Erratum for Kim et al., “Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance”

ABSTRACT The recently emerged pathogenic yeast Candida auris is a major concern for human health, because it is easily transmissible, difficult to eradicate from hospitals, and highly drug resistant. Most C. auris isolates are resistant to the widely used antifungal drug fluconazole due to mutations in the target enzyme Erg11 and high activity of efflux pumps, such as Cdr1. In the well-studied, distantly related yeast Candida albicans, overexpression of drug efflux pumps also is a major mechanism of acquired fluconazole resistance and caused by gain-of-function mutations in the zinc cluster transcription factors Mrr1 and Tac1. In this study, we investigated a possible involvement of related transcription factors in efflux pump expression and fluconazole resistance of C. auris. The C. auris genome contains three genes encoding Mrr1 homologs and two genes encoding Tac1 homologs, and we generated deletion mutants lacking these genes in two fluconazole-resistant strains from clade III and clade IV. Deletion of TAC1b decreased the resistance to fluconazole and voriconazole in both strain backgrounds, demonstrating that the encoded transcription factor contributes to azole resistance in C. auris strains from different clades. CDR1 expression was not or only minimally affected in the mutants, indicating that Tac1b can confer increased azole resistance by a CDR1-independent mechanism. IMPORTANCE Candida auris is a recently emerged pathogenic yeast that within a few years after its initial description has spread all over the globe. C. auris is a major concern for human health, because it can cause life-threatening systemic infections, is easily transmissible, and is difficult to eradicate from hospital environments. Furthermore, C. auris is highly drug resistant, especially against the widely used antifungal drug fluconazole. Mutations in the drug target and high activity of efflux pumps are associated with azole resistance, but it is not known how drug resistance genes are regulated in C. auris. We have investigated the potential role of several candidate transcriptional regulators in the intrinsic fluconazole resistance of C. auris and identified a transcription factor that contributes to the high resistance to fluconazole and voriconazole of two C. auris strains from different genetic clades, thereby providing insight into the molecular basis of drug resistance of this medically important yeast.

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Genetic Analysis of Azole Resistance in the Darlington Strain of Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.44.11.2985-2990.2000 ◽

2000 ◽

Vol 44 (11) ◽

pp. 2985-2990 ◽

Cited By ~ 54

Author(s):

Hiroshi Kakeya ◽

Yoshitsugu Miyazaki ◽

Haruko Miyazaki ◽

Katherine Nyswaner ◽

Brian Grimberg ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Amino Acids ◽

Candida Albicans ◽

Amino Acid ◽

Genetic Analysis ◽

Expression System ◽

Single Copy ◽

Gene Replacement ◽

Azole Resistance ◽

High Level

ABSTRACT High-level azole resistance in the Darlington strain ofCandida albicans was investigated by gene replacement inC. albicans and expression in Saccharomyces cerevisiae. We sequenced the ERG11 gene, which encodes the sterol C14α-demethylase, from our copy of the Darlington strain. Both alleles contained the histidine for tyrosine substitution at position 132 (Y132H) reported in Darlington by others, but we also found a threonine-for-isoleucine substitution (I471T) not previously reported in the C. albicans ERG11. The encoded I471T change in amino acids conferred azole resistance when overexpressed alone and increased azole resistance when added to the Y132H amino acid sequence in an S. cerevisiae expression system. Replacement of one copy of ERG11 in an azole-susceptible strain of C. albicans with a single copy of the Darlington ERG11 resulted in expression of the integrated copy and a modest increase in azole resistance. The profound azole resistance of the Darlington strain is the result of multiple mutations.

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Rapid Detection of ERG11-Associated Azole Resistance and FKS-Associated Echinocandin Resistance in Candida auris

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01811-18 ◽

2018 ◽

Vol 63 (1) ◽

Cited By ~ 20

Author(s):

Xin Hou ◽

Annie Lee ◽

Cristina Jiménez-Ortigosa ◽

Milena Kordalewska ◽

David S. Perlin ◽

...

Keyword(s):

Rapid Assessment ◽

Melting Curve ◽

Rapid Identification ◽

Molecular Diagnostic ◽

Melting Curve Analysis ◽

Azole Resistance ◽

Candida Auris ◽

Content Type ◽

Echinocandin Resistance

ABSTRACT Candida auris is an emerging multidrug-resistant yeast that can cause serious invasive infections. The accurate and rapid assessment of antifungal resistance is important for effective patient management. A novel and highly accurate diagnostic platform was established for the rapid identification of ERG11 mutations conferring azole resistance and FKS1 mutations associated with echinocandin resistance in C. auris. Using allele-specific molecular beacons and DNA melting curve analysis following asymmetric PCR, a duplex ERG11 assay and a simplex FKS1 HS1 assay were developed to identify the most prominent resistance-associated mutations (Y132F and K143R in ERG11; S639F in FKS1 HS1) within 2 h. Assays were validated by testing a panel of 94 C. auris clinical isolates in a blind manner. The molecular diagnostic results from the assays were 100% concordant with DNA sequencing results. This platform has the potential to overcome the deficiencies of existing in vitro susceptibility-based assays to identify azole- and/or echinocandin-resistant C. auris, and thus, it holds promise as a surrogate diagnostic method to direct antifungal therapy more effectively.

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Novel ERG11 and TAC1b Mutations Associated with Azole Resistance in Candida auris

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02663-20 ◽

2021 ◽

Vol 65 (5) ◽

Author(s):

Jizhou Li ◽

Alix T. Coste ◽

Maroussia Liechti ◽

Daniel Bachmann ◽

Dominique Sanglard ◽

...

Keyword(s):

Invasive Candidiasis ◽

Efflux Pump ◽

Cumulative Effect ◽

Resistance Mechanisms ◽

Azole Resistance ◽

Candida Auris ◽

Content Type ◽

Drug Classes ◽

In The Wild

ABSTRACT Candida auris is a novel Candida species that has spread in all continents, causing nosocomial outbreaks of invasive candidiasis. C. auris has the ability to develop resistance to all antifungal drug classes. Notably, many C. auris isolates are resistant to the azole drug fluconazole, a standard therapy for invasive candidiasis. Azole resistance in C. auris can result from mutations in the azole target gene ERG11 and/or overexpression of the efflux pump Cdr1. TAC1 is a transcription factor controlling CDR1 expression in C. albicans. The role of TAC1 homologs in C. auris (TAC1a and TAC1b) remains to be better defined. In this study, we compared sequences of ERG11, TAC1a, and TAC1b between a fluconazole-susceptible and five fluconazole-resistant C. auris isolates of clade IV. Among four of the resistant isolates, we identified similar genotypes with concomitant mutations in ERG11 (F444L) and TAC1b (S611P). The simultaneous deletion of tandemly arranged TAC1a/TAC1b resulted in a decrease of MIC for fluconazole. Introduction of the ERG11 and TAC1b mutations separately and/or combined in the wild-type azole-susceptible isolate resulted in a significant increase of azole resistance with a cumulative effect of the two combined mutations. Interestingly, CDR1 expression was not significantly affected by TAC1a/TAC1b deletion or by the presence of the TAC1b S611P mutation, suggesting the existence of Tac1-dependent and Cdr1-independent azole resistance mechanisms. In conclusion, we demonstrated the role of two previously unreported mutations responsible for azole resistance in C. auris, which were a common signature among four azole-resistant isolates of clade IV.

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Antifungal Resistance: Specific Focus on Multidrug Resistance in Candida auris and Secondary Azole Resistance in Aspergillus fumigatus

Journal of Fungi ◽

10.3390/jof4040129 ◽

2018 ◽

Vol 4 (4) ◽

pp. 129 ◽

Cited By ~ 16

Author(s):

Sevtap Arikan-Akdagli ◽

Mahmoud Ghannoum ◽

Jacques Meis

Keyword(s):

Aspergillus Fumigatus ◽

Molecular Mechanisms ◽

Multidrug Resistant ◽

Fungal Species ◽

Antifungal Resistance ◽

Current Status ◽

Clinical Implications ◽

Azole Resistance ◽

Candida Auris

Antifungal resistance is a topic of concern, particularly for specific fungal species and drugs. Among these are the multidrug-resistant Candida auris and azole-resistant Aspergillus fumigatus. While the knowledge on molecular mechanisms of resistance is now accumulating, further data are also available for the clinical implications and the extent of correlation of in vitro resistance to clinical outcomes. This review article summarizes the epidemiology of C. auris infections, animal models focusing on the activity of novel antifungal compounds in C. auris infections, virulence factors, and the mechanisms of antifungal resistance for this multi-resistant Candida species. Regarding A. fumigatus, the significance of azoles in the treatment of A. fumigatus infections, reference methods available for the detection of resistance in vitro, molecular mechanisms of secondary azole resistance, routes of acquisition, and clinical implications of in vitro resistance are covered to provide guidance for the current status of azole resistance in A. fumigatus.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03921-14 ◽

2014 ◽

Vol 58 (11) ◽

pp. 6904-6912 ◽

Cited By ~ 26

Author(s):

Sanjoy Paul ◽

Thomas B. Bair ◽

W. Scott Moye-Rowley

Keyword(s):

Abc Transporter ◽

Candida Glabrata ◽

High Throughput Sequencing ◽

Target Genes ◽

Primary Response ◽

Azole Resistance ◽

Mrna Levels ◽

Content Type ◽

Transcriptional Activator Protein ◽

Azole Tolerance

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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Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

mBio ◽

10.1128/mbio.02529-18 ◽

2019 ◽

Vol 10 (1) ◽

pp. e02529-18 ◽

Cited By ~ 17

Author(s):

Sang Hu Kim ◽

Kali R. Iyer ◽

Lakhansing Pardeshi ◽

José F. Muñoz ◽

Nicole Robbins ◽

...

Keyword(s):

Drug Resistance ◽

Fungal Pathogen ◽

Fungal Pathogens ◽

Filamentous Growth ◽

Antifungal Drugs ◽

Clinical Isolates ◽

Azole Resistance ◽

Candida Auris ◽

Content Type ◽

Insight Into

ABSTRACT Candida auris is an emerging fungal pathogen and a serious global health threat as the majority of clinical isolates display elevated resistance to currently available antifungal drugs. Despite the increased prevalence of C. auris infections, the mechanisms governing drug resistance remain largely elusive. In diverse fungi, the evolution of drug resistance is enabled by the essential molecular chaperone Hsp90, which stabilizes key regulators of cellular responses to drug-induced stress. Hsp90 also orchestrates temperature-dependent morphogenesis in Candida albicans, a key virulence trait. However, the role of Hsp90 in the pathobiology of C. auris remains unknown. In order to study regulatory functions of Hsp90 in C. auris, we placed HSP90 under the control of a doxycycline-repressible promoter to enable transcriptional repression. We found that Hsp90 is essential for growth in C. auris and that it enables tolerance of clinical isolates with respect to the azoles, which inhibit biosynthesis of the membrane sterol ergosterol. High-level azole resistance was independent of Hsp90 but dependent on the ABC transporter CDR1, deletion of which resulted in abrogated resistance. Strikingly, we discovered that C. auris undergoes a morphogenetic transition from yeast to filamentous growth in response to HSP90 depletion or cell cycle arrest but not in response to other cues that induce C. albicans filamentation. Finally, we observed that this developmental transition is associated with global transcriptional changes, including the induction of cell wall-related genes. Overall, this report provides a novel insight into mechanisms of drug tolerance and resistance in C. auris and describes a developmental transition in response to perturbation of a core regulator of protein homeostasis. IMPORTANCE Fungal pathogens pose a serious threat to public health. Candida auris is an emerging fungal pathogen that is often resistant to commonly used antifungal drugs. However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored. In this study, we adapted a conditional expression system to modulate the transcription of an essential gene, HSP90, which regulates antifungal resistance and virulence in diverse fungal pathogens. We showed that Hsp90 is essential for growth in C. auris and is important for tolerance of the clinically important azole antifungals, which block ergosterol biosynthesis. Further, we established that the Cdr1 efflux transporter regulates azole resistance. Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling. Overall, this work provides a novel insight into mechanisms regulating azole resistance in C. auris and uncovers a distinct developmental program regulated by Hsp90.

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