Candida glabrata Transcription Factor Rpn4 Mediates Fluconazole Resistance through Regulation of Ergosterol Biosynthesis and Plasma Membrane Permeability

Based on the previous identification of Rpn4 as a member of the multidrug resistance network in several yeast species, the role of the uncharacterized CgRPN4 gene in azole resistance was determined. Figure 1A shows that deleting CgRPN4 increases sensitivity to 6 different azole drugs compared to the KUE100 wild-type strain. Interestingly, the overexpression of CgRPN4 in the L5U1 wild-type C. glabrata strain increased its tolerance to fluconazole and ketoconazole (Fig. 1B). Besides susceptibility to azole antifungals, phenotypes to antifungals from other families, amphotericin B and flucytosine (5-FC), were tested. Susceptibility to the fungicide mancozeb was also tested. Other than antimycotic agents, susceptibility to temperature, osmotic stress, and oxidative stress was also tested. The deletion of CgRPN4 was seen to increase susceptibility only to hydrogen peroxide (Fig. 1A). These results indicate that CgRPN4 plays a primary role in mediating azole resistance.

The MICs of several antifungals were also used to quantitatively assess drug susceptibility variability between the wild-type strain and the derived Δcgrpn4 mutant (Table 1). Consistent with previous results, no MIC differences were attained for amphotericin B or 5-FC. The susceptibility profiles against the imidazole ketoconazole and the triazoles fluconazole, itraconazole, and posaconazole were also determined. The deletion of CgRPN4 led to an 8-fold increase in susceptibility to ketoconazole and posaconazole and a 2-fold increase in susceptibility to itraconazole. The clinical outcome of these data cannot be directly estimated due to the absence of breakpoints for these antifungals; however, the observed shifts in susceptibility support a relevant role of CgRpn4 in multiple-azole resistance. The deletion of the TF also resulted in a 4-fold increase in susceptibility to fluconazole. Both strains are categorized as intermediately susceptible to fluconazole, with CgRpn4 playing a measurable role in the susceptibility profile.

We used transcriptional profiling to determine the response of the KUE100 wild-type strain to treatment with the previously determined inhibitory concentration of 150 mg/liter fluconazole. Notably, this concentration of fluconazole is higher than that attained by MIC assays, as it was found to be an inhibitory concentration under the conditions used for cell growth to perform the remaining experiments. Resistance measurements can vary according to the experimental conditions that are used (e.g., cell density, agitation, temperature, and medium), and cell density has been correlated with antifungal resistance (38). Control experiments were performed with reduced cell and fluconazole concentrations to validate the same inhibitory effect with drug concentrations more closely related to those found under physiological conditions (see Fig. S1 in the supplemental material). The fact that the remaining experiments were conducted using a much higher cell concentration than the one used in MIC assays might justify the need for a higher concentration of the antifungal. The expression levels of 44 genes were altered in C. glabrata cells following exposure to fluconazole for 1 h (log₂-fold change of greater than 0.5 or less than −0.5; Benjamini-Hochberg-adjusted P value of <0.05) (Table 2; Table S1). The expression levels of 29 (66%) genes were increased, whereas the expression levels of 15 (34%) were decreased. The genes fall into 11 functional groups: drug resistance, sterol metabolism, and intracellular traffic, which are associated exclusively with the upregulated genes; lipid and fatty acid metabolism; nitrogen metabolism; carbon metabolism; heme homeostasis; mitochondrial function; the stress response; the cytoskeleton/cell cycle; and unknown function. As expected, the expression of the multidrug resistance transporter-encoding gene CDR1, a well-known biomarker of azole resistance (39–41), was upregulated 1.75-fold in fluconazole-challenged cells. Additionally, 41% of the upregulated genes are related to sterol metabolism, including components of the ergosterol biosynthetic pathway (ERG1, ERG2, ERG3, ERG4, ERG5, and ERG6) and the azole target ERG11. The upregulation of ERG11 in response to azole stress in C. glabrata and other Candida spp. has been reported previously (42–45), as has the upregulation of additional ERG genes in cases of azole resistance (46, 47). Two more genes with predicted roles in ergosterol biosynthesis, namely, HES1 and CYB5, are also present in this group. The upregulation of the ergosterol pathway is therefore likely a core response to mild fluconazole stress.

In turn, genes involved in the heme biosynthetic pathway are upregulated (HEM13 and HEM14), while a gene involved in heme degradation is downregulated (HMX1). This profile suggests that the increased synthesis of heme, a vital prosthetic group of Erg11, may be required to accompany the increased expression of Erg11, aiming at increased levels of functional Erg11 molecules.

In order to study the role of CgRpn4 in the response of C. glabrata to fluconazole, gene expression changes occurring upon fluconazole exposure in the Δcgrpn4 mutant strain were compared to those observed in the wild-type strain. The expressions of 80 genes were found to be activated by CgRpn4, while 132 genes were found to be repressed, possibly in an indirect fashion, upon fluconazole exposure (Table S2). The most prevalent functional groups activated by CgRpn4 include the proteasome and ubiquitination, lipid and fatty acid metabolism, and the stress response (Fig. 2A), whereas repressed genes are enriched in cell wall organization and carbon metabolism (Fig. 2B).

The comparison of this regulon with differentially expressed genes in fluconazole-challenged wild-type cells enabled the identification of 18 genes that respond to fluconazole only when CgRPN4 is present (Table 2). The most enriched functional group in this set comprises genes from the ergosterol biosynthetic pathway, namely, ERG1, ERG2, ERG3, and ERG11. A putative gene involved in the regulation of ergosterol synthesis in S. cerevisiae (HES1) (48, 49) is also found in this set. Interestingly, two genes involved in heme biosynthesis are also present: HEM13 and HEM14. As described previously, heme is a key prosthetic group of several ergosterol biosynthesis enzymes, and increased Hem13 protein levels were detected in an azole-resistant strain, concurrently with Erg11 (41). It is important to note that the upregulation of ergosterol biosynthesis is the most dramatic response to fluconazole stress and that CgRpn4 functions as an activator of ergosterol and heme biosynthesis (Fig. 3A). Moreover, the activation of ERG and HEM genes constitutes a specific role of this TF during fluconazole stress, as CgRpn4 does not regulate their basal expression (Table S3). Altogether, these data indicate that the activation of the ergosterol biosynthetic pathway, mediated by CgRpn4, could be a major regulatory mechanism of azole antifungal resistance. The transcriptome data were validated by real-time PCR (RT-PCR) quantification of CgERG11 expression in the wild type and the Δcgrpn4 mutant. The attained CgERG11 activation after 1 h of fluconazole exposure is comparable to the expression levels determined by the transcriptomics approach, as was the decreased fold change in the mutant strain (Fig. 3B).

CgRpn4 was found to regulate the expression of several additional genes irrespective of fluconazole treatment. The largest functional group includes proteasome and ubiquitination genes. This group comprises exclusively genes activated by CgRpn4, in accordance with its role in both C. albicans and S. cerevisiae as an activator of proteasome genes (27, 28, 50). The high enrichment of proteasome subunit-encoding genes in the CgRpn4 regulon strongly indicates that its physiological function is conserved with other species.

Genes encoding five multidrug resistance transporters are repressed by CgRpn4: the major facilitator superfamily (MFS) transporters encoded by CAGL0L10912g, CAGL0B02343g, TPO1_1, and QDR2 as well as the ABC transporter YOR1. Of these, YOR1, TPO1_1, and QDR2 have been implicated in azole resistance (18, 19, 22). This indicates that CgRpn4 does not activate the expression of drug transporters, reinforcing its role as an activator of ergosterol biosynthesis as the main mechanism of fluconazole resistance.

In order to examine possible CgRpn4 activation mechanisms, we investigated its subcellular localization. CgRpn4 was fused to green fluorescent protein (GFP), expressed via the pGREG576_MTI_CgRPN4 plasmid in C. glabrata cells grown to mid-exponential phase in basal medium (BM) supplemented with 50 μM Cu₂SO₄ to induce fusion protein expression, and then transferred to fresh medium (control) or to fresh medium containing 150 mg/liter fluconazole. After 1 h of incubation, cells were inspected by fluorescence microscopy.

In untreated C. glabrata cells, the CgRpn4_GFP fusion protein is distributed throughout the whole cell, with some level of nuclear signal (Fig. 4A). After fluconazole stress, an enrichment of nuclear localization was observed (Fig. 4B and C). Fluconazole treatment therefore changes the relative distribution of CgRpn4 to the nucleus. The activation of its target genes could be partially dependent on the translocation of the transcription factor. In S. cerevisiae, Rpn4 protein levels are regulated by the proteasome in a negative-feedback loop. Therefore, a decrease in the CgRpn4_GFP signal could occur due to the activation of proteasome genes by the transcription factor. However, our localization data appear to show steady levels of CgRpn4 production. To evaluate if the levels of CgRpn4 are being affected by a negative-feedback loop, Western blotting was performed before and after fluconazole treatment (Fig. 4D). The CgRpn4_GFP fusion protein could be detected at similar levels under both experimental conditions, which is consistent with the localization data. This shows that CgRpn4 plasmid-driven production contributes to the protein steady state.

Transcriptomics analysis of the KUE100 wild-type C. glabrata strain during fluconazole stress revealed the significant activation of the ergosterol biosynthesis pathway mediated by CgRpn4. This led us to hypothesize that CgRpn4 may contribute to preserving ergosterol levels in C. glabrata upon fluconazole exposure.

C. glabrata cells were grown to the mid-exponential phase and transferred to fresh medium (control) or fresh medium containing 150 mg/liter fluconazole. After 4 h and 12 h of incubation, cells were collected, total ergosterol was extracted, and its levels were quantified by high-performance liquid chromatography (HPLC).

Deleting CgRPN4 does not affect ergosterol levels in untreated cells (Fig. 5A). The absence of CgRPN4 reduces ergosterol levels upon fluconazole stress for 4 h or 12 h. Ergosterol levels in the wild type decreased only after 12 h and to levels comparable to those of the deletion strain, which indicates a relevant role of the transcription factor in this mechanism. Altogether, these data implicate CgRpn4 in the maintenance of the ergosterol content upon early fluconazole exposure. Moreover, these results show that CgRpn4, through transcriptional regulation of the ergosterol biosynthesis pathway, has a measurable effect on ergosterol levels during fluconazole stress, which contributes to antifungal resistance.

Cell permeability in response to fluconazole was investigated using the fluorescent probe propidium iodide (PI), and the possible participation of CgRpn4 was evaluated. Upon 1 h of exposure to fluconazole, C. glabrata cell permeability increases significantly (Fig. 5B). This observation is probably related to the inhibition of Erg11 by fluconazole, described previously to lead to the accumulation of the toxic sterol DMCDD that permeabilizes the plasma membrane (51, 52). The permeability of untreated Δcgrpn4 cells is not significantly different from that of wild-type cells. However, the permeability of Δcgrpn4 cells is significantly higher than that of the wild-type strain following fluconazole stress (Fig. 5B). These data indicate that fluconazole increases C. glabrata cell permeability and that CgRpn4 contributes to controlling its maintenance during fluconazole exposure, presumably by the upregulation of ergosterol biosynthesis.

The role of CgRPN4 in ergosterol biosynthesis and plasma membrane permeability led us to investigate if this could be related to the increased azole susceptibility observed in the Δcgrpn4 mutant. Consistent with the observed susceptibility and cell permeability phenotypes, the Δcgrpn4 deletion mutant was found to accumulate 2.5-fold more radiolabeled fluconazole than the wild-type strain after 20 min of exposure to the antifungal (Fig. 5C). These results suggest that CgRpn4 activity mediates C. glabrata resistance to fluconazole by reducing its intracellular accumulation in yeast cells, possibly as a result of its regulation of ergosterol levels and membrane permeability.

In order to identify possible CgRpn4 DNA recognition sites, the promoters of CgRpn4-activated genes during fluconazole stress were searched for enriched motifs using DREME (53). Excluding TATA box sequences, 4 overrepresented motifs were found (Fig. 6A). Interestingly, one of the identified motifs (TGGCAAA) is identical or nearly identical to a core region of the ScRpn4 and CaRpn4 consensus (GGTGGCAAA and GAAGGCAAAA, respectively) found in promoters of proteasome genes (28, 50). This indicates that there is a high level of conservation in the promoter sequences recognized by Rpn4 across these species.

We found a nearly identical motif (TTGCAAA) located at positions −363 to −356 (Fig. 6B) of CgERG11. To determine if this motif is required for the activation of CgERG11, the CgERG11 promoter was placed upstream of the lacZ reporter gene, and site-directed mutagenesis was used to disrupt the putative binding site.

Upon 1 h of fluconazole exposure, lacZ expression driven by the CgERG11 promoter increased 2-fold in comparison to control conditions (Fig. 6C), which is in accordance with our transcriptomics data. When the TTGCAAA motif in the CgERG11 promoter was disrupted by 2 nucleotide substitutions (from TTGCAAA to TAACAAA), lacZ expression was reduced by 20-fold compared to the wild-type promoter under control conditions and even more so after fluconazole exposure (59-fold) (Fig. 6C). These results indicate that the identified motif is required for CgERG11 basal expression and especially CgERG11 activation during fluconazole stress. Chromatin immunoprecipitation (ChIP) followed by RT-PCR was used to establish a direct link between CgRpn4 and the identified CgERG11 promoter motif using a CgRpn4_c-Myc fusion protein expressed via the pGREG526_PDC1_CgRPN4 plasmid. The results show CgRpn4 can bind the CgERG11 promoter in the region containing the TTGCAAA motif (Fig. 6D). The promoter is bound by CgRpn4 under control conditions and increases its promoter occupancy during fluconazole stress, strongly suggesting direct CgRpn4 binding to this essential motif in the CgERG11 promoter.

Candida glabrata parental strains KUE100 (58) and L5U1 (cgura3Δ0 cgleu2Δ0) (59) and derived single-deletion mutants were batch cultured at 30°C at 250 rpm in basal medium (BM) (20 g/liter glucose, 2.7 g/liter ammonium sulfate, and 1.7 g/liter yeast nitrogen base [YNB] without ammonia or amino acids) or RPMI 1640–2% glucose medium at pH 7.0. For BM assays using L5U1 transformed with either the pGREG576 or pGREG526 plasmid (harboring a URA3 selection marker), the medium was supplemented with 60 mg/liter leucine. The plasmids pGREG576 and pGREG526 were obtained from the Drag&Drop collection (60). The Saccharomyces cerevisiae BY4741 strain (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) was batch cultured at 30°C at 250 rpm in yeast extract-peptone-dextrose (YPD) medium (20 g/liter glucose, 20 g/liter peptone, 10 g/liter yeast extract).

The deletion of C. glabrata RPN4 addressed in this study was carried out in the parental strain KUE100 using the method described previously by Ueno et al. (61). The primers used are presented in Table S4 in the supplemental material.

The pGREG576 and pGREG526 plasmids from the Drag&Drop collection were used as described previously to clone and express the C. glabrata open reading frame (ORF) CAGL0K01727g (18, 22, 62–65), giving rise to pGREG576_CgRPN4 or pGREG526_CgRPN4. The GAL1 promoter present in each plasmid was replaced by the copper-inducible MTI C. glabrata promoter (pGREG576_CgRPN4) or the constitutive PDC1 C. glabrata promoter (pGREG526_CgRPN4) (66), giving rise to the pGREG576_MTI_CgRPN4 or pGREG526_PDC1_CgRPN4 plasmid. The primers used are presented in Table S4. The recombinant plasmids were obtained through homologous recombination in S. cerevisiae and verified by DNA sequencing.

The susceptibility of the parental strain KUE100 to inhibitory concentrations of the selected drugs was compared to that of the deletion mutant KUE100_Δcgrpn4 by spot assays. The ability of CgRPN4 gene expression to increase wild-type resistance to the tested chemical stresses was also examined in the URA3⁻ L5U1 C. glabrata strain using the pGREG576_MTI_CgRPN4 centromeric plasmid.

Cell suspension preparation and spot assays were carried as described previously (18, 22, 62–65). The tested drugs included the following compounds, used in the specified concentration ranges that were found to exert inhibitory growth effects: the azole antifungal drugs ketoconazole (10 to 60 mg/liter), fluconazole (100 to 250 mg/liter), miconazole (0.10 to 0.50 mg/liter), itraconazole (15 to 30 mg/liter), clotrimazole (2.5 to 15 mg/liter), and tioconazole (0.30 to 0.70 mg/liter) (all from Sigma). Antifungals from other families included the polyene amphotericin B (0.05 to 0.20 mg/liter), the pyrimidine analog 5-FC (0.10 to 0.30 mg/liter), and the broad-activity fungicide mancozeb (0.5 to 1.5 mg/liter) (all from Sigma). The following osmotic and oxidative stresses were tested at levels found to exert inhibitory growth effects: NaCl (0.5 to 2 M) (from Panreac) and H₂O₂ (7.5 to 15 mM) (from Sigma), respectively. The MIC values of each antifungal were determined according to the EUCAST susceptibility testing method (67). Drugs not present in the reference method (e.g., ketoconazole) were used according to the same experimental procedures. The MIC assays were performed in 96-well plates containing RPMI 1640–2% glucose medium with the appropriate drug concentrations. Cells grown for 18 h were used to create an initial inoculum containing 5 × 10⁶ CFU/ml in distilled water, which was subsequently used to prepare a 5 × 10⁵-CFU/ml working suspension. The plates were then inoculated with the cell suspension to a final inoculum density of 2.5 × 10⁵ CFU/ml and incubated at 37°C for 24 h. Final cell growth was assessed by absorbance measurement at a 530-nm wavelength.

The subcellular localization of the CgRpn4 protein was determined based on the observation of L5U1 C. glabrata cells transformed with the pGREG576_MTI_CgRPN4 plasmid. These cells express the CgRpn4_GFP fusion protein, whose localization may be determined using fluorescence microscopy as described previously (18, 22, 62–65). C. glabrata cell suspensions were prepared in BM supplemented with leucine and 50 μM CuSO₄, until a standard culture optical density at 600 nm (OD₆₀₀) of 0.5 was reached, and transferred to the same medium with or without fluconazole. After 1 h of incubation, 1 drop of NucRed Live 647 was added to 1 ml of 4 × 10⁷ cells/ml, and cell suspensions were incubated in the dark with orbital agitation (30 min at 250 rpm). Cells were centrifuged (17,500 × g for 5 min), washed twice, and resuspended in phosphate-buffered saline (PBS) for final aliquots of 10⁷ cells/ml. The distribution of the CgRpn4_GFP fusion protein in yeast cells was determined by fluorescence microscopy with a Zeiss Axioplan microscope (Carl Zeiss MicroImaging) using excitation and emission wavelengths of 395 and 509 nm (GFP) or 541 and 686 nm (NucRed). Fluorescence images were captured using a cooled Zeiss AxioCam 503 color camera (Carl Zeiss Microscopy). For the determination of nuclear signal intensity, fluorescence intensity was defined as the average pixel-by-pixel intensity in the selected region of interest (nucleus) after the deduction of the cytoplasm pixel intensity. A minimum of 100 cells per experiment were used. The fluorescence images were background corrected by using dark-current images.

C. glabrata strains KUE100 and KUE100_Δcgrpn4 were grown in BM until mid-exponential phase. Subsequently, cells were transferred to fresh medium (control) or fresh medium containing 150 mg/liter fluconazole and harvested after 1 h of incubation. Total RNA was isolated using an Ambion RiboPure yeast RNA kit according to the manufacturer’s instructions.

Strand-specific RNA-seq library preparation and sequencing were carried out as a paid service by the next-generation sequencing (NGS) core of the Oklahoma Medical Research Foundation, Oklahoma City, OK. Paired-end reads (Illumina HiSeq 3000 PE150, 2 by 150 bp, with 2 Gb of clean data) were obtained from KUE100 and KUE100_Δcgrpn4. Two replicates of each sample were obtained from three independent RNA isolations, which were subsequently pooled. Sample reads were trimmed using Skewer (v0.2.2) (68) and aligned to the C. glabrata CBS138 reference genome, obtained from the Candida Genome Database (CGD) (http://www.candidagenome.org/), using TopHat (v2.1.1) (69) with the parameters –p 12 (number of threads), –g 1 (maximum number of times that a read can be mapped to the genome), –b2-very-sensitive (preset option), and –library-type fr-firststrand (to account for strand specificity). HTSeq (v0.7.1) (70) was used to count mapped reads per ORF. Differentially expressed genes were identified using DESeq2 (71), with an adjusted P value threshold of 0.05 and log₂-fold change thresholds of −0.5 and 0.5. Default parameters in DESeq2 were used. Candida albicans and Saccharomyces cerevisiae homologs were obtained from the Candida Genome Database and Saccharomyces Genome Database (SGD) (https://www.yeastgenome.org/), respectively.

Ergosterol was extracted from cells using methods adapted from the ones described previously by Gong et al. (72) and carried out as described previously (73). Cells were cultivated in RPMI 1640–2% glucose medium with orbital agitation (250 rpm) until a standard culture OD₆₀₀ of 5.0 was reached, harvested by centrifugation, and resuspended in 5 ml of methanol. One milliliter of a solution of 1 mg/ml of cholesterol (Sigma) was added as an internal standard to estimate the ergosterol extraction yield. Homogenization was carried out with glass beads for 30 s, followed by incubation at 320 rpm for 1 h. Each sample was then centrifuged, and 1.7 ml of the supernatant was collected, clarified, and stored until HPLC analysis. The extracts were separated in a 250-mm by 4-mm C₁₈ column (LiChroCART Purospher Star RP-18 end-capped 5-μm column) at 30°C. Samples were eluted in 100% methanol at a flow rate of 1 ml/min. The detection of cholesterol and ergosterol was performed using a UV-visible (UV-Vis) detector set at 282 and 210 nm, respectively. Under the conditions used, the retention time of cholesterol was 15.4 ± 0.4 min, while ergosterol was eluted at 12.5 ± 0.2 min. Subsequent quantification of the two lipids was performed using appropriate calibration curves. The results are shown as micrograms of ergosterol per milligram of wet cell weight.

Plasma membrane permeability was assessed by the passive uptake of propidium iodide (PI) (20 mM in dimethyl sulfoxide [DMSO]; Invitrogen). C. glabrata cell suspensions from strains KUE100 and KUE100_Δcgrpn4 were prepared in BM until a standard culture OD₆₀₀ of 0.5 was reached and transferred to the same medium with or without 150 mg/liter fluconazole. After 1 h of incubation, PI was added to 1 ml of 4 × 10⁷ cells/ml to a final concentration of 20 μM, and cell suspensions were incubated in the dark with orbital agitation (15 min at 250 rpm). Cells exposed to PI were centrifuged (17,500 × g for 5 min), washed twice, and resuspended in PBS for final aliquots of 10⁷ cells/ml. PI fluorescence was detected by fluorescence microscopy with a Zeiss Axioplan microscope (Carl Zeiss MicroImaging), using excitation and emission wavelengths of 536 and 595 nm, respectively. Fluorescence images were captured using a cooled Zeiss AxioCam 503 color camera (Carl Zeiss Microscopy), and the images were analyzed with ZEN lite software from Zeiss Microscopy. The cell-to-cell fluorescence intensity was defined as the average pixel-by-pixel intensity in the selected region of interest, and a minimum of 100 cells per experiment were used. The fluorescence images were background corrected by using dark-current images.

[³H]fluconazole transport assays were carried out as described previously for other radiolabeled compounds (18, 22, 62–65). The internal accumulation of fluconazole was determined by calculating the ratio of the radiolabeled fluconazole measured within the yeast cells to that in the external medium (intracellular/extracellular). The parental strain KUE100 and the mutant strain KUE100_Δcgrpn4 were grown in BM until mid-exponential phase and harvested by filtration. Cells were washed and resuspended in BM to obtain dense cell suspensions (OD₆₀₀ = 0.5, equivalent to approximately 1.57 mg [dry weight] ml⁻¹). Readily, 0.1 μM [³H]fluconazole (1 mCi/ml) (Moravek Inc.) and 150 mg/liter of unlabeled fluconazole were added to the cell suspensions. Incubation proceeded for an additional 20 min. The intracellular accumulation of labeled fluconazole was monitored by filtering 200 μl of the cell suspension, at adequate time intervals, through prewetted glass microfiber filters (Whatman GF/C). The filters were washed with ice-cold TM buffer [0.1 M 2-(N-morpholino)ethanesulfonic acid (Sigma)/41 mM Tris (Sigma)], and radioactivity was measured in a Beckman LS 5000TD scintillation counter. Extracellular [³H]fluconazole was estimated by radioactivity assessment of 50 μl of the supernatant. Nonspecific [³H]fluconazole adsorption to the filters and to the cells (less than 5% of the total radioactivity) was assessed and taken into consideration. To calculate the intracellular concentration of labeled fluconazole, the internal cell volume (V_i) of the exponential cells, grown in the absence of the drug and used for accumulation assays, was considered constant and equal to 2.5 mg (dry weight) μl⁻¹ (74).

The promoters (bp −1000 to −1) upstream of the coding regions of genes whose expression was found to be activated by CgRpn4 were retrieved using the Retrieve Upstream Sequence tool from PathoYeastract (75). The obtained sequences were submitted to DREME (MEME suite) (53) for the discovery of enriched sequences, using default parameters. The identified motifs were then cross-checked with ScRpn4 and CaRpn4 motifs retrieved from the TF-Consensus List tool from YEASTRACT (76, 77) and PathoYeastract (75), respectively.

The pYPE354 plasmid was used as described previously to clone and express the lacZ reporter gene (18). pYEP354 contains the yeast selectable marker URA3 and the bacterial selectable marker ampR. CgERG11 promoter DNA was generated by PCR using genomic DNA extracted from the sequenced CBS138 C. glabrata strain and primers presented in Table S4. The first primer contains a region with homology within the beginning of the CgERG11 promoter and a recognition site for the EcoRI restriction enzyme, flanked by additional bases. The second primer contains a region with homology within the end of the CgERG11 promoter and the beginning of the CgERG11 coding sequence and a recognition site for the PstI restriction enzyme, flanked by additional bases. The amplified fragment was ligated into the pYEP354 vector (T4 ligase; New England BioLabs), previously cut with the same restriction enzymes, to obtain the pYEP354_CgERG11prom_lacZ plasmid. The putative CgRpn4 consensus sequence in the CgERG11 promoter was mutated by site-directed mutagenesis using the primers listed in Table S4. The designed primers contain two mutations within the potential consensus sequence, resulting in the production of the mutated consensus sequence by PCR amplification to obtain the pYEP354_mut_CgERG11prom_lacZ plasmid. The original template was then degraded by DpnI digestion.

The transcript levels of the CgERG11 or the lacZ reporter gene encoding β-galactosidase were determined by quantitative real-time PCR (RT-PCR). For CgERG11 expression measurements, the KUE100 and KUE100_Δcgrpn4 strains were grown in BM until mid-exponential phase, transferred to fresh medium (control) or fresh medium containing 150 mg/liter fluconazole (OD600 = 0.5), and harvested after 1 h of incubation. Samples were immediately frozen at −80°C until RNA extraction. For lacZ expression measurements, L5U1 cells transformed with the pYEP354_CgERG11prom_lacZ or pYEP354_mut_CgERG11prom_lacZ plasmid were grown in BM supplemented with leucine until mid-exponential phase. Fluconazole exposure, cell harvesting, and storage were performed as mentioned above. For total RNA extraction, the hot-phenol method was applied (78). RT-PCR was performed as described previously (18, 22, 62–65). The synthesis of cDNA for RT-PCR experiments, from total RNA samples, was performed using the MultiScribe reverse transcriptase kit (Applied Biosystems) and the 7500 RT-PCR thermal cycler block (Applied Biosystems) according to the manufacturer’s instructions. The quantity of cDNA for the following reactions was kept at around 10 ng. The subsequent RT-PCR step was carried out using SYBR green (NZYTech) reagents with default parameters established by the manufacturer and the primers in Table S4. The CgRDN25 gene transcript levels were used as an internal reference.

For Western blot analysis, L5U1 C. glabrata cells transformed with the pGREG576_MTI_CgRPN4 plasmid were grown to mid-exponential phase in BM supplemented with leucine and 50 μM Cu₂SO₄ to induce fusion protein expression and then transferred to fresh medium (control) or fresh medium containing 150 mg/liter fluconazole. After 1 h of incubation, cells were collected and washed twice with cold PBS (pH 7.4). For protein extraction, a TCA (trichloroacetic acid) precipitation protocol was used. Briefly, cell pellets were resuspended in resuspension buffer (1.85 M NaOH, 7.5% 2-mercaptoethanol) by vortexing, followed by a 2-min incubation on ice (5 times). Proteins were precipitated with 25% TCA using 5 cycles of vortexing and 2 min of incubation on ice, followed by centrifugation. The pellets were washed with acetone and resuspended in Laemmli buffer at 65°C for 20 min (with rotation), followed by centrifugation, and the supernatant containing the protein extract was collected. Samples were separated on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins were transferred to an Amersham Protran 0.2-μm nitrocellulose membrane (GE Healthcare). Immunoblotting was performed using a 1:200 dilution of mouse IgG anti-GFP antibody (catalog number sc-9996; Santa Cruz Biotechnology Inc.) and a 1:1,000 dilution of anti-mouse IgG BP-HRP (binding protein conjugated to horseradish peroxidase) (catalog number sc-516102; Santa Cruz Biotechnology Inc.). Detection was performed using Fusion Solo (Vilber) following incubation with an enhanced chemiluminescence (ECL) substrate.

L5U1 C. glabrata cells transformed with the pGREG526_PDC1_CgRPN4 plasmid were grown to mid-exponential phase in BM supplemented with leucine until 100 OD units were reached and then transferred to fresh medium (control) or fresh medium containing 150 mg/liter fluconazole. After 1 h of incubation, cells were cross-linked with 1% formaldehyde (15 min), and cross-linking was quenched with 340 mM glycine (10 min). Cells were collected, washed twice with cold PBS (pH 7.4), and frozen immediately at −80°C. Cell pellets were lysed with 0.5-mm zirconia beads (Invitrogen) in lysis buffer (50 mM HEPES [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.5% NP-40, 0.5 mM dithiothreitol [DTT]) by vortexing for 20 s and incubation on ice for 2 min (4 times). Cell debris was pelleted by centrifugation, and the lysate was collected. Lysates were sonicated four times for 2 min followed by 2 min on ice using a Bioruptor water bath sonicator. Sonicated lysates were cleared by centrifugation, and 5% of the sample was collected for use as the input (sonicated chromatin, not immunoprecipitated). For immunoprecipitation (IP), chromatin was incubated overnight at 4°C (with rotation) with protein G SureBeads (Bio-Rad) coupled with IgG anti-c-Myc antibody (catalog number sc-40; Santa Cruz Biotechnology Inc.). Beads were washed according to the manufacturer’s instructions, and the samples were eluted at 65°C for 20 min in Tris-EDTA (TE) buffer with 0.5% SDS. Input and immunoprecipitated samples were incubated overnight at 65°C for cross-linking reversal. DNA was purified with the NZYGelPure kit (NZYTech). Quantification of a specific CgERG11 promoter region was performed by RT-PCR using 5 μl of the DNA preparation from each reaction mixture. The primers used are listed in Table S4. Immunoprecipitated samples were processed together with the input samples, and the amplification product was normalized to the total template used. Based on methods reported previously (79, 80), the ratio of the immunoprecipitated promoter to the input (IP/input) was calculated, and ratios in fluconazole samples were compared to the ratio in the control samples at the specific locus of the CgERG11 promoter.

All experiments represent the averages from two or more independent experiments, with at least two technical replicates each. Error bars represent standard deviations. Statistical analyses were performed using one-way analysis of variance (ANOVA) with Tukey’s correction (comparisons of more than 2 groups) or two-tailed t tests with Welch’s correction (comparisons of 2 groups). Significance levels are indicated in the figure legends (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Raw data are available at the Gene Expression Omnibus (GEO) under accession number GSE129349.