State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, People's Republic of China.
University of Chinese Academy of Sciences, Beijing, 100049, People's Republic of China.
Curr Genet. 2019 Jun;65(3):735-745. doi: 10.1007/s00294-018-0924-7. Epub 2019 Jan 2.
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.
唑类药物是临床和农业中控制真菌感染最广泛使用的抗真菌药物。真菌可以通过快速激活许多基因的转录来适应唑类药物的压力，其中一些基因可以提高对唑类药物的抗性。我们曾报道转录因子 CCG-8 是 Neurospora crassa 和 Fusarium verticillioides 适应抗真菌唑类药物压力的新调节剂。在这项研究中，我们进一步以 N. crassa 为模型研究了 CCG-8 促进真菌适应唑类药物压力的机制。虽然 ccg-8 的缺失使 N. crassa 对唑类药物敏感，但 ccg-8 过表达菌株比野生型更能抵抗唑类药物，这进一步证实了 ccg-8 在适应抗真菌唑类药物中的积极作用。液相色谱-质谱分析显示，ccg-8 的缺失导致麦角固醇生物合成减少，细胞内有毒固醇 14α-甲基-3,6-二醇和酮康唑（KTC）积累增加，而 ccg-8 过表达菌株细胞内酮康唑积累减少。为了分析 CCG-8 对唑类药物外排的影响，我们测试了预测的多药转运蛋白对唑类药物抗性的贡献，发现 CDR4 是 N. crassa 中唑类药物外排的主要贡献者。有趣的是，在 ccg-8 缺失突变体中过表达 cdr4 或 erg11 恢复了其敏感表型，而过表达 cdr4 可以降低细胞内 KTC 的水平。然而，ccg-8 和 cdr4 的双突变体比每个单突变体更敏感，这表明药物外排泵 CDR4 在 ccg-8 缺失突变体中对细胞内唑类药物积累的贡献较小，而 CCG-8 可能调节药物摄取。总之，我们的结果表明，CCG-8 通过调节细胞内药物积累在 N. crassa 的唑类药物适应性反应中发挥关键作用。
