Fungal Zn(II) ₂ Cys ₆ Transcription Factor ADS-1 Regulates Drug Efflux and Ergosterol Metabolism under Antifungal Azole Stress

Abstract

Antifungal azoles are the most widely used antifungal drugs in clinical and agricultural practice. Fungi can mount adaptive responses to azole stress by modifying the transcript levels of many genes, and the responsive mechanisms to azoles are the basis for fungi to develop azole resistance. In this study, we identified a new Zn(II)₂Cys₆ transcription factor, ADS-1, with a positive regulatory function in transcriptional responses to azole stress in the model filamentous fungal species <i>Neurospora crassa</i> Under ketoconazole (KTC) stress, the <i>ads-1</i> transcript level was significantly increased in <i>N. crassa</i> Deletion of <i>ads-1</i> increased susceptibility to different azoles, while its overexpression increased resistance to these azoles. The <i>cdr4</i> gene, which encodes the key azole efflux pump, was positively regulated by ADS-1. Deletion of <i>ads-1</i> reduced the transcriptional response by <i>cdr4</i> to KTC stress and increased cellular KTC accumulation under KTC stress, while <i>ads-1</i> overexpression had the opposite effect. ADS-1 also positively regulated the transcriptional response by <i>erg11</i>, which encodes the azole target lanosterol 14α-demethylase for ergosterol biosynthesis, to KTC stress. After KTC treatment, the <i>ads-1</i> deletion mutant had less ergosterol but accumulated more lanosterol than the wild type, while <i>ads-1</i> overexpression had the opposite effect. Homologs of ADS-1 are widely present in filamentous fungal species of Ascomycota but not in yeasts. Deletion of the gene encoding an ADS-1 homolog in <i>Aspergillus flavus</i> also increased susceptibility to KTC and itraconazole (ITZ). Besides, deletion of <i>A. flavus</i><i>ads-1</i> (Af<i>ads-1</i>) significantly reduced the transcriptional responses by genes encoding homologs of CDR4 and ERG11 in <i>A. flavus</i> to KTC stress, and the deletion mutant accumulated more KTC but less ergosterol. Taken together, these findings demonstrate that the function and regulatory mechanism of ADS-1 homologs among different fungal species in azole responses and the basal resistance of azoles are highly conserved.