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

The ability to acquire azole resistance is an emblematic trait of the fungal pathogen Candida glabrata. Understanding the molecular basis of azole resistance in this pathogen is crucial for designing more suitable therapeutic strategies. This study shows that the C. glabrata transcription factor (TF) CgRpn4 is a determinant of azole drug resistance. RNA sequencing during fluconazole exposure revealed that CgRpn4 regulates the expression of 212 genes, activating 80 genes and repressing, likely in an indirect fashion, 132 genes.

Investigation of the transcriptional regulation of antifungal resistance in C. glabrata is focused mostly on the role of the transcription factor (TF) CgPdr1, regarded as a master regulator of clinical acquisition of resistance to azole antifungals by activating the expression of multidrug resistance transporters (17)(18)(19)(20)(21)(22). In C. albicans, CaTac1 plays a similar role by regulating the expression of multidrug resistance transporters (23)(24)(25). However, the level of sequence similarity between CaTac1 and CgPdr1 is low. Apart from these pathways, knowledge about regulatory networks involved in the response to antifungal stress is scarce, especially for C. glabrata (26).
In this study, we identified the transcription factor Rpn4 as a regulator of fluconazole resistance in C. glabrata. In Saccharomyces cerevisiae, the Rpn4 homolog regulates the expression of proteasome genes, affecting proteasome activity and ubiquitin-mediated proteolysis (27)(28)(29)(30), and contributes to the unfolded protein response (UPR) (31). Rpn4 has also been identified as a member of the multidrug resistance network in S. cerevisiae (32,33). Consistently, the deletion of ScRPN4 results in increased sensitivity to fluconazole (34,35) and clotrimazole (36). Moreover, the expression of ScRPN4 and, consequently, the ubiquitin-proteasome system is regulated by the multidrug resistance regulators Pdr1 and Pdr3 (29). In C. albicans, RPN4 is also a regulator of proteasome genes, and its absence results in enhanced sensitivity to fluconazole (37). To the best of our knowledge, despite the apparent role of Rpn4 in azole resistance phenotypes, its actual role and mechanistic insights into antifungal resistance have not been addressed.
Given the identification of CgRPN4 as a new determinant of azole drug resistance in C. glabrata, its role in the transcriptome-wide response to fluconazole was assessed. The determination of Rpn4 target genes enabled the identification of a new regulatory pathway toward azole resistance, which may provide the molecular basis of Rpn4mediated azole resistance in pathogenic and nonpathogenic yeasts.

RESULTS
The transcription factor CgRPN4 is a determinant of fluconazole resistance in Candida glabrata. 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.
Transcriptome-wide changes in response to fluconazole stress in C. glabrata. 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 (log 2 -fold change of greater than 0.5 or less than Ϫ0.5; Benjamini-Hochberg-adjusted P value of Ͻ0.05) ( 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 wellknown biomarker of azole resistance (39)(40)(41), was upregulated 1.75-fold in fluconazolechallenged 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)(43)(44)(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.
Role of CgRpn4 in transcriptome-wide changes occurring in response to fluconazole in C. glabrata. 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 fluconazolechallenged 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   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. CgRpn4 is activated upon fluconazole stress, leading to its nuclear accumulation. 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 midexponential phase in basal medium (BM) supplemented with 50 M Cu 2 SO 4 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.
CgRPN4 plays a role in the maintenance of ergosterol levels, membrane permeability, and fluconazole accumulation. 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.
Determination of promoter recognition motifs by CgRpn4 and direct regulation of CgERG11 expression. 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 Ϫ363 to Ϫ356 (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 sitedirected 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.

DISCUSSION
C. glabrata is an emerging fungal pathogen with an impressive ability to acquire resistance to azole antifungal drugs. An understanding of the molecular basis of this phenotype is crucial for designing better-suited therapeutic approaches to tackle infections caused by C. glabrata.
In this study, we present a novel pathway used by C. glabrata to respond and adapt to azole drugs under the control of the transcription factor CgRpn4. Deletion of CgRPN4 increases C. glabrata susceptibility to multiple azole drugs. According to the latest EUCAST breakpoints, the attained MIC values represent decreased susceptibility to fluconazole, although not a shift from resistant to intermediate, as the parental strain used exhibits an intermediate fluconazole susceptibility profile. In the closely related yeast S. cerevisiae and in the pathogenic species C. albicans, Rpn4 is a regulator of proteasome genes (27,28,50). Defects in proteasome levels can potentially impact a wide range of phenotypes and play a role in the general stress response. To test this possibility, the susceptibility of the Δcgrpn4 deletion mutant to additional antifungal drugs and other stress agents was assessed. Notably, the results indicate that CgRpn4 functions mainly as a regulator of azole resistance.
RNA sequencing (RNA-seq)-based transcriptomics revealed that Rpn4 is also a regulator of proteasome genes in C. glabrata, thus unveiling a highly conserved physiological function among yeast species. Additionally, the activation of ERG genes, including CgERG11, was identified as a key underlying mechanism of CgRpn4-mediated fluconazole resistance. These findings are supported by our data as well as data from other studies showing ERG gene upregulation during azole stress (42)(43)(44)(45)(46)(47). Moreover, the upregulation of CgERG11 was found to be dependent on a promoter motif recognized and bound by CgRpn4. Together with the transcriptomics data, the essential role of the TTGCAAA motif in CgERG11 activation and the direct binding of CgRpn4 (especially under fluconazole exposure) to the CgERG11 promoter element provide evidence for a prevalent regulatory role in ergosterol biosynthesis regulation leading to fluconazole resistance.
The activation of the ergosterol biosynthesis pathways was seen to have a measurable impact on the ergosterol levels of C. glabrata during fluconazole exposure. The deletion of CgRPN4 leads to a reduction of ergosterol levels in the presence of fluconazole, indicating that the activation of ergosterol biosynthesis genes during fluconazole stress may be part of a compensatory mechanism to counteract ergosterol synthesis targeted by the antifungal. A similar mechanism has been reported for C. albicans, where fluconazole-tolerant strains present higher levels of intermediate metabolites of the sterol biosynthetic pathway and ergosterol ester, which was associated with the upregulation of ergosterol biosynthetic pathway genes (54). Furthermore, a reduction in the content of sphingolipids or ergosterol results in enhanced susceptibility to drugs (55)(56)(57). Consistent with this hypothesis, C. glabrata cells are more permeable and accumulate more intracellular fluconazole when CgRPN4 is deleted, which is in good agreement with the role of this TF in the activation of ergosterol biosynthesis. In S. cerevisiae, ScRPN4 is transcriptionally regulated under various stresses, and ScRpn4 protein levels are also controlled by proteasome activity in a negativefeedback loop (27). In our study, constant protein steady-state levels of CgRpn4 were detected, although it is possible that the efficient expression of the CgRpn4 protein by our expression system hindered the ability to detect turnover changes. As these data relate to the protein stability of CgRpn4, a possible feedback loop at the transcriptional regulation of CgRPN4 remains to be established.
Here, we show the role of the transcription factor CgRpn4 in mediating fluconazole resistance in C. glabrata. Through transcriptional control over the ergosterol biosynthesis pathway, CgRpn4 regulates ergosterol levels in the plasma membrane, thus affecting cell permeability and fluconazole accumulation. Additionally, CgRpn4 exerts direct control over CgERG11 expression, reinforcing its role as a relevant player in C. glabrata fluconazole resistance, which is likely conserved among other yeast species.
Based on these results, we propose that CgRpn4 is a promising target to tackle the acquisition of azole drug resistance.
Disruption of CgRPN4. 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.
Cloning of the C. glabrata CgRPN4 gene (ORF CAGL0K01727g). 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)(63)(64)(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.
Antifungal susceptibility assays. 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 Ϫ 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)(63)(64)(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 H 2 O 2 (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 ϫ 10 6 CFU/ml in distilled water, which was subsequently used to prepare a 5 ϫ 10 5 -CFU/ml working suspension. The plates were then inoculated with the cell suspension to a final inoculum density of 2.5 ϫ 10 5 CFU/ml and incubated at 37°C for 24 h. Final cell growth was assessed by absorbance measurement at a 530-nm wavelength.
CgRpn4 subcellular localization assessment. 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)(63)(64)(65). C. glabrata cell suspensions were prepared in BM supplemented with leucine and 50 M CuSO 4 , until a standard culture optical density at 600 nm (OD 600 ) 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 ϫ 10 7 cells/ml, and cell suspensions were incubated in the dark with orbital agitation (30 min at 250 rpm). Cells were centrifuged (17,500 ϫ g for 5 min), washed twice, and resuspended in phosphatebuffered saline (PBS) for final aliquots of 10 7 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.
Total RNA extraction. 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.
Library preparation and gene expression analysis. 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 log 2 -fold change thresholds of Ϫ0.5 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 quantification. 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 OD 600 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 C 18 column (LiChroCART Purospher Star RP-18 end-capped 5-m 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 Ϯ 0.4 min, while ergosterol was eluted at 12.5 Ϯ 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. 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 OD 600 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 ϫ 10 7 cells/ml to a final concentration of 20 M, and cell suspensions were incubated in the dark with orbital agitation (15 min at 250 rpm). Cells exposed to PI were centrifuged (17,500 ϫ g for 5 min), washed twice, and resuspended in PBS for final aliquots of 10 7 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.
[ 3 H]fluconazole accumulation assays. [ 3 H]fluconazole transport assays were carried out as described previously for other radiolabeled compounds (18,22,(62)(63)(64)(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 (OD 600 ϭ 0. 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) l Ϫ1 (74).
In silico prediction of overrepresented sequences in CgRpn4-activated promoters. The promoters (bp Ϫ1000 to Ϫ1) 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.
Cloning of the CgERG11 promoter and site-directed mutagenesis. 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