Transcription factor CCG-8 plays a pivotal role in azole adaptive responses of Neurospora crassa by regulating intracellular azole accumulation.

Azoles are the most widely used antifungals for controlling fungal infections in clinic and agriculture. Fungi can adapt to azole stress by rapidly activating the transcription of a number of genes, and some of these genes can elevate resistance to azoles. We had reported the transcription factor CCG-8 as a new regulator in the adaptation to antifungal azole stress in Neurospora crassa and Fusarium verticillioides. In this study, we further investigate the mechanisms by which CCG-8 promotes fungal adaptation to azole stress using N. crassa as a model. While deletion of ccg-8 made N. crassa hypersensitive to azoles, ccg-8 overexpression strain was more resistant to azoles than wild type, which further confirmed the positive role of ccg-8 in the adaptation to antifungal azoles. Liquid chromatography-mass spectrometry analysis showed that deletion of ccg-8 resulted in decrease of ergosterol biosynthesis, and high accumulation of toxic sterol 14α-methyl-3,6-diol and ketoconazole (KTC) in the cells, whereas intracellular accumulation of ketoconazole was decreased in the ccg-8 overexpression strain as compared to wild type. For analyzing the effect of CCG-8 on azole export, we tested the contribution of predicted multidrug transporters to azole resistance and found that CDR4 is the major contributor for azole efflux in N. crassa. Interestingly, overexpression of cdr4 or erg11 in the ccg-8 deletion mutant restored its hypersensitive phenotype and overexpression of cdr4 can reduce the level of intracellular KTC. However, the double mutant of ccg-8 and cdr4 was more sensitive than each single mutant, suggesting that drug efflux pump CDR4 plays less contribution for intracellular azole accumulation in the ccg-8 deletion mutant, and CCG-8 may regulate drug uptake. Together, our results revealed that CCG-8 plays a pivotal role in azole adaptive responses of N. crassa by regulating the drug accumulation in the cells.