Abstract

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.

Highlights

  • Antifungal drugs are limited to three classes of compounds that are routinely used in the clinic [1]

  • The amino terminal 254 residues define a Zn2 Cys6 zinc cluster-containing DNA-binding domain (DBD) while the extreme carboxy-terminal 138 amino acids specify the major transactivation domain (TAD). These two critical regions are separated by 715 amino acids that encode the central regulatory domain (CRD) of this factor

  • Loss of the central regulatory domain led to the production of a form of Pdr1 that was so active that it was lethal when present as the only copy of this gene in the cell [10]

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Summary

Introduction

Antifungal drugs are limited to three classes of compounds that are routinely used in the clinic [1]. D1082G allele into the Δ255–968 form of the PDR1 gene led to a 2-log increase in transformation efficiency when compared to the same clone containing the wildtype D1082 form as the wild-type TAD caused toxicity in this context [10]. This finding prompted us to characterize the behavior of this altered form of the Δ255–968 Pdr in more detail. To compare Δ255–968 D1082G and Δ255–968 Pdr forms, plasmids containing each of these genes or the empty vector alone were introduced into PDR1 cells Both the Δ255–968 forms of Pdr drove high level fluconazole resistance, the Δ255–968 D1082G Pdr was reduced compared to Δ255–968 Pdr (Fig 4C). Chromatin was sheared with E220 Focused-ultrasonicator (Covaris) under the following conditions: peak incident power (W): 175, duty factor: 20%, cycles per burst: 200, treatment time (sec): 720, temperature ( ̊C): 7, sample volume (μl): 130, under the presence of E220-intensifier

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