Fungal Zn(II)2Cys6 Transcription Factor ADS-1 Regulates Drug Efflux and Ergosterol Metabolism under Antifungal Azole Stress

Abstract

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 Neurospora crassa. Under ketoconazole (KTC) stress, the ads-1 transcript level was significantly increased in N. crassa. Deletion of ads-1 increased susceptibility to different azoles, while its overexpression increased resistance to these azoles. The cdr4 gene, which encodes the key azole efflux pump, was positively regulated by ADS-1. Deletion of ads-1 reduced the transcriptional response by cdr4 to KTC stress and increased cellular KTC accumulation under KTC stress, while ads-1 overexpression had the opposite effect. ADS-1 also positively regulated the transcriptional response by erg11, which encodes the azole target lanosterol 14α-demethylase for ergosterol biosynthesis, to KTC stress. After KTC treatment, the ads-1 deletion mutant had less ergosterol but accumulated more lanosterol than the wild type, while ads-1 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 Aspergillus flavus also increased susceptibility to KTC and itraconazole (ITZ). Besides, deletion of A. flavus ads-1 (Afads-1) significantly reduced the transcriptional responses by genes encoding homologs of CDR4 and ERG11 in A. flavus 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.