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.
Randomized trials have shown that the transplantation of filgrastim-mobilized peripheral-blood stem cells from HLA-identical siblings accelerates engraftment but increases the risks of acute and ...chronic graft-versus-host disease (GVHD), as compared with the transplantation of bone marrow. Some studies have also shown that peripheral-blood stem cells are associated with a decreased rate of relapse and improved survival among recipients with high-risk leukemia.
We conducted a phase 3, multicenter, randomized trial of transplantation of peripheral-blood stem cells versus bone marrow from unrelated donors to compare 2-year survival probabilities with the use of an intention-to-treat analysis. Between March 2004 and September 2009, we enrolled 551 patients at 48 centers. Patients were randomly assigned in a 1:1 ratio to peripheral-blood stem-cell or bone marrow transplantation, stratified according to transplantation center and disease risk. The median follow-up of surviving patients was 36 months (interquartile range, 30 to 37).
The overall survival rate at 2 years in the peripheral-blood group was 51% (95% confidence interval CI, 45 to 57), as compared with 46% (95% CI, 40 to 52) in the bone marrow group (P=0.29), with an absolute difference of 5 percentage points (95% CI, -3 to 14). The overall incidence of graft failure in the peripheral-blood group was 3% (95% CI, 1 to 5), versus 9% (95% CI, 6 to 13) in the bone marrow group (P=0.002). The incidence of chronic GVHD at 2 years in the peripheral-blood group was 53% (95% CI, 45 to 61), as compared with 41% (95% CI, 34 to 48) in the bone marrow group (P=0.01). There were no significant between-group differences in the incidence of acute GVHD or relapse.
We did not detect significant survival differences between peripheral-blood stem-cell and bone marrow transplantation from unrelated donors. Exploratory analyses of secondary end points indicated that peripheral-blood stem cells may reduce the risk of graft failure, whereas bone marrow may reduce the risk of chronic GVHD. (Funded by the National Heart, Lung, and Blood Institute-National Cancer Institute and others; ClinicalTrials.gov number, NCT00075816.).
Purpose To provide evidence-based guidance on the use of platelet transfusion in people with cancer. This guideline updates and replaces the previous ASCO platelet transfusion guideline published ...initially in 2001. Methods ASCO convened an Expert Panel and conducted a systematic review of the medical literature published from September 1, 2014, through October 26, 2016. This review builds on two 2015 systematic reviews that were conducted by the AABB and the International Collaboration for Transfusion Medicine Guidelines. For clinical questions that were not addressed by the AABB and the International Collaboration for Transfusion Medicine Guidelines (the use of leukoreduction and platelet transfusion in solid tumors or chronic, stable severe thrombocytopenia) or that were addressed partially (invasive procedures), the ASCO search extended back to January 2000. Results The updated ASCO review included 24 more recent publications: three clinical practice guidelines, eight systematic reviews, and 13 observational studies. Recommendations The most substantial change to a previous recommendation involved platelet transfusion in the setting of hematopoietic stem-cell transplantation. Based on data from randomized controlled trials, adult patients who undergo autologous stem-cell transplantation at experienced centers may receive a platelet transfusion at the first sign of bleeding, rather than prophylactically. Prophylactic platelet transfusion at defined platelet count thresholds is still recommended for pediatric patients undergoing autologous stem-cell transplantation and for adult and pediatric patients undergoing allogeneic stem-cell transplantation. Other recommendations address platelet transfusion in patients with hematologic malignancies or solid tumors or in those who undergo invasive procedures. Guidance is also provided regarding the production of platelet products, prevention of Rh alloimmunization, and management of refractoriness to platelet transfusion ( www.asco.org/supportive-care-guidelines and www.asco.org/guidelineswiki ).
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.
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.
A critical risk to the continued success of antifungal chemotherapy is the acquisition of resistance; a risk exacerbated by the few classes of effective antifungal drugs. Predictably, as the use of ...these drugs increases in the clinic, more resistant organisms can be isolated from patients. A particularly problematic form of drug resistance that routinely emerges in the major fungal pathogens is known as multidrug resistance. Multidrug resistance refers to the simultaneous acquisition of tolerance to a range of drugs via a limited or even single genetic change. This review will focus on recent progress in understanding pathways of multidrug resistance in fungi including those of most medical relevance. Analyses of multidrug resistance in Saccharomyces cerevisiae have provided the most detailed outline of multidrug resistance in a eukaryotic microorganism. Multidrug resistant isolates of S. cerevisiae typically result from changes in the activity of a pair of related transcription factors that in turn elicit overproduction of several target genes. Chief among these is the ATP-binding cassette (ABC)-encoding gene PDR5. Interestingly, in the medically important Candida species, very similar pathways are involved in acquisition of multidrug resistance. In both C. albicans and C. glabrata, changes in the activity of transcriptional activator proteins elicits overproduction of a protein closely related to S. cerevisiae Pdr5 called Cdr1. The major filamentous fungal pathogen, Aspergillus fumigatus, was previously thought to acquire resistance to azole compounds (the principal antifungal drug class) via alterations in the azole drug target-encoding gene cyp51A. More recent data indicate that pathways in addition to changes in the cyp51A gene are important determinants in A. fumigatus azole resistance. We will discuss findings that suggest azole resistance in A. fumigatus and Candida species may share more mechanistic similarities than previously thought.
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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