转录因子ADS-1介导的对唑类抗真菌药物的适应性响应机制

The antifungal azoles, including imidazoles and triazoles, are the major drugs for fungal infections. Some azoles are also applied as pesticides. Azoles disrupt ergosterol biosynthesis by inhibiting the enzyme 14α-demethylase. Fungi are able to adapt to azole stress by altering transcriptional levels of many genes. Overexpression of some azole-responsive genes, such as azole target gene ERG11 or azole pumps encoding genes, has been demonstrated to be able to increase azole resistance in many fungi. Thus, the transcriptional response is an adaptive response to azole stress, which promote fungi to survive under azole stress. Our group found a novel transcription factor named as ADS-1 (antifungal drug sensitive-1). Its coding gene ads-1 increases transcriptional levels under ketoconazole stress. Deletion of ads-1 resulted in hypersusceptibility to antifungal azoles while its overexpression make N. crassa more resistant to azoles. ADS-1 homologs widely present in filamentous fungi. Deletion of ADS-1 homologue encoding genes fvads-1 in Fusarium verticillioides and afads-1 in Aspergillus flavus also make these two fungi hypersensitive to antifungal azoles. Thus, ADS-1 homologs are functional among different fungal species. In this study, we propose to investigate the mechanism by which ADS-1 promote fungal adaptation under azoles using N. crassa as a model. We will identify the genes regulated by ADS-1 by RNA-seq followed with result confirmation by qRT-PCR. We then test the azole susceptibility of gene knockout mutants of ADS-1-regulated genes to determine the roles of ADS-1-regulated genes in azole adaptation. We will also identify genome-wide DNA regions where ADS-1 binds with ChIP-seq. Based one the DNA sequence analysis, we will conclude the consensus motifs recognized and bound by ADS-1. In addition, we will analyze the relationship among ADS-1 and other transcription factors including CCG-8 and ADS-4. Finally, the role and mechanisms of ADS-1 in the network of azole response will be revealed.

唑类药物是麦角甾醇的合成抑制剂，是临床和农业上使用最广泛的抗真菌药物。在唑类药物胁迫下，真菌很多基因会出现适应性的转录响应，促进真菌在药物环境下生存。ADS-1是我们发现的一个新转录因子。ADS-1的缺失会使粗糙脉孢菌对唑类药物的敏感性增强，而过表达能使粗糙脉孢菌对唑类药物的耐受性增强。ADS-1的同源蛋白在丝状真菌中普遍存在，将其轮枝镰刀菌、黄曲霉中ADS-1的同源蛋白编码基因敲除之后，突变体对唑类药物的敏感性也增强，说明该转录因子的功能保守。ADS-1及其同源蛋白在抗真菌药物胁迫响应中的作用与机制尚未见报道。本项目将以粗糙脉孢菌为模式材料，鉴定转录因子ADS-1所调控的基因，明确这些基因在真菌对唑类抗真菌药物的适应性响应中作用，鉴定ADS-1所结合的DNA序列的共性特征，分析ADS-1与其它转录因子在唑类药物胁迫响应过程中的分工与协作关系，最终揭示ADS-1对唑类响应的机制。

唑类药物是临床和农业中最常用的抗真菌药物，但在其使用过程中很容易产生耐药性。真菌对唑类抗真菌药物胁迫的响应是耐药性产生的基础，解析其响应调控机制对揭示耐药性的成因与研发新抗真菌药物具有重要价值。而目前，这一研究在丝状真菌中较为匮乏。本课题以模式丝状真菌粗糙脉孢菌（Neurospora crassa）为研究对象，确定了一个新的Zn(II)2Cys6转录因子ADS-1（Antifungal Drug Sensitive-1）在响应唑类药物胁迫中的正向调控作用。ADS-1 对唑类药物存在转录响应，其缺失增加了对多种唑类药物的敏感性，而其高表达则增加了对这些唑类药物的抗性。通过RNA-seq、表型分析和化学分析，我们明确了ADS-1的调控机制，即通过正向调控唑类药物外排泵基因cdr4来影响唑类药物的体内积累，并通过正向调控唑类药物靶标基因erg11的转录响应来影响唑类药物胁迫下的甾醇稳态。蛋白保守性分析表明，ADS-1的同源蛋白广泛存在于子囊菌门的丝状真菌中。功能分析显示，在病原真菌黄曲霉（Aspergillus flavus）和轮枝镰刀菌（Fusarium verticillioides）中，ADS-1同源蛋白编码基因也对唑类药物存在转录响应，且其敲除可提高对唑类药物的敏感性。此外，机制分析表明，黄曲霉和轮枝镰刀菌中ADS-1同源蛋白同样通过调控药物外排泵和唑类药物靶点编码基因分别调控着药物的体内积累和甾醇稳态，说明ADS-1在功能和调控机制上高度保守。在上述研究的基础上，我们根据调控的基因分析出了ADS-1结合的DNA基序NGNANNCGAN，并通过ChIP-PCR在粗糙脉孢菌中进行了验证，确定了ADS-1对CDR4和ERG11的直接调控。综上所述，本课题确定了ADS-1这一保守的转录因子对唑类药物胁迫响应的调控作用及其调控机制，加深了对唑类药物胁迫响应调控网络的理解，为抗真菌药物的使用和研发提供了新的理论基础。