Positive autoregulation and repression of transactivation are key regulatory features of the Candida glabrata Pdr1 transcription factor

• Published in: Molecular microbiology Vol. 107; no. 6; pp. 747 - 764
• Main Authors: Khakhina, Svetlana, Simonicova, Lucia, Moye‐Rowley, W. Scott
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.03.2018
• Subjects: Antifungal Agents - pharmacology; ATP-Binding Cassette Transporters - metabolism; Binding Sites; Candida glabrata; Candida glabrata - genetics; Candida glabrata - metabolism; Deletion mutant; Derivatives; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Drug resistance; Drug Resistance, Fungal; Fluconazole - pharmacology; Fungi; Fungicides; Gene expression; Gene Expression Regulation, Fungal - drug effects; Genes; Homology; Mutation; Promoter Regions, Genetic; Resistance factors; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Site-directed mutagenesis; Trans-Activators - metabolism; Transcription factors; Transcription Factors - genetics; Transcription Factors - metabolism; Transcriptional Activation - drug effects; Zinc
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.13913

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.