蛋白激酶STK-17调控真菌对唑类药物的胁迫响应与耐受机制

The increasing emergence of antifungal azole resistance in clinic and agriculture in recent years has received much concern. The understanding of drug resistance mechanisms will provide more theoretical basis for the treatment of drug resistance and development of antifungal drugs. Our group found the protein kinase STK-17 participating in azole response and resistance. Its coding gene stk-17 increased transcriptional levels under ketoconazole stress. Deletion or active site mutation of STK-17 in Neurospora crassa and Fusarium verticillioides caused hypersensitive to antifungal azoles. STK-17 is a new regulator in the upstream signaling pathway of responses and resistance to antifungal azole stress. However, the regulatory mechanisms of this kinase and its homologs in azole response and resistance are unknown. In this study, we propose to investigate the mechanism by which STK-17 promote fungal adaptation under azoles using N. crassa as a model. We will explore the direct target proteins and phosphorylation motif of STK-17 and their roles in azole response and resistance, by using proteomic and phosphoproteomic methods in combination with mutant analysis, in vitro phosphorylation assay and drug sensitivity test. We will also investigate the key genes and biological processes regulated by STK-17. Furthermore, the roles of the kinase STK-17 in regulating ergosterol biosynthesis and intracellular drug accumulation will also be dissected. Finally, the role and mechanisms of STK-17 in the network of azole response and resistance will be revealed.

近年来临床和农业病原真菌对使用最广泛的唑类药物耐受性问题日趋严重，耐药机制的解析将为真菌耐药性治理及新抗真菌药物的研发提供重要理论依据。我们前期的研究发现了一个保守的受唑类药物胁迫响应的蛋白激酶STK-17，其缺失或活性位点突变导致粗糙脉孢菌和轮枝镰刀菌对唑类药物的超敏感。作为真菌对唑类药物胁迫响应调控的上游信号途径中新发现的一个关键调控因子，STK-17的作用机制尚不清楚。本项目将以粗糙脉孢菌为材料，采用磷酸蛋白谱、点突变、体外磷酸化及药敏分析，明确STK-17直接磷酸化的蛋白与位点，以及靶蛋白磷酸化修饰在真菌对唑类药物耐受性中的作用；通过差异表达谱与遗传分析，解析STK-17所调控的关键基因与生物学过程；通过HPLC-MS/MS与转录分析，探究STK-17对真菌麦角甾醇合成与体内唑类药物积累的影响。综合以上研究结果，揭示STK-17在真菌对唑类药物胁迫响应与耐受调控网络中的作用及机制。

真菌耐药性问题尤其是对应用最为广泛的唑类药物的耐药性在全球范围内日趋严重，给真菌感染和真菌病害的防控带来巨大挑战。真菌耐药机制的解析将为真菌病害的防治及新抗真菌药物的研发提供重要理论依据。我们前期新发现蛋白激酶STK-17是真菌对唑类药物胁迫响应上游信号途径中的一个关键调控因子。本项目旨在解析STK-17在真菌对唑类药物胁迫响应与耐受调控网络中的作用与机制，我们按计划完成了项目的研究目标。本项目以粗糙脉孢菌为研究对象，进一步明确了STK-17作为保守的激酶是一个全新的唑类药物胁迫响应调控因子。STK-17的敲除或者功能丧失均会导致粗糙脉孢菌对唑类药物、阿莫洛芬、特比萘芬等其它麦角甾醇合成抑制剂的超敏感。此外，STK-17还影响了对氧化胁迫的敏感性，但这和其影响唑类药物敏感性无关。通过RNA-seq、表型分析、化学分析和遗传互作分析，我们明确了STK-17通过调控细胞膜的稳态，调控唑类药物靶标基因erg11和其它麦角甾醇合成基因的表达来影响唑类药物胁迫下的甾醇稳态，以及调控非药泵CDR4依赖的唑类药物的体内积累而调控真菌对唑类药物的胁迫响应。通过磷酸化蛋白质组，我们发现stk-17的敲除影响了包括CDR4、CCG-8和FWD-1等多个和唑类药物耐药性相关的蛋白的磷酸化。STK-17通过一个保守的磷酸化基序MxRxxS/T来影响下游蛋白的磷酸化。综上所述，我们明晰了蛋白激酶STK-17调控真菌对唑类药物的胁迫响应的机制，加深了我们对真菌耐药机制的理解。STK-17在真菌中广泛存在且高度保守，并调控了生长发育、胁迫响应等诸多重要过程。我们的结果也为抗真菌药物的使用和研发提供了新的理论基础。