转录因子PDR1调控PUP1调节光滑假丝酵母菌氧化应激介导其对唑类药物耐药机制研究

Candida glabrata has been a predominant pathogen causing invasive infectious disease and its resistance to azole antifungals leads to the increase difficulty for efficient clinical treatment. In our preliminary experiments, mutations in transcription factor PDR1, upregulated expression of mitochondrial-related gene PUP1 and impairment of oxidation respiratory function are found in azole-resistant C.glabrata. Studies on adaptability of oxidative stress of C.glabrata protected from antifungals have attracted more and more attentions recently. Especially, the transcriptor factor PDR1 gene mutation-mediated mechanism became a hot topic. However, it's still unclear whether PUP1, including two Pleiotropic Drug Responsive Element （PDRE）sequences in its promoter, was regulated by transcriptory factor PDR1 and was realted to the respiratory status. In this project, two aspects are to be performed. Firstly, we'll describe the interactions beteween PDR1 and PUP1 and elucidate the regulation mechanism by construction of PUP1 promoter expression vetor and chromatin immunoprecipitation assay. The second purpose of our study is to demonstrate the impact of PDR1 and PUP1 on oxidation respiratory, activity of enzymes invovled in resporatory pathways and azole-susceptibility by analysis of gene knock-out and reversion isolates. Our goal is to discuss the molecular mechanism of the transcription factor PDR1 regualting PUP1 mediating the azole-resistance based on a broad point of stress response. These studies are expected to provide new target for the novel antifungal drug design and development.

光滑假丝酵母菌已成为侵袭性真菌感染的重要病原体，其对唑类抗真菌药物的耐药易感性给临床治疗带来很大困难。本课题组研究发现其唑类耐药株有转录因子PDR1突变、线粒体相关蛋白PUP1表达上调及氧化呼吸功能减弱。近年来假丝酵母菌适应性氧化应激抵抗药物作用的机制日益受到关注。PDR1突变介导光滑假丝酵母菌唑类耐药机制是研究热点。启动子含有PDRE序列的PUP1是否受PDR1调控，PUP1又是否与氧化代谢相关？本课题拟通过构建启动子表达载体，采用染色质免疫共沉淀技术，明确PDR1与PUP1之间的相互作用；分别构建PDR1、PUP1基因敲除株和回复株，分析菌株氧化呼吸功能，线粒体呼吸链相关酶的活性以及药物敏感性，探讨PDR1、PUP1对氧化呼吸及药物敏感性的影响。本研究从全新的氧化应激的角度阐明光滑假丝酵母菌转录因子PDR1调控PUP1介导其对唑类耐药的分子机制，为新型抗真菌药物研发提供新的靶点。

近年来，侵袭性真菌的感染率不断上升。临床上，光滑假丝酵母菌感染率仅次于白假丝酵母菌已经成为引起深部真菌感染的重要病原体。光滑假丝酵母菌对唑类临床抗真菌药物有先天耐药的倾向，增加了临床治疗的难度。. 本课题组对临床分离的38株光滑假丝酵母菌进行了药物敏感性分析，分离出17株唑类耐药株，发现其PDR1基因均有有义突变，其中未见L344S、P927S、G943S突变的报道。各敏感和耐药菌株间PDR1 mRNA水平无显著变化，然而唑类耐药菌株PUP1表达均显著高于敏感菌株，提示唑类耐药菌株中PDR1的有义突变影响了PUP1基因的表达水平。同时，我们发现17株光滑假丝酵母菌唑类耐药株线粒体膜电位、细胞内ROS水平及ATP含量均显著低于唑类敏感菌株（P<0.05），表明耐药菌株线粒体呼吸功能弱于敏感菌株，PDR1突变可以使光滑假丝酵母菌处于低氧化代谢状态。PDR1敲除菌株中PUP1的表达水平明显下调，而含有PDR1突变位点的菌株PUP1表达明显升高，表明PUP1为PDR1的下游调控基因。然而PUP1的敲除并不影响光滑假丝酵母菌对唑类药物的敏感性，因此PUP1与光滑假丝酵母菌对唑类药物的敏感性之间并无直接联系。同时，本课题组构建了Pdr1蛋白氨基端和羧基端携带Flag标签的菌株，发现Pdr1蛋白羧基端3xFLAG融合蛋白修饰诱导PDR1基因和Pdr1蛋白表达下调且介导菌株对唑类药物的敏感性升高。这表示Pdr1蛋白羧基端3xFLAG融合蛋白修饰后，其部分蛋白功能失活。这种蛋白功能的部分失活与转录共激活因子Gal11A无关，Pdr1蛋白羧基端3xFLAG融合蛋白修饰后，Pdr1蛋白与Gal11A仍存在相互作用。同时我们证明了GAL11A的缺失会造成Pdr1无法激活下游药物外排泵基因的表达从而造成菌株耐药性降低。. 本研究从氧化代谢的角度阐述了转录因子PDR1介导光滑假丝酵母菌耐药的分子机制，明确了转录因子Pdr1调控PUP1的表达，表明了PUP1对光滑假丝酵母菌氧化呼吸功能的影响，同时也阐明了Pdr1蛋白功能部分缺失造成光滑假丝酵母菌对唑类药物敏感的分子通路，为临床治疗光滑假丝酵母菌感染寻找新的药物作用靶点提供有力的实验基础和理论依据。