript levels.

Our present analysis of CaCDR1 up-regulation in clinical AS isolates revealed that the tested inducers activate a transient and reversible up-regulation of CaCDR1 which is solely contributed by its transcriptional activation. The reporter gene transcription and TRO assays confirmed the enhanced transcriptional rate of CaCDR1 in induced AS isolates (Figures 2a and 2b). This also matched well with the occurrence of drug and steroid responsive DRE sequence in its proximal promoter [10]. Subsequent, kinetic analysis revealed that induced transcriptional up-regulation of CaCDR1 was reversible as its expression reverted back to basal level within 2 h exposure of cells to the inducers (Figures 3a–3c). The overexpressed CaCDR1 as well as CaCDR2 probably lead to enhanced efflux of tested inducers resulting in its reduced intracellular concentration. We hypothesized that there might exist a negative feedback mechanism where the inducer itself shuts down its effect; however, we do not have data to support this notion. This is indeed in contrast to MDR phenotype of corresponding AR isolates, where sustained higher CaCDR1 transcript levels are maintained not only by its transcriptional activation but also by post-transcriptional events involving enhanced stability and hyperadenylation of its transcript [18].

Although transcriptional regulation seems to be the major control point, post-transcriptional mechanism such as the mRNA stability has also been shown to regulate the transient-induced MDR phenotype in many human cell lines. For example, T-lymphoblastoid MDR cell line (CCRF VCR 100) show relatively increased mRNA stability of hMDR1 after actinomycin D exposure, with no change in its transcription rate [30]. In leukaemic cell lines, an increased mRNA level of hMDR1 on transient exposure to a variety of cytotoxic drugs, during liver regeneration, and on depletion of mitochondrial DNA in HCT-8 colon cancer cell lines was reported to be solely due to its enhanced mRNA stability [22,31,32]. In another study, CHX induction increased the length of 3′-UTR of class II P-gp transcript levels; however, whether this led to an enhanced mRNA stability was not examined in that study [33]. Notably, an increase in EhPgp5 mRNA levels of resistant phenotype of Entamoeba histolytica trophozoites at high emetine concentration was contributed by its transcriptional activation as well as by enhanced mRNA t½, along with the lengthening of poly(A) tail [34]. However, our post-transcriptional analysis of induced AS strains revealed no change in CaCDR1 mRNA stability (Figures 4a and 4b) or in its 3′-UTR polyadenylation status (Table 4). Thus, the effects of inducers in up-regulating CaCDR1 transcript levels are restricted solely to its transcriptional activation.

From the matched pair of AS and AR strains, it appears likely that a stepwise accumulation of mutation(s)/recombination(s) could occur within AS isolates during azole chemotherapy, resulting in an enhanced transcript levels of CaCDR1. That a hyperactive gain-of-function mutation (N977D) in TAC1 causes CaCDR1 up-regulation in one of our studied AR isolates (DSY296) would support this notion [35]. Notably, CaMDR1 and CaERG11 overexpression in other AR isolates have also been reported to be due to hyperactive mutations in their corresponding TFs MRR1 (P683S, G997V) and UPC2 (G648D, A643T) respectively [3638]. Earlier Karababa et al. [39] had compared gene expression profiles of FPZ-induced AS isolates and CaCDR1 overexpressing AR isolates and showed that in vitro-induced gene expression only partly mimics the expression profile of AR isolates [39]. Thus, there are differences in regulatory mechanisms between induced versus acquired drug resistance of clinical AS and AR isolates respectively. In this context, our results show differences in regulations that exist between induced AS and uninduced AR isolates at the level of a single major MDR gene, CaCDR1. Our results, therefore, not only make a first-time distinction between induced and acquired activation of CaCDR1 transcript levels but also opens possibilities for the development of therapeutic strategies to target either in vitro or in vivo developed drug-resistance in C. albicans. In this context, it should be noted that the intervention of overexpressing P-gp in MDR cell lines by natural and synthetic inhibitors has already been reported to be due to its specific transcriptional down-regulation [4043].

Abbreviations

     
  • AR

    azole-resistant

  •  
  • AS

    azole-susceptible

  •  
  • β-EST

    β-oestradiol

  •  
  • CHX

    cycloheximide

  •  
  • DEPC

    diethyl pyrocarbonate

  •  
  • DRE

    drug response element

  •  
  • FIC

    fractional inhibitory concentration

  •  
  • FICI

    FIC index

  •  
  • FLC

    fluconazole

  •  
  • FPZ

    fluphenazine

  •  
  • KTC

    ketaonazole

  •  
  • MCZ

    miconazole

  •  
  • MDR

    multi-drug resistance

  •  
  • MIC

    minimal inhibitory concentration

  •  
  • MIC80

    MIC for 80% of input cells

  •  
  • ORF

    open reading frame

  •  
  • PAP

    poly(A) polymerase

  •  
  • PAT

    polyadenylation test

  •  
  • P-gp

    P-glycoprotein

  •  
  • poly(A)+

    polyadenylated

  •  
  • PRG

    progesterone

  •  
  • RACE

    rapid amplification of cDNA ends

  •  
  • R6G

    rhodamine6G

  •  
  • RT–PCR

    reverse transcription–PCR

  •  
  • t½

    half-life

  •  
  • TAC1

    transcriptional activator of CDR genes 1

  •  
  • TF

    transcription factor

  •  
  • TRO

    transcription run on

  •  
  • UTR

    untranslated region

  •  
  • WT

    wild-type

  •  
  • YEPD

    yeast extract peptone dextrose

We thank Joachim Morschhäuser (Institut für Molekulare Infektionsbiologie, Universität Würzburg, Germany) and Dominique Sanglard (Institute of Microbiology, University Hospital Lausanne, Switzerland) for providing the clinical isolates Gu4/Gu5 and DSY294/DSY296 respectively. Thiolutin (CP-4092) and FLC were gifts from Pfizer (Groton, CT, U.S.A.) and Ranbaxy Laboratories (New Delhi, India) respectively.

FUNDING

The work presented in this paper has been supported in part by grants to R.P. from Indo-Swiss [INT/SWISS/P-31/2009]; Department of Science and Technology [SR/SO/BB-34/2008]; and Department of Biotechnology [BT/PR9100/Med/29/03/2007 and BT/PR11158/BRB/10/640/2008].

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Supplementary data