Identification of Genomic Binding Sites for Candida glabrata Pdr1 Transcription Factor in Wild-Type and ρ0Cells

ABSTRACTThe fungal pathogenCandida glabratais an emerging cause of candidiasis in part owing to its robust ability to acquire tolerance to the major clinical antifungal drug fluconazole. Similar to the related speciesCandida albicans,C. glabratamost typically gains azole tolerance via transcriptional induction of a suite of resistance genes, including a locus encoding an ABCG-type ATP-binding cassette (ABC) transporter that is referred to asCDR1inCandidaspecies. InC. glabrata,CDR1expression is controlled primarily by the activity of a transcriptional activator protein called Pdr1. Strains exhibiting reduced azole susceptibility often contain substitution mutations inPDR1that in turn lead to elevated mRNA levels of target genes with associated azole resistance. Pdr1 activity is also induced upon loss of the mitochondrial genome status and upon challenge by azole drugs. While extensive analyses of the transcriptional effects of Pdr1 have identified a number of genes that are regulated by this factor, we cannot yet separate direct from indirect target genes. Here we used chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) to identify the promoters and associated genes directly regulated by Pdr1. These genes include many that are shared with the yeastSaccharomyces cerevisiaebut others that are unique toC. glabrata, including the ABC transporter-encoding locusYBT1, genes involved in DNA repair, and several others. These data provide the outline for understanding the primary response genes involved in production of Pdr1-dependent azole resistance inC. glabrata.

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Loss of Mitochondrial Functions Associated with Azole Resistance in Candida glabrata Results in Enhanced Virulence in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01271-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1852-1860 ◽

Cited By ~ 76

Author(s):

Sélène Ferrari ◽

Maurizio Sanguinetti ◽

Flavia De Bernardis ◽

Riccardo Torelli ◽

Brunella Posteraro ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Abc Transporter ◽

Candida Glabrata ◽

Selective Advantage ◽

Azole Resistance ◽

Content Type ◽

Mitochondrial Functions ◽

Abc Transporter Genes

ABSTRACTMitochondrial dysfunction is one of the possible mechanisms by which azole resistance can occur inCandida glabrata. Cells with mitochondrial DNA deficiency (so-called “petite mutants”) upregulate ATP binding cassette (ABC) transporter genes and thus display increased resistance to azoles. Isolation of suchC. glabratamutants from patients receiving antifungal therapy or prophylaxis has been rarely reported. In this study, we characterized two sequential and relatedC. glabrataisolates recovered from the same patient undergoing azole therapy. The first isolate (BPY40) was azole susceptible (fluconazole MIC, 4 μg/ml), and the second (BPY41) was azole resistant (fluconazole MIC, >256 μg/ml). BPY41 exhibited mitochondrial dysfunction and upregulation of the ABC transporter genesC. glabrata CDR1(CgCDR1),CgCDR2, andCgSNQ2. We next assessed whether mitochondrial dysfunction conferred a selective advantage during host infection by testing the virulence of BPY40 and BPY41 in mice. Surprisingly, even within vitrogrowth deficiency compared to BPY40, BPY41 was more virulent (as judged by mortality and fungal tissue burden) than BPY40 in both systemic and vaginal murine infection models. The increased virulence of the petite mutant correlated with a drastic gain of fitness in mice compared to that of its parental isolate. To understand this unexpected feature, genome-wide changes in gene expression driven by the petite mutation were analyzed by use of microarrays duringin vitrogrowth. Enrichment of specific biological processes (oxido-reductive metabolism and the stress response) was observed in BPY41, all of which was consistent with mitochondrial dysfunction. Finally, some genes involved in cell wall remodelling were upregulated in BPY41 compared to BPY40, which may partially explain the enhanced virulence of BPY41. In conclusion, this study shows for the first time that mitochondrial dysfunction selectedin vivounder azole therapy, even if strongly affectingin vitrogrowth characteristics, can confer a selective advantage under host conditions, allowing theC. glabratamutant to be more virulent than wild-type isolates.

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AtrR Is an Essential Determinant of Azole Resistance in Aspergillus fumigatus

mBio ◽

10.1128/mbio.02563-18 ◽

2019 ◽

Vol 10 (2) ◽

Cited By ~ 5

Author(s):

Sanjoy Paul ◽

Mark Stamnes ◽

Grace Heredge Thomas ◽

Hong Liu ◽

Daisuke Hagiwara ◽

...

Keyword(s):

Transcription Factor ◽

Aspergillus Fumigatus ◽

High Throughput ◽

Transcriptional Control ◽

High Throughput Sequencing ◽

Target Genes ◽

Azole Resistance ◽

Promoter Regions ◽

Mobility Shift ◽

Content Type

ABSTRACT Aspergillosis associated with azole-resistant Aspergillus fumigatus has a mortality rate that can approach 90% in certain patient populations. The best-understood avenue for azole resistance involves changes in the cyp51A gene that encodes the target of azole drugs, lanosterol α-14 demethylase. The most common azole resistance allele currently described is a linked change corresponding to a change in the coding sequence of cyp51A and a duplication of a 34-bp region in the promoter leading to a tandem repeat (TR). Our previous studies identified a positively acting transcription factor called AtrR that binds to the promoter of cyp51A as well as that of an important membrane transporter protein gene called abcG1. In this work, we characterize two different mutant alleles of atrR, either an overproducing or an epitope-tagged form, causing constitutive activation of this factor. Using an epitope-tagged allele of atrR for chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq), the genomic binding sites for AtrR were determined. Close to 900 genes were found to have an AtrR response element (ATRE) in their promoter regions. Transcriptome evaluation by RNA sequencing (RNA-seq) indicated that both alleles led to elevated transcription of a subset of target genes. An electrophoretic mobility shift assay and DNase I protection mapping localized the ATREs in both the abcG1 and cyp51A promoters. The ATRE in cyp51A was located within the 34-bp repeat element. Virulence in a murine model was compromised when AtrR was either deleted or overproduced, indicating that the proper dosage of this factor is key for pathogenesis. IMPORTANCE Aspergillus fumigatus is the major filamentous fungal pathogen in humans. Infections associated with A. fumigatus are often treated with azole drugs, but resistance to these antifungal agents is increasing. Mortality from aspergillosis associated with azole-resistant fungi is extremely high. Previous work has identified transcriptional control of the azole drug target-encoding gene cyp51A as an important contributor to resistance in A. fumigatus. Here, we demonstrate that the transcription factor AtrR binds to a region in the cyp51A promoter that is associated with alleles of this gene conferring clinically important azole resistance. Using high-throughput genomic technologies, we also uncover a large suite of target genes controlled by AtrR. These data indicate that AtrR coordinately regulates many different processes involved in drug resistance, metabolism, and virulence. Our new understanding of AtrR function provides important new insight into the pathogenesis of A. fumigatus.

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Vacuolar Sequestration of Azoles, a Novel Strategy of Azole Antifungal Resistance Conserved across Pathogenic and Nonpathogenic Yeast

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01347-18 ◽

2019 ◽

Vol 63 (3) ◽

Cited By ~ 9

Author(s):

Nitesh Kumar Khandelwal ◽

Mohd Wasi ◽

Remya Nair ◽

Meghna Gupta ◽

Mohit Kumar ◽

...

Keyword(s):

Abc Transporter ◽

Atp Hydrolysis ◽

Consensus Sequence ◽

Antifungal Resistance ◽

Azole Resistance ◽

Casein Kinase I ◽

Pathogenic Yeast ◽

Content Type ◽

Novel Strategy ◽

Vacuolar Sequestration

ABSTRACT Target alteration and overproduction and drug efflux through overexpression of multidrug transporters localized in the plasma membrane represent the conventional mechanisms of azole antifungal resistance. Here, we identify a novel conserved mechanism of azole resistance not only in the budding yeast Saccharomyces cerevisiae but also in the pathogenic yeast Candida albicans. We observed that the vacuolar-membrane-localized, multidrug resistance protein (MRP) subfamily, ATP-binding cassette (ABC) transporter of S. cerevisiae, Ybt1, could import azoles into vacuoles. Interestingly, the Ybt1 homologue in C. albicans, Mlt1p, could also fulfill this function. Evidence that the process is energy dependent comes from the finding that a Mlt1p mutant version made by converting a critical lysine residue in the Walker A motif of nucleotide-binding domain 1 (required for ATP hydrolysis) to alanine (K710A) was not able to transport azoles. Additionally, we have shown that, as for other eukaryotic MRP subfamily members, deletion of the conserved phenylalanine amino acid at position 765 (F765Δ) results in mislocalization of the Mlt1 protein; this mislocalized protein was devoid of the azole-resistant attribute. This finding suggests that the presence of this protein on vacuolar membranes is an important factor in azole resistance. Further, we report the importance of conserved residues, because conversion of two serines (positions 973 and 976, in the regulatory domain and in the casein kinase I [CKI] consensus sequence, respectively) to alanine severely affected the drug resistance. Hence, the present study reveals vacuolar sequestration of azoles by the ABC transporter Ybt1 and its homologue Mlt1 as an alternative strategy to circumvent drug toxicity among pathogenic and nonpathogenic yeasts.

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Role of ATP-Binding-Cassette Transporter Genes in High-Frequency Acquisition of Resistance to Azole Antifungals in Candida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.4.1174-1183.2001 ◽

2001 ◽

Vol 45 (4) ◽

pp. 1174-1183 ◽

Cited By ~ 169

Author(s):

Dominique Sanglard ◽

Francoise Ischer ◽

Jacques Bille

Keyword(s):

Abc Transporter ◽

High Frequency ◽

Candida Glabrata ◽

Antifungal Agents ◽

Clinical Isolates ◽

Azole Resistance ◽

Atp Binding ◽

Transporter Gene ◽

Atp Binding Cassette

ABSTRACT Candida glabrata has been often isolated from AIDS patients with oropharyngeal candidiasis treated with azole antifungal agents, especially fluconazole. We recently showed that the ATP-binding-cassette (ABC) transporter gene CgCDR1 was upregulated in C. glabrata clinical isolates resistant to azole antifungal agents (D. Sanglard, F. Ischer, D. Calabrese, P. A. Majcherczyk, and J. Bille, Antimicrob. Agents Chemother. 43:2753–2765, 1999). Deletion of CgCDR1 in C. glabrata rendered the null mutant hypersusceptible to azole derivatives and showed the importance of this gene in mediating azole resistance. We observed that wild-type C. glabrata exposed to fluconazole in a medium containing the drug at 50 μg/ml developed resistance to this agent and other azoles at a surprisingly high frequency (2 × 10−4 to 4 × 10−4). We show here that this high-frequency azole resistance (HFAR) acquired in vitro was due, at least in part, to the upregulation ofCgCDR1. The CgCDR1 deletion mutant DSY1041 could still develop HFAR but in a medium containing fluconazole at 5 μg/ml. In the HFAR strain derived from DSY1041, a distinct ABC transporter gene similar to CgCDR1, calledCgCDR2, was upregulated. This gene was slightly expressed in clinical isolates but was upregulated in strains with the HFAR phenotype. Deletion of both CgCDR1 and CgCDR2suppressed the development of HFAR in a medium containing fluconazole at 5 μg/ml, showing that both genes are important mediators of resistance to azole derivatives in C. glabrata. We also show here that the HFAR phenomenon was linked to the loss of mitochondria in C. glabrata. Mitochondrial loss could be obtained by treatment with ethidium bromide and resulted in acquisition of resistance to azole derivatives without previous exposure to these agents. Azole resistance obtained in vitro by HFAR or by agents stimulating mitochondrial loss was at least linked to the upregulation of both CgCDR1 and CgCDR2.

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Heterogeneous Expression of the Virulence-Related Adhesin Epa1 between Individual Cells and Strains of the Pathogen Candida glabrata

Eukaryotic Cell ◽

10.1128/ec.05232-11 ◽

2011 ◽

Vol 11 (2) ◽

pp. 141-150 ◽

Cited By ~ 33

Author(s):

Samantha C. Halliwell ◽

Matthew C. A. Smith ◽

Philippa Muston ◽

Sara L. Holland ◽

Simon V. Avery

Keyword(s):

Gene Expression ◽

Epithelial Cells ◽

Candida Glabrata ◽

Mrna Levels ◽

Content Type ◽

Gene Expression Noise ◽

Heterogeneous Expression ◽

Gene Expression Heterogeneity ◽

Yeast Genes

ABSTRACTWe investigated the relevance of gene expression heterogeneity to virulence properties of a major fungal pathogen,Candida glabrata. The organism's key virulence-associated factors include glycosylphosphatidylinositol-anchored adhesins, encoded subtelomerically by theEPAgene family. Individual-cell analyses of expression revealed very striking heterogeneity for Epa1, an adhesin that mediates ∼95% of adherence to epithelial cellsin vitro. The heterogeneity in Epa1 was markedly greater than that known for other yeast genes. Sorted cells expressing high or low levels of Epa1 exhibited high and low adherence to epithelial cells, indicating a link between gene expression noise and potential virulence. The phenotypes of sorted subpopulations reverted to mixed phenotypes within a few generations. Variation in single-cell Epa1 protein and mRNA levels was correlated, consistent with transcriptional regulation of heterogeneity. Sir-dependent transcriptional silencing was the primary mechanism driving heterogeneous Epa1 expression inC. glabrataBG2, but not in CBS138 (ATCC 2001). Inefficient silencing in the latter strain was not due to a difference inEPA1sequence or (sub)telomere length and was overcome by ectopicSIR3expression. Moreover, differences between strains in the silencing dependence ofEPA1expression were evident across a range of clinical isolates, with heterogeneity being the greatest in strains whereEPA1was subject to silencing. The study shows how heterogeneity can impact the virulence-related properties ofC. glabratacell populations, with potential implications for microbial pathogenesis more broadly.

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The Candida glabrata Upc2A transcription factor is a global regulator of antifungal drug resistance pathways

PLoS Genetics ◽

10.1371/journal.pgen.1009582 ◽

2021 ◽

Vol 17 (9) ◽

pp. e1009582

Author(s):

Bao Gia Vu ◽

Mark A. Stamnes ◽

Yu Li ◽

P. David Rogers ◽

W. Scott Moye-Rowley

Keyword(s):

Transcription Factor ◽

Membrane Protein ◽

Candida Glabrata ◽

Target Genes ◽

Protein Biosynthesis ◽

Antifungal Drugs ◽

Azole Resistance ◽

Mutant Form ◽

Pathogenic Yeast ◽

Genes Encoding

The most commonly used antifungal drugs are the azole compounds, which interfere with biosynthesis of the fungal-specific sterol: ergosterol. The pathogenic yeast Candida glabrata commonly acquires resistance to azole drugs like fluconazole via mutations in a gene encoding a transcription factor called PDR1. These PDR1 mutations lead to overproduction of drug transporter proteins like the ATP-binding cassette transporter Cdr1. In other Candida species, mutant forms of a transcription factor called Upc2 are associated with azole resistance, owing to the important role of this protein in control of expression of genes encoding enzymes involved in the ergosterol biosynthetic pathway. Recently, the C. glabrata Upc2A factor was demonstrated to be required for normal azole resistance, even in the presence of a hyperactive mutant form of PDR1. Using genome-scale approaches, we define the network of genes bound and regulated by Upc2A. By analogy to a previously described hyperactive UPC2 mutation found in Saccharomyces cerevisiae, we generated a similar form of Upc2A in C. glabrata called G898D Upc2A. Analysis of Upc2A genomic binding sites demonstrated that wild-type Upc2A binding to target genes was strongly induced by fluconazole while G898D Upc2A bound similarly, irrespective of drug treatment. Transcriptomic analyses revealed that, in addition to the well-described ERG genes, a large group of genes encoding components of the translational apparatus along with membrane proteins were responsive to Upc2A. These Upc2A-regulated membrane protein-encoding genes are often targets of the Pdr1 transcription factor, demonstrating the high degree of overlap between these two regulatory networks. Finally, we provide evidence that Upc2A impacts the Pdr1-Cdr1 system and also modulates resistance to caspofungin. These studies provide a new perspective of Upc2A as a master regulator of lipid and membrane protein biosynthesis.

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Investigation of Multiple Resistance Mechanisms in Voriconazole-Resistant Aspergillus flavus Clinical Isolates from a Chest Hospital Surveillance in Delhi, India

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01928-17 ◽

2018 ◽

Vol 62 (3) ◽

Cited By ~ 20

Author(s):

Cheshta Sharma ◽

Rakesh Kumar ◽

Nitin Kumar ◽

Aradhana Masih ◽

Dinesh Gupta ◽

...

Keyword(s):

Aspergillus Flavus ◽

Target Genes ◽

Empirical Therapy ◽

Homology Model ◽

Binding Pocket ◽

Resistance Mechanisms ◽

Drug Binding ◽

Whole Genome Sequence ◽

Azole Resistance ◽

Content Type

ABSTRACT Invasive and allergic infections by Aspergillus flavus are more common in tropical and subtropical countries. The emergence of voriconazole (VRC) resistance in A. flavus impacts the management of aspergillosis, as azoles are used as the first-line and empirical therapy. We screened 120 molecularly confirmed A. flavus isolates obtained from respiratory and sinonasal specimens in a chest hospital in Delhi, India, for azole resistance using the CLSI broth microdilution (CLSI-BMD) method. Overall, 2.5% ( n = 3/120) of A. flavus isolates had VRC MICs above epidemiological cutoff values (>1 μg/ml). The whole-genome sequence analysis of three non-wild-type (WT) A. flavus isolates with high VRC MICs showed polymorphisms in azole target genes ( cyp51A , cyp51B , and cyp51C ). Further, four novel substitutions (S196F, A324P, N423D, and V465M) encoded in the cyp51C gene were found in a single non-WT isolate which also exhibited overexpression of cyp51 ( cyp51A , - B , and - C ) genes and transporter genes, namely, MDR1 , MDR2 , atrF , and mfs1 . The homology model of the non-WT isolate suggests that substitutions S196F and N423D exhibited major structural and functional effects on cyp51C drug binding. The substrate (drug) may not be able to bind to binding pocket due to changes in the pocket size or closing down or narrowing of cavities in drug entry channels. Notably, the remaining two VRC-resistant A. flavus isolates, including the one which had a pan-azole resistance phenotype (itraconazole and posaconazole), did not show upregulation of any of the analyzed target genes. These results suggest that multiple target genes and mechanisms could simultaneously contribute to azole resistance in A. flavus .

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Candida glabrata Med3 Plays a Role in Altering Cell Size and Budding Index To Coordinate Cell Growth

Applied and Environmental Microbiology ◽

10.1128/aem.00781-18 ◽

2018 ◽

Vol 84 (15) ◽

Cited By ~ 1

Author(s):

Hui Liu ◽

Lulin Kong ◽

Yanli Qi ◽

Xiulai Chen ◽

Liming Liu

Keyword(s):

Cell Growth ◽

Cell Size ◽

Candida Glabrata ◽

Mrna Level ◽

Growth Defect ◽

Mrna Levels ◽

Acetyl Coa ◽

Content Type ◽

Quantitative Expression ◽

Transcriptomics Data

ABSTRACT Candida glabrata is a promising microorganism for the production of organic acids. Here, we report deletion and quantitative-expression approaches to elucidate the role of C. glabrata Med3AB (CgMed3AB), a subunit of the mediator transcriptional coactivator, in regulating cell growth. Deletion of CgMed3AB caused an 8.6% decrease in final biomass based on growth curve plots and 10.5% lower cell viability. Based on transcriptomics data, the reason for this growth defect was attributable to changes in expression of genes involved in pyruvate and acetyl-coenzyme A (CoA)-related metabolism in a Cgmed3abΔ strain. Furthermore, the mRNA level of acetyl-CoA synthetase was downregulated after deleting Cgmed3ab, resulting in 22.8% and 21% lower activity of acetyl-CoA synthetase and cellular acetyl-CoA, respectively. Additionally, the mRNA level of CgCln3, whose expression depends on acetyl-CoA, was 34% lower in this strain. As a consequence, the cell size and budding index in the Cgmed3abΔ strain were both reduced. Conversely, overexpression of Cgmed3ab led to 16.8% more acetyl-CoA and 120% higher CgCln3 mRNA levels, as well as 19.1% larger cell size and a 13.3% higher budding index than in wild-type cells. Taken together, these results suggest that CgMed3AB regulates cell growth in C. glabrata by coordinating homeostasis between cellular acetyl-CoA and CgCln3. IMPORTANCE This study demonstrates that CgMed3AB can regulate cell growth in C. glabrata by coordinating the homeostasis of cellular acetyl-CoA metabolism and the cell cycle cyclin CgCln3. Specifically, we report that CgMed3AB regulates the cellular acetyl-CoA level, which induces the transcription of Cgcln3, finally resulting in alterations to the cell size and budding index. In conclusion, we report that CgMed3AB functions as a wheel responsible for driving cellular acetyl-CoA metabolism, indirectly inducing the transcription of Cgcln3 and coordinating cell growth. We propose that Mediator subunits may represent a vital regulatory target modulating cell growth in C. glabrata.

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Evidence that Ergosterol Biosynthesis Modulates Activity of the Pdr1 Transcription Factor inCandida glabrata

mBio ◽

10.1128/mbio.00934-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 7

Author(s):

Bao Gia Vu ◽

Grace Heredge Thomas ◽

W. Scott Moye-Rowley

Keyword(s):

Transcription Factor ◽

Candida Glabrata ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Atp Binding ◽

Pathogenic Yeast ◽

Content Type ◽

Atp Binding Cassette Transporter ◽

Encoding Gene ◽

Atp Binding Cassette

ABSTRACTA crucial limitation in antifungal chemotherapy is the limited number of antifungal drugs currently available. Azole drugs represent the most commonly used chemotherapeutic, and loss of efficacy of these drugs is a major risk factor in successful treatment of a variety of fungal diseases.Candida glabratais a pathogenic yeast that is increasingly found associated with bloodstream infections, a finding likely contributed to by its proclivity to develop azole drug resistance.C. glabrataoften acquires azole resistance via gain-of-function (GOF) mutations in the transcription factor Pdr1. These GOF forms of Pdr1 drive elevated expression of target genes, including the ATP-binding cassette transporter-encodingCDR1locus. GOF alleles ofPDR1have been extensively studied, but little is known of how Pdr1 is normally regulated. Here we test the idea that reduction of ergosterol biosynthesis (as occurs in the presence of azole drugs) might trigger activation of Pdr1 function. Using two different means of genetically inhibiting ergosterol biosynthesis, we demonstrated that Pdr1 activity and target gene expression are elevated in the absence of azole drug. Blocks at different points in the ergosterol pathway lead to Pdr1 activation as well as to induction of other genes in this pathway. Delivery of the signal from the ergosterol pathway to Pdr1 involves the transcription factor Upc2A, anERGgene regulator. We show that Upc2A binds directly to thePDR1andCDR1promoters. Our studies argue for a physiological link between ergosterol biosynthesis and Pdr1-dependent gene regulation that is not restricted to efflux of azole drugs.IMPORTANCEA likely contributor to the increased incidence of non-albicanscandidemias involvingCandida glabratais the ease with which this yeast acquires azole resistance, in large part due to induction of the ATP-binding cassette transporter-encoding geneCDR1. Azole drugs lead to induction of Pdr1 transactivation, with a central model being that this factor binds these drugs directly. Here we provide evidence that Pdr1 is activated without azole drugs by the use of genetic means to inhibit expression of azole drug target-encoding geneERG11. These acute reductions in Erg11 levels lead to elevated Pdr1 activity even though no drug is present. A key transcriptional regulator of theERGpathway, Upc2A, is shown to directly bind to thePDR1andCDR1promoters. We interpret these data as support for the view that Pdr1 function is responsive to ergosterol biosynthesis and suggest that this connection reveals the normal physiological circuitry in which Pdr1 participates.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00554-20 ◽

2020 ◽

Vol 64 (9) ◽

Cited By ~ 2

Author(s):

Pedro Pais ◽

Raquel Califórnia ◽

Mónica Galocha ◽

Romeu Viana ◽

Mihaela Ola ◽

...

Keyword(s):

Transcription Factor ◽

Plasma Membrane ◽

Candida Glabrata ◽

Cell Permeability ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Binding Motif ◽

Plasma Membrane Permeability ◽

Content Type ◽

Content Understanding

ABSTRACT 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. Targets comprise several proteasome and ergosterol biosynthesis genes, including ERG1, ERG2, ERG3, and ERG11. The localization of CgRpn4 to the nucleus increases upon fluconazole stress. Consistent with a role in ergosterol and plasma membrane homeostasis, CgRpn4 is required for the maintenance of ergosterol levels upon fluconazole stress, which is associated with a role in the upkeep of cell permeability and decreased intracellular fluconazole accumulation. We provide evidence that CgRpn4 directly regulates ERG11 expression through the TTGCAAA binding motif, reinforcing the relevance of this regulatory network in azole resistance. In summary, CgRpn4 is a new regulator of the ergosterol biosynthesis pathway in C. glabrata, contributing to plasma membrane homeostasis and, thus, decreasing azole drug accumulation.

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