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Fluconazole resistance and CDR1 expression in Candida albicans mediated by the hyperactive Tac1-5 transcriptional
Wen Jun Lim1, Brenda Lee2, Zahra Farrington2
1Division of Oral Biosciences, Dublin Dental University Hospital, University of Dublin, Trinity College Dublin, Dublin, Ireland.
Abstract:
Candida albicans is an opportunistic fungal pathogen associated with superficial and systemic infections in humans. Azole antifungal resistance in C. albicans is of clinical concern, and both oral and systemic Candida infections can be difficult to treat due to the lack of alternative antifungal drugs. Expression of a hyperactive form of the transcription factor Tac1 is a major contributor to azole resistance in C. albicans isolates resulting in the increased expression of the azole efflux pump Cdr1. In this study, we investigated whether the Mediator tail component Med2, encoded by the expanded (n=14) TLO gene family of C. albicans, was required for Tac1 activity. A homozygous TAC1-5 gain-of-function point mutation was introduced into WT, tloΔ and med3Δ strains of C. albicans which enables them to express hyperactive Tac1. qRT-PCR analysis revealed that tloΔ-TAC1-5 had reduced basal and fluphenazine-induced CDR1 expression relative to WT-TAC1-5 strains and exhibited reduced levels of resistance to fluconazole and terbinafine. Individual copies of representatives from each of the alpha, beta and gamma TLO clades were reintroduced into tloΔ-TAC1-5 to investigate their ability to restore Tac1-activated resistance. These studies show that alpha and beta TLO genes could restore fluconazole resistance in the tloΔ-TAC1-5 background, whereas gamma clade genes did not result in any detectable phenotypic complementation. Transcript profiling showed that reintroduction of TLOα1 led to increased expression of TAC1-5-activated genes such as CDR1. Further analysis using ChIP-qPCR revealed that Tloα1 localizes to the drug response element which is the site where Tac1 binds to the CDR1 promoter. These data have identified that the TLO gene family is required for the expression of Tac1-mediated fluconazole resistance. However, this effect is confined to members of the alpha and beta, but not the gamma, TLO clades.
Insights
The TLO gene family in Candida albicans is essential for Tac1-mediated azole antifungal resistance. Alpha and beta TLO genes restore resistance, while gamma clade genes do not, highlighting specific clade roles in drug resistance.
Area of Science:
- Medical Mycology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Candida albicans is a major cause of fungal infections.
- Azole antifungal resistance is a growing clinical problem.
- Hyperactive Tac1 transcription factor drives azole resistance via CDR1 upregulation.
Purpose of the Study:
- Investigate the role of the Mediator tail component Med2, encoded by the TLO gene family, in Tac1 activity.
- Determine if TLO genes are required for Tac1-mediated azole resistance.
Main Methods:
- Generated TAC1-5 gain-of-function mutants in Candida albicans.
- Utilized qRT-PCR to analyze CDR1 gene expression.
- Performed phenotypic analysis of antifungal resistance.
- Reintroduced individual TLO genes to assess complementation.
- Conducted transcript profiling and ChIP-qPCR.
Main Results:
- tloΔ-TAC1-5 strains showed reduced CDR1 expression and azole resistance.
- Alpha and beta TLO genes restored fluconazole resistance in tloΔ-TAC1-5 mutants.
- Gamma clade TLO genes did not restore resistance.
- TLOα1 reintroduction increased TAC1-5-activated gene expression.
- Tloα1 localizes to the CDR1 promoter's drug response element.
Conclusions:
- The TLO gene family is crucial for Tac1-mediated fluconazole resistance in Candida albicans.
- This function is specific to alpha and beta TLO clades, not the gamma clade.
- Identifies a novel role for specific TLO clades in regulating antifungal drug resistance.
