Fluconazole resistance and CDR1 expression in Candida albicans mediated by the hyperactive Tac1-5 transcriptional activator requires Tlo proteins.

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.