Untargeted Metabolomics of Nicotiana tabacum Grown in United States and India Characterizes the Association of Plant Metabolomes With Natural Climate and Geography.

Climate change and geography affect all the living organisms. To date, the effects of climate and geographical factors on plant metabolome largely remain open for worldwide and local investigations. In this study, we designed field experiments with tobacco (Nicotiana tabacum) in India versus USA and used untargeted metabolomics to understand the association of two weather factors and two different continental locations with respect to tobacco metabolism. Field research stations in Oxford, North Carolina, USA, and Rajahmundry, Andhra Pradesh India were selected to grow a commercial tobacco genotype (K326) for 2 years. Plant growth, field management, and leaf curing followed protocols standardized for tobacco cultivation. Gas chromatography-mass spectrometry based unbiased profiling annotated 171 non-polar and 225 polar metabolites from cured tobacco leaves. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) showed that two growing years and two field locations played primary and secondary roles affecting metabolite profiles, respectively. PCA and Pearson analysis, which used nicotine, 11 other groups of metabolites, two locations, temperatures, and precipitation, revealed that in North Carolina, temperature changes were positively associated with the profiles of sesquiterpenes, diterpenes, and triterpenes, but negatively associated with the profiles of nicotine, organic acids of tricarboxylic acid, and sugars; in addition, precipitation was positively associated with the profiles of triterpenes. In India, temperature was positively associated with the profiles of benzenes and polycyclic aromatic hydrocarbons, but negatively associated with the profiles of amino acids and sugar. Further comparative analysis revealed that nicotine levels were affected by weather conditions, nevertheless, its trend in leaves was independent of two geographical locations and weather changes. All these findings suggested that climate and geographical variation significantly differentiated the tobacco metabolism.

Assessment of germination, phytochemicals, and transcriptional responses to ethephon priming in soybean [Glycine max (L.) Merrill].

The present work aims to dissect the underlying signaling pathways associated with soybean [Glycine max (L.) Merrill] seed hormo-priming with ethephon (Eth). Our results demonstrated that soybean germination improved significantly upon Eth priming (Ethp). Phytohormone quantification shows relative enhanced endogenous gibberellin A4 (GA4) levels concomitant with impaired biogenesis and signaling of auxin, viz., indole acetic acid (IAA) and abscisic acid (ABA). Phytochemical analysis revealed relative reduced levels of individual and total raffinose family oligosaccharide (RFO) components, starch, soluble sugars, and sucrose concomitant with enhanced levels of reducing sugars, glucose, cellular ATP, and acetyl-CoA pools. Secondary metabolite analysis revealed the activation of the mevalonate (MVA) pathway with a concomitant suppression of the plastidal 2-methyl-d-erythritol-4-phosphate/1-deoxy-d-xylulose-5-phosphate (MEP/DOX) and phenylpropanoid pathways, substantiated by relative reduced levels of total phenolics, tannins, and proanthocyanidin. Ethp also enhances the in vitro antioxidative activity (viz., 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability and ferric reducing antioxidant power (FRAP)) and endogenous antioxidants levels (viz., flavonoids, isoflavones, ß-carotene, vitamin C, and vitamin E). Further quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed transcriptional pattern of representative genes in agreement with these metabolic alterations.

Control of Plasma Membrane Permeability by ABC Transporters.

ATP-binding cassette transporters Pdr5 and Yor1 from Saccharomyces cerevisiae control the asymmetric distribution of phospholipids across the plasma membrane as well as serving as ATP-dependent drug efflux pumps. Mutant strains lacking these transporter proteins were found to exhibit very different resistance phenotypes to two inhibitors of sphingolipid biosynthesis that act either late (aureobasidin A [AbA]) or early (myriocin [Myr]) in the pathway leading to production of these important plasma membrane lipids. These pdr5Δ yor1 strains were highly AbA resistant but extremely sensitive to Myr. We provide evidence that these phenotypic changes are likely due to modulation of the plasma membrane flippase complexes, Dnf1/Lem3 and Dnf2/Lem3. Flippases act to move phospholipids from the outer to the inner leaflet of the plasma membrane. Genetic analyses indicate that lem3Δ mutant strains are highly AbA sensitive and Myr resistant. These phenotypes are fully epistatic to those seen in pdr5Δ yor1 strains. Direct analysis of AbA-induced signaling demonstrated that loss of Pdr5 and Yor1 inhibited the AbA-triggered phosphorylation of the AGC kinase Ypk1 and its substrate Orm1. Microarray experiments found that a pdr5Δ yor1 strain induced a Pdr1-dependent induction of the entire Pdr regulon. Our data support the view that Pdr5/Yor1 negatively regulate flippase function and activity of the nuclear Pdr1 transcription factor. Together, these data argue that the interaction of the ABC transporters Pdr5 and Yor1 with the Lem3-dependent flippases regulates permeability of AbA via control of plasma membrane protein function as seen for the high-affinity tryptophan permease Tat2.

In vitro effect of malachite green on Candida albicans involves multiple pathways and transcriptional regulators UPC2 and STP2.

In this study, we show that a chemical dye, malachite green (MG), which is commonly used in the fish industry as an antifungal, antiparasitic, and antibacterial agent, could effectively kill Candida albicans and non-C. albicans species. We have demonstrated that Candida cells are susceptible to MG at a very low concentration (MIC that reduces growth by 50% [MIC(50)], 100 ng ml(-1)) and that the effect of MG is independent of known antifungal targets, such as ergosterol metabolism and major drug efflux pump proteins. Transcriptional profiling in response to MG treatment of C. albicans cells revealed that of a total of 207 responsive genes, 167 genes involved in oxidative stress, virulence, carbohydrate metabolism, heat shock, amino acid metabolism, etc., were upregulated, while 37 genes involved in iron acquisition, filamentous growth, mitochondrial respiration, etc., were downregulated. We confirmed experimentally that Candida cells exposed to MG resort to a fermentative mode of metabolism, perhaps due to defective respiration. In addition, we showed that MG triggers depletion of intracellular iron pools and enhances reactive oxygen species (ROS) levels. These effects could be reversed by the addition of iron or antioxidants, respectively. We provided evidence that the antifungal effect of MG is exerted through the transcription regulators UPC2 (regulating ergosterol biosynthesis and azole resistance) and STP2 (regulating amino acid permease genes). Taken together, our transcriptome, genetic, and biochemical results allowed us to decipher the multiple mechanisms by which MG exerts its anti-Candida effects, leading to a metabolic shift toward fermentation, increased generation of ROS, labile iron deprivation, and cell necrosis.

Molecular determinants of transient and reversible induced up-regulation of CaCDR1 in azole susceptible clinical isolates of Candida albicans.

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 ß-EST (ß-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 Δcdr1/Δcdr2 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.

Overcoming the heterologous bias: an in vivo functional analysis of multidrug efflux transporter, CgCdr1p in matched pair clinical isolates of Candida glabrata.

We have taken advantage of the natural milieu of matched pair of azole sensitive (AS) and azole resistant (AR) clinical isolates of Candida glabrata for expressing its major ABC multidrug transporter, CgCdr1p for structure and functional analysis. This was accomplished by tagging a green fluorescent protein (GFP) downstream of ORF of CgCDR1 and integrating the resultant fusion protein at its native chromosomal locus in AS and AR backgrounds. The characterization confirmed that in comparison to AS isolate, CgCdr1p-GFP was over-expressed in AR isolates due to its hyperactive native promoter and the GFP tag did not affect its functionality in either construct. We observed that in addition to Rhodamine 6 G (R6G) and Fluconazole (FLC), a recently identified fluorescent substrate of multidrug transporters Nile Red (NR) could also be expelled by CgCdr1p. Competition assays with these substrates revealed the presence of overlapping multiple drug binding sites in CgCdr1p. Point mutations employing site directed mutagenesis confirmed that the role played by unique amino acid residues critical to ATP catalysis and localization of ABC drug transporter proteins are well conserved in C. glabrata as in other yeasts. This study demonstrates a first in vivo novel system where over-expression of GFP tagged MDR transporter protein can be driven by its own hyperactive promoter of AR isolates. Taken together, this in vivo system can be exploited for the structure and functional analysis of CgCdr1p and similar proteins wherein the artefactual concerns encountered in using heterologous systems are totally excluded.

Regulation of yeast nutrient permease endocytosis by ATP-binding cassette transporters and a seven-transmembrane protein, RSB1.

Ceramide is produced by the condensation of a long chain base with a very long chain fatty acid. In Saccharomyces cerevisiae, one of the two major long chain bases is called phytosphingosine (PHS). PHS has been shown to cause toxicity in tryptophan auxotrophic strains of yeast because this bioactive ceramide precursor causes diversion of the high affinity tryptophan permease Tat2 to the vacuole rather than the plasma membrane. Loss of the integral membrane protein Rsb1 increased PHS sensitivity, which was suggested to be due to this protein acting as an ATP-dependent long chain base efflux protein. More recent experiments demonstrated that loss of the genes encoding the ATP-binding cassette transporter proteins Pdr5 and Yor1 elevated PHS tolerance. This increased resistance was suggested to be due to increased expression of RSB1. Here, we provide an alternative view of PHS resistance influenced by Rsb1 and Pdr5/Yor1. Rsb1 has a seven-transmembrane domain topology more consistent with that of a regulatory protein like a G-protein-coupled receptor rather than a transporter. Importantly, an rsb1Δ cell does not exhibit higher internal levels of PHS compared with isogenic wild-type cells. However, tryptophan transport is increased in pdr5Δ yor1 strains and reduced in rsb1Δ cells. Localization and vacuolar degradation of Tat2 are affected in these genetic backgrounds. Finally, internalization of FM4-64 dye suggests that loss of Pdr5 and Yor1 slows normal endocytic rates. Together, these data argue that Rsb1, Pdr5, and Yor1 regulate the endocytosis of Tat2 and likely other membrane transporter proteins.

Analysis of physico-chemical properties of substrates of ABC and MFS multidrug transporters of pathogenic Candida albicans.

In this study, we have explored the structure activity relationships of substrates of two major, promiscuous, multidrug transporters of an opportunistic human pathogen Candida albicans namely, CaCdr1p and CaMdr1p. To differentiate between substrates and non-substrates, the susceptibilities of the Saccharomyces cerevisiae strains over-expressing CaCdr1p or CaMdr1p were determined for 67 structurally diverse xenobiotics. A comparison of physico-chemical indices of these tested compounds enabled identification of molecular descriptors such as, degree of hydrophobicity (MLogP), geometrical descriptor (DISPv), molecular edge descriptor (MDEC.12 and MDEC.13) and 3D-Morse descriptors, that allowed their segregation into substrates and non-substrates for both the transporter proteins. Taken together, present study provides first evidence of chemical basis of substrate specificities of two clinically relevant multidrug transporters of an opportunistic human pathogen C. albicans.

Synergistic anticandidal activity of pure polyphenol curcumin I in combination with azoles and polyenes generates reactive oxygen species leading to apoptosis.

We have shown previously that pure polyphenol curcumin I (CUR-I) shows antifungal activity against Candida species. By employing the chequerboard method, filter disc and time-kill assays, in the present study we demonstrate that CUR-I at non-antifungal concentration interacts synergistically with azoles and polyenes. For this, pure polyphenol CUR-I was tested for synergy with five azole and two polyene drugs - fluconazole (FLC), miconazole, ketoconazole (KTC), itraconazole (ITR), voriconazole (VRC), nystatin (NYS) and amphotericin B (AMB) - against 21 clinical isolates of Candida albicans with reduced antifungal sensitivity, as well as a drug-sensitive laboratory strain. Notably, there was a 10-35-fold drop in the MIC(80) values of the drugs when CUR-I was used in combination with azoles and polyenes, with fractional inhibitory concentration index (FICI) values ranging between 0.09 and 0.5. Interestingly, the synergistic effect of CUR-I with FLC and AMB was associated with the accumulation of reactive oxygen species, which could be reversed by the addition of an antioxidant such as ascorbic acid. Furthermore, the combination of CUR-I and FLC/AMB triggered apoptosis that could also be reversed by ascorbic acid. We provide the first evidence that pure CUR-I in combination with azoles and polyenes represents a novel therapeutic strategy to improve the activity of common antifungals.

Morphogenic regulator EFG1 affects the drug susceptibilities of pathogenic Candida albicans.

This study shows that the morphogenic regulator EFG1 level affects the drug susceptibilities of Candida albicans when grown on solid growth media. The Deltaefg1 mutant showed sensitivity particularly to those drugs that target ergosterol or its metabolism. Efg1p disruption showed a gene-dosage effect on drug susceptibilities and resulted in enhanced susceptibility to drugs in the homozygous mutant as compared with the wild type, heterozygous and revertant strains. The enhanced sensitivity to drugs was independent of the status of ATP-binding cassette and MFS multidrug efflux pumps of C. albicans. The Deltaefg1 mutant displayed increased membrane fluidity that coincided with the downregulation of ERG11 and upregulation of OLE1 and ERG3, leading to enhanced passive diffusion of drugs. Interestingly, Deltaefg1 mutant cells displayed enhanced levels of endogenous ROS levels. Notably, the higher levels of ROS in the Deltaefg1 mutant could be reversed by the addition of antioxidants. However, the restoration of ROS levels did not reverse the drug sensitivities of the Deltaefg1 mutant. Taken together, we, for the first time, establish a new role to EFG1 in affecting the drug susceptibilities of C. albicans cells, independent of ROS and known drug efflux mechanisms.

PAP1 [poly(A) polymerase 1] homozygosity and hyperadenylation are major determinants of increased mRNA stability of CDR1 in azole-resistant clinical isolates of Candida albicans.

Using genetically matched azole-susceptible (AS) and azole-resistant (AR) clinical isolates of Candida albicans, we recently demonstrated that CDR1 overexpression in AR isolates is due to its enhanced transcriptional activation and mRNA stability. This study examines the molecular mechanisms underlying enhanced CDR1 mRNA stability in AR isolates. Mapping of the 3' untranslated region (3' UTR) of CDR1 revealed that it was rich in adenylate/uridylate (AU) elements, possessed heterogeneous polyadenylation sites, and had putative consensus sequences for RNA-binding proteins. Swapping of heterologous and chimeric lacZ-CDR1 3' UTR transcriptional reporter fusion constructs did not alter the reporter activity in AS and AR isolates, indicating that cis-acting sequences within the CDR1 3' UTR itself are not sufficient to confer the observed differential mRNA decay. Interestingly, the poly(A) tail of the CDR1 mRNA of AR isolates was approximately 35-50 % hyperadenylated as compared with AS isolates. C. albicans poly(A) polymerase (PAP1), responsible for mRNA adenylation, resides on chromosome 5 in close proximity to the mating type-like (MTL) locus. Two different PAP1 alleles, PAP1-a/PAP1-alpha, were recovered from AS (MTL-a/MTL-alpha), while a single type of PAP1 allele (PAP1-alpha) was recovered from AR isolates (MTL-alpha/MTL-alpha). Among the heterozygous deletions of PAP1-a (Deltapap1-a/PAP1-alpha) and PAP1-alpha (PAP1-a/Deltapap1-alpha), only the former led to relatively enhanced drug resistance, to polyadenylation and to transcript stability of CDR1 in the AS isolate. This suggests a dominant negative role of PAP1-a in CDR1 transcript polyadenylation and stability. Taken together, our study provides the first evidence, to our knowledge, that loss of heterozygosity at the PAP1 locus is linked to hyperadenylation and subsequent increased stability of CDR1 transcripts, thus contributing to enhanced drug resistance.

Antifungal curcumin induces reactive oxygen species and triggers an early apoptosis but prevents hyphae development by targeting the global repressor TUP1 in Candida albicans.

In the present study, we have investigated the antifungal effects of a natural polyphenol, CUR (curcumin), against albicans and non-albicans species of Candida and have shown its ability to inhibit the growth of all the tested strains. The inhibitory effects of CUR were independent of the status of the multidrug efflux pump proteins belonging to either ABC transporter (ATP-binding cassette transporter) or MFS (major facilitator) superfamilies of transporters. By using a systemic murine model of infection, we established that CUR and piperine, when administered together, caused a significant fungal load reduction (1.4log10) in kidneys of Swiss mice. Additionally, CUR raised the levels of ROS (reactive oxygen species), which, as revealed by annexin V-FITC labelling, triggered early apoptosis in Candida cells. Coincident with the raised ROS levels, mRNAs of tested oxidative stress-related genes [CAP1 (Candida albicans AP-1), CaIPF7817 (putative NADH-dependent flavin oxidoreductase), SOD2 (superoxide dismutase 2), GRP2 (NADPH-dependent methyl glyoxal reductase) and CAT1 (catalase 1)] were also elevated. The growth inhibitory effects of CUR could be reversed by the addition of natural and synthetic antioxidants. Notably, independent of ROS status, polyphenol CUR prevented hyphae development in both liquid and solid hypha-inducing media by targeting the global suppressor TUP1 (thymidine uptake 1). Taken together, our results provide the first evidence that CUR acts as an antifungal agent, via generation of oxidative stress, and inhibits hyphae development by targeting TUP1.

Differential dynamics of membrane proteins in yeast.

Lateral diffusion of lipids and proteins in yeast plasma membranes has been reported to be anomalously slow, and implicated as a possible reason for polarization in yeast. In order to gain insight into the observed slow diffusion in yeast membranes, we explored lateral diffusion of two proteins of different origin. We compared lateral dynamics of the Candida drug resistance protein-1 (Cdr1p), and the human serotonin(1A) receptor (5-HT(1A)R) by fluorescence recovery after photobleaching (FRAP). Our results show that while Cdr1p-GFP displays slow diffusion, the diffusion of 5-HT(1A)R-EYFP is significantly faster. Interestingly, upon ergosterol depletion, the mobility of Cdr1p-GFP did not exhibit appreciable change, while 5-HT(1A)R-EYFP mobility showed an increase. On the other hand, upon actin cytoskeleton destabilization, the mobile fraction of 5-HT(1A)R-EYFP showed considerable increase, while the mobility of Cdr1p-GFP was not altered. Our results represent the first report on the dynamics of the important drug resistance protein Cdr1p and provide novel insight on diffusion of membrane proteins in yeast membranes.

Curcumin modulates efflux mediated by yeast ABC multidrug transporters and is synergistic with antifungals.

Curcumin (CUR), a natural product of turmeric, from rhizomes of Curcuma longa, is a known agent of reversal of drug resistance phenotypes in cancer cells overexpressing ATP-binding cassette (ABC) transporters, viz., ABCB1, ABCG2, and ABCC1. In the present study, we evaluated whether CUR could also modulate multidrug transporters of yeasts that belong either to the ABC family or to the major facilitator superfamily (MFS). The effect of CUR on multidrug transporter proteins was demonstrated by examining rhodamine 6G (R6G) efflux in Saccharomyces cerevisiae cells overexpressing the Candida albicans ABC transporters Cdr1p and Cdr2p (CaCdr1p and CaCdr2p, respectively) and the MFS transporters CaMdr1p and S. cerevisiae Pdr5p. CUR decreased the extracellular concentration of R6G in ABC transporter-expressing cells but had no effect on methotrexate efflux mediated through the MFS transporter CaMdr1p. CUR competitively inhibited R6G efflux and the photolabeling of CaCdr1p by [(125)I]iodoarylazidoprazosin, a drug analogue of the substrate prazosin (50% inhibitory concentration, 14.2 microM). Notably, the mutant variants of CaCdr1p that displayed abrogated efflux of R6G also showed reduced modulation by CUR. Drug susceptibility testing of ABC protein-expressing cells by spot assays and checkerboard tests revealed that CUR was selectively synergistic with drug substrates such as R6G, ketoconazole, itraconazole, and miconazole but not with fluconazole, voriconazole, anisomycin, cycloheximide, or FK520. Taken together, our results provide the first evidence that CUR modulates only ABC multidrug transporters and could be exploited in combination with certain conventional antifungal drugs to reverse multidrug resistance in Candida cells.

MFS transportome of the human pathogenic yeast Candida albicans.

BACKGROUND: The major facilitator superfamily (MFS) is one of the two largest superfamilies of membrane transporters present ubiquitously in bacteria, archaea, and eukarya and includes members that function as uniporters, symporters or antiporters. We report here the complete transportome of MFS proteins of a human pathogenic yeast Candida albicans. RESULTS: Computational analysis of C. albicans genome enabled us to identify 95 potential MFS proteins which clustered into 17 families using Saier's Transport Commission (TC) system. Among these SP, DHA1, DHA2 and ACS represented major families consisting of 22, 22, 9 and 16 members, respectively. Family designations in C. albicans were validated by subjecting Saccharomyces cerevisiae genome to TC system. Based on the published available genomics/proteomics data, 87 of the putative MFS genes of C. albicans were found to express either at mRNA or protein levels. We checked the expression of the remaining 8 genes by using RT-PCR and observed that they are not expressed under basal growth conditions implying that either these 8 genes are expressed under specific growth conditions or they may be candidates for pseudogenes. CONCLUSION: The in silico characterisation of MFS transporters in Candida albicans genome revealed a large complement of MFS transporters with most of them showing expression. Considering the clinical relevance of C. albicans and role of MFS members in antifungal resistance and nutrient transport, this analysis would pave way for identifying their physiological relevance.

Transcriptional activation and increased mRNA stability contribute to overexpression of CDR1 in azole-resistant Candida albicans.

Many azole-resistant (AR) clinical isolates of Candida albicans display increased expression of the drug transporters CDR1 and CDR2. In this study, we evaluate the molecular mechanisms that contribute to the maintenance of constitutively high CDR1 transcript levels in two matched pairs of azole-susceptible (AS) and AR clinical isolates of C. albicans. To address this, we use reporter constructs of GFP and lacZ fused either to the CDR1 promoter (P CDR1-GFP/lacZ; transcriptional fusion) or to the CDR1 open reading frame (P CDR1-CDR1-GFP/lacZ; translational fusion) integrated at the native CDR1 locus. It is observed that expression of the two reporter genes as a transcriptional fusion in the AR isolates is higher than that in matched AS isolates. However, the difference in the reporter activity between the AS and AR isolates is even greater for the translational fusions, indicating that the sequences within the CDR1 coding region also contribute to its increased expression in AR isolates. Further analysis of these observations by transcription run-on assays demonstrated a approximately 5- to 7-fold difference in the transcription initiation rates for the AR isolates from those for their respective matched AS isolates. Measurement of mRNA stability showed that the half-life of CDR1 mRNA in the AR isolates was threefold higher than that in the corresponding AS isolates. Our results demonstrate that both increased CDR1 transcription and enhanced CDR1 mRNA stability contribute to the overexpression of CDR1 in AR C. albicans isolates.

Expression of the CDR1 efflux pump in clinical Candida albicans isolates is controlled by a negative regulatory element.

Resistance to azole antifungal drugs in clinical isolates of the human fungal pathogen Candida albicans is often caused by constitutive overexpression of the CDR1 gene, which encodes a multidrug efflux pump of the ABC transporter superfamily. To understand the relevance of a recently identified negative regulatory element (NRE) in the CDR1 promoter for the control of CDR1 expression in the clinical scenario, we investigated the effect of mutation or deletion of the NRE on CDR1 expression in two matched pairs of azole-sensitive and resistant clinical isolates of C. albicans. Expression of GFP or lacZ reporter genes from the wild type CDR1 promoter was much higher in the azole-resistant C. albicans isolates than in the azole-susceptible isolates, reflecting the known differences in CDR1 expression in these strains. Deletion or mutation of the NRE resulted in enhanced reporter gene expression in azole-sensitive strains, but did not further increase the already high CDR1 promoter activity in the azole-resistant strains. In agreement with these findings, electrophoretic mobility shift assays showed a reduced binding to the NRE of nuclear extracts from the resistant C. albicans isolates as compared with extracts from the sensitive isolates. These results demonstrate that the NRE is involved in maintaining CDR1 expression at basal levels and that this repression is overcome in azole-resistant clinical C. albicans isolates, resulting in constitutive CDR1 overexpression and concomitant drug resistance.