Transcription factor CCG-8 as a new regulator in the adaptation to antifungal azole stress

Antifungal azoles are widely used for controlling fungal infections. Fungi are able to change the expression of many genes when they adapt to azole stress, and increased expression of some of these genes can elevate resistance to azoles. However, the regulatory mechanisms behind transcriptional adaption to azoles in filamentous fungi are poorly understood. In this study, we found that deletion of the transcription factor gene ccg-8, which is known to be a clock-controlled gene, made Neurospora crassa hypersensitive to azoles. A comparative genome-wide analysis of the responses to ketoconazole of the wild type and the ccg-8 mutant revealed that the transcriptional responses to ketoconazole of 78 of the 488 transcriptionally ketoconazole-upregulated genes and the 427 transcriptionally ketoconazole-downregulated genes in the wild type were regulated by CCG-8. Ketoconazole sensitivity testing of all available knockout mutants for CCG-8-regulated genes revealed that CCG-8 contributed to azole adaption by regulating the ketoconazole responses of many genes, including the target gene (erg11), an azole transporter gene (cdr4), a hexose transporter gene (hxt13), a stress response gene (locus number NCU06317, named kts-1), two transcription factor genes (NCU01386 [named kts-2] and fsd-1/ndt80), four enzyme-encoding genes, and six unknown-function genes. CCG-8 also regulated phospholipid synthesis in N. crassa in a manner similar to that of its homolog in Saccharomyces cerevisiae, Opi1p. However, there was no cross talk between phospholipid synthesis and azole resistance in N. crassa. CCG-8 homologs are conserved and are common in filamentous fungi. Deletion of the CCG-8 homolog-encoding gene in Fusarium verticillioides (Fvccg-8) also made this fungus hypersensitive to antifungal azoles.