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

<i>Candida albicans</i> is an opportunistic fungal pathogen associated with superficial and systemic infections in humans. Azole antifungal resistance in <i>C. albicans</i> is of clinical concern, and both oral and systemic <i>Candida</i> 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 <i>C. albicans</i> 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) <i>TLO</i> gene family of <i>C. albicans</i>, was required for Tac1 activity. A homozygous <i>TAC1-5</i> gain-of-function point mutation was introduced into WT, <i>tlo</i>Δ and <i>med3</i>Δ strains of <i>C. albicans</i> which enables them to express hyperactive Tac1. qRT-PCR analysis revealed that <i>tlo</i>Δ-<i>TAC1-5</i> had reduced basal and fluphenazine-induced <i>CDR1</i> expression relative to WT-<i>TAC1-5</i> strains and exhibited reduced levels of resistance to fluconazole and terbinafine. Individual copies of representatives from each of the alpha, beta and gamma <i>TLO</i> clades were reintroduced into <i>tlo</i>Δ-<i>TAC1-5</i> to investigate their ability to restore Tac1-activated resistance. These studies show that alpha and beta <i>TLO</i> genes could restore fluconazole resistance in the <i>tlo</i>Δ-<i>TAC1-5</i> background, whereas gamma clade genes did not result in any detectable phenotypic complementation. Transcript profiling showed that reintroduction of <i>TLO</i>α<i>1</i> led to increased expression of <i>TAC1-5</i>-activated genes such as <i>CDR1</i>. Further analysis using ChIP-qPCR revealed that Tloα1 localizes to the drug response element which is the site where Tac1 binds to the <i>CDR1</i> promoter. These data have identified that the <i>TLO</i> 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, <i>TLO</i> clades.