A second route of resistance involves increased expression of loci encoding ATP-binding cassette (ABC) transporters, which are thought to efflux drugs and prevent toxic intracellular concentrations ...from being achieved 2. ABC, ATP-binding cassette; SRE, sterol response elements. https://doi.org/10.1371/journal.ppat.1008819.g001 Experiments in Saccharomyces cerevisiae had supported a model in which azole drugs can act directly as activators of transcription factors that are known to induce expression of ABC transporter-encoding genes 7. Coupled with extensive previous data demonstrating that pharmacological inhibition of Erg11 by azole drugs led to induction of the CgPdr1/CDR1 pathway, these genetic experiments provided evidence that the reduction of Erg11 activity was the common feature triggering activation of downstream genes, including those encoding drug efflux pumps. Using available ChIP-seq data from A. fumigatus AtrR 6, C. albicans Upc2 14 and ChIP-seq data from C. glabrata Upc2A (B. Vu and M. Stamnes, Personal Communication), I converted the genes from each fungus into their nonsyntenic S. cerevisiae homologues.
Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a ...gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.
Summary
Resistance to azole drugs, the major clinical antifungal compounds, is most commonly due to gain‐of‐function (GOF) substitution mutations in a gene called PDR1 in the fungal pathogen Candida ...glabrata. PDR1 encodes a zinc cluster‐containing transcription factor. GOF forms of Pdr1 drive high level expression of downstream target gene expression with accompanying azole resistance. PDR1 has two homologous genes in Saccharomyces cerevisiae, called ScPDR1 and ScPDR3. This study provides evidence that the PDR1 gene in C. glabrata represents a blend of the properties found in the two S. cerevisiae genes. We demonstrated that GOF Pdr1 derivatives are overproduced at the protein level and less stable than the wild‐type protein. Overproduction of wild‐type Pdr1 increased target gene expression but to a lesser extent than GOF derivatives. Site‐directed mutagenesis of Pdr1 binding sites in the PDR1 promoter provided clear demonstration that autoregulation of PDR1 is required for its normal function. An internal deletion mutant of Pdr1 lacking its central regulatory domain behaved as a hyperactive transcription factor that was lethal unless conditionally expressed. A full understanding of the regulation of Pdr1 will provide a new avenue of interfering with azole resistance in C. glabrata.
Candida glabrata Pdr1 is the central transcriptional regulator driving azole resistance in this yeast. We provide evidence that the central domain of this factor confers negative regulation on the activity of Pdr1. Loss of this central domain generated a mutant protein that was found to be toxic when conditionally expressed in cells as the sole form of Pdr1. Mutant, hyperactive forms of Pdr1 were also determined to be unstable relative to the wild‐type protein.
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.
Fluconazole is one of the most commonly used antifungals today. A result of this has been the inevitable selection of fluconazole-resistant organisms. This is an especially acute problem in the ...pathogenic yeast
. Elevated minimal inhibitory concentrations for fluconazole in
are frequently associated with substitution mutations within the Zn2Cys6 zinc cluster-containing transcription factor-encoding gene
. These mutant Pdr1 regulators drive constitutively high expression of target genes like
that encodes an ATP-binding cassette transporter thought to act as a drug efflux pump. Exposure of
to fluconazole induced expression of both Pdr1 and
, although little is known of the molecular basis underlying the upstream signals that trigger Pdr1 activation. Here, we show that the protein phosphatase calcineurin is required for fluconazole-dependent induction of Pdr1 transcriptional regulation. Calcineurin catalytic activity is required for normal Pdr1 regulation, and a hyperactive form of this phosphatase can decrease susceptibility to the echinocandin caspofungin but does not show a similar change for fluconazole susceptibility. Loss of calcineurin from strains expressing two different gain-of-function forms of Pdr1 also caused a decrease in
expression and increased fluconazole susceptibility, demonstrating that even these hyperactive Pdr1 regulatory mutants cannot bypass the requirement for calcineurin. Our data implicate calcineurin activity as a link tying azole and echinocandin susceptibility together via the control of transcription factor activity.IMPORTANCEDrug-resistant microorganisms are a problem in the treatment of all infectious diseases; this is an especially acute problem with fungi due to the existence of only three major classes of antifungal drugs, including the azole drug fluconazole. In the pathogenic yeast
, mutant forms of a transcription factor called Pdr1 are commonly associated with decreased fluconazole susceptibility and poor clinical outcomes. Here, we identify a protein phosphatase called calcineurin that is required for fluconazole-dependent induction of Pdr1 transcriptional activation and associated drug susceptibility. Gain-of-function mutant forms of Pdr1 still required the presence of calcineurin to confer normally decreased fluconazole susceptibility. Previous studies showed that calcineurin controls susceptibility to the echinocandin class of antifungal drugs, and our data demonstrate that this protein phosphatase is also required for normal azole drug susceptibility. Calcineurin plays a central role in susceptibility to two of the three major classes of antifungal drugs in
.
Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction ...in ergosterol production, an essential fungal membrane sterol. Many
species, such as Candida albicans, develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata, these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the
gene. Here, we generated C. glabrata
mutations that were analogous to azole resistance alleles from C. albicans
. Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of
imposed a fitness cost that was not observed with hyperactive
alleles. Crucially, the presence of the DM
allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in
activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata.
A common need for microbial cells is the ability to respond to potentially toxic environmental insults. Here we review the progress in understanding the response of the yeast Saccharomyces cerevisiae ...to two important environmental stresses: heat shock and oxidative stress. Both of these stresses are fundamental challenges that microbes of all types will experience. The study of these environmental stress responses in S. cerevisiae has illuminated many of the features now viewed as central to our understanding of eukaryotic cell biology. Transcriptional activation plays an important role in driving the multifaceted reaction to elevated temperature and levels of reactive oxygen species. Advances provided by the development of whole genome analyses have led to an appreciation of the global reorganization of gene expression and its integration between different stress regimens. While the precise nature of the signal eliciting the heat shock response remains elusive, recent progress in the understanding of induction of the oxidative stress response is summarized here. Although these stress conditions represent ancient challenges to S. cerevisiae and other microbes, much remains to be learned about the mechanisms dedicated to dealing with these environmental parameters.
While azole drugs targeting the biosynthesis of ergosterol are effective antifungal agents, their extensive use has led to the development of resistant organisms. Infections involving azole-resistant ...forms of the filamentous fungus
are often associated with genetic changes in the
gene encoding the lanosterol α14 demethylase target enzyme. Both a sequence duplication in the
promoter (TR34) and a substitution mutation in the coding sequence (L98H) are required for the full expression of azole resistance. A mechanism commonly observed in pathogenic yeast such as
involves gain-of-function mutations in transcriptional regulatory proteins that induce expression of genes encoding ATP-binding cassette (ABC) transporters. We and others have found that an ABC transporter protein called Cdr1B (here referred to as AbcG1) is required for wild-type azole resistance in
Here, we test the genetic relationship between the TR34 L98H allele of
and an
null mutation. Loss of AbcG1 from a TR34 L98H
-containing strain caused a large decrease in the azole resistance of the resulting double-mutant strain. We also generated antibodies that enabled the detection of both the wild-type and L98H forms of the Cyp51A protein. The introduction of the L98H lesion into the
gene led to a decreased production of immunoreactive enzyme, suggesting that this mutant protein is unstable. Our data confirm the importance of AbcG1 function during azole resistance even in a strongly drug-resistant background.
Summary
The primary route for development of azole resistance in the fungal pathogen Candida glabrata is acquisition of a point mutation in the PDR1 gene. This locus encodes a transcription factor ...that upon mutation drives high level expression of a range of genes including the ATP‐binding cassette transporter‐encoding gene CDR1. Pdr1 activity is also elevated in cells that lack the mitochondrial genome (ρ° cells), with associated high expression of CDR1 driving azole resistance. To gain insight into the mechanisms controlling activity of Pdr1, we expressed a tandem affinity purification (TAP)‐tagged form of Pdr1 in both wild‐type (ρ+) and ρ° cells. Purified proteins were analyzed by multidimensional protein identification technology mass spectrometry identifying a protein called Bre5 as a factor that co‐purified with TAP‐Pdr1. In Saccharomyces cerevisiae, Bre5 is part of a deubiquitinase complex formed by association with the ubiquitin‐specific protease Ubp3. Genetic analyses in C. glabrata revealed that loss of BRE5, but not UBP3, led to an increase in expression of PDR1 and CDR1 at the transcriptional level. These studies support the view that Bre5 acts as a negative regulator of Pdr1 transcriptional activity and behaves as a C. glabrata‐specific modulator of azole resistance.
Bre5 is a known deubiquitinase subunit in Saccharomyces cerevisiae. We demonstrate that Bre5 is a negative regulator of Pdr1‐dependent transcription in Candida glabrata, although this function is not conserved in S. cerevisiae. Induction of Pdr1 transcriptional activation is associated with decreased association of Bre5 with Pdr1 and we suggest that this represents a ubiquitin independent role for this protein.
DNA recognition by the HapX transcription factor from Aspergillus species requires the presence of a heterotrimeric DNA-binding protein called the CCAAT-binding complex (CBC). In this issue of ...Structure, Huber and colleagues illuminate the structural basis for the multivalent binding of the CBC, HapX, and the DNA target site.
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