白念珠菌对唑类耐药和汉防己甲素逆转耐药过程中线粒体氧化磷酸化的主要机制

Our previous studies demonstrated that the changes of energy metabolism, in which oxidative phosphorylation is a critical link, are key mechanism underlying azole resistance and tetrandrine-mediated reversal of resistance in Candida albicans. The aim of this project is to reveal the mechanism by investigation of the major steps in oxidative phosphorylation, and ATP synthase, the key enzyme of it. To systematically reveal the the relationship between azole resistance and tetrandrine-mediated reversal of resistance in C. albicans and each step of oxidative phosphorylation, fluconazole-sensitive or fluconazole-resistant substrains of C. albicans isolated from a single patient would be used; the electron transport pathway, phosphorylation of ADP, and Oxidative phosphorylation coupling are suppressed by using of electron transport inhibitors, oxidative phosphorylation inhibitors, and uncoupling agents, respectively. Following this, strains with genes knock-out and overexpression of ATP1 and ATP2, both of which are important coding genes for ATP synthase in C. albicans, would be constructed by STA1-Flipper and overexpression strategies, and observed for differences in phenotype (growth, drug resistance and virulence, etc), proteomics, metabonomics, and other aspects before and after drug administration to discover novel functions as well as mechanisms of ATP synthase in drug resistance and reversible response. Because of the specific structure of ATP synthase in C. albicans, our proposed approach will be great scientific significance for clarifying the novel functions of oxidative phosphorylation in drug resistance, and finding drugs that can act on the specific target, combining anti-fungal therapy and reversing drug resistance.

我们前期发现，能量代谢变化影响白念珠菌对唑类耐药和汉防己甲素逆转耐药的过程。氧化磷酸化是能量代谢的关键环节。为了揭示其机制，本项目针对氧化磷酸化主要环节及其末端酶和关键酶ATP合酶进行研究。拟采用同一亲本来源的氟康唑敏感或耐药菌株，以呼吸链抑制剂、氧化磷酸化抑制剂和解偶联剂分别抑制呼吸链电子传递、ADP磷酸化及其偶联，系统揭示白念珠菌耐药及其逆转与氧化磷酸化各环节的关系。以STA1-Flipper基因敲除和异位高表达术分别构建白念珠菌ATP合酶重要编码基因ATP1、ATP2缺失株和高表达株，观察其用药前后表型、耐药基因表达、蛋白质组学和代谢组学等变化，揭示ATP合酶在耐药及其逆转中的新机制。由于白念珠菌ATP合酶结构特异，项目完成后将极有希望发现氧化磷酸化（尤其ATP合酶）在耐药过程中的新功能，并进而发现作用于该靶点的药物，为抗真菌药物（及其增效剂）的研发提供新思路，具有重要科学意义。

白念珠菌是人类致死性真菌性感染最重要的病原体之一，系统感染时药物疗效较差，且有耐药现象出现。. 本项目主要研究内容：①线粒体氧化磷酸化参与白念珠菌对唑类耐药、汉防己甲素（TET）逆转耐药过程的系统证据与主要机制；②ATP合酶重要编码基因对唑类药物耐药、TET逆转耐药的影响机制。. 研究发现：（1）针对呼吸链电子传递、ADP磷酸化及偶联三个环节，在抑制剂SHAM/AA/Oligomycin/FCCP作用下，不同耐药水平的白念珠菌株（加入或不加TET）①抑制氧化呼吸链后显著增加耐药株对氟康唑敏感性，细胞内Rh123蓄积增多、外排减少，耐药基因CDR1/CDR2表达下调，线粒体功能受损，细胞凋亡显著增多；②TET联合氟康唑显著提高耐药株敏感性，促进内源性ROS产生，升高SOD及CAT酶活性，下调ROS清除相关因子及氧化应激相关基因表达，增加细胞凋亡，提高对凋亡因子的激活作用。依此，获得了线粒体氧化磷酸化途径参与白念珠菌耐药、TET逆转耐药的证据和机制。（2）构建白念珠菌ATP合酶编码基因（ATP1、ATP2、ATP16）缺失株，采用CLSI M27-A3微量法及点板实验等测定其药敏变化，发现上述基因缺失后耐药性改变（特别是非发酵碳源培养基中，耐药明显增强），获得ATP合酶重要编码基因与耐药相关的直接证据。（3）令人振奋的是，白念珠菌ATP2、ATP16缺失后虽不明显影响生长繁殖和ATP产生、细胞壁成分相关因子，但无法对小鼠造成致死性感染，且菌丝形成少、生物膜形成及活性低、在巨噬细胞中存活的能力低。（4）这些基因可通过碳源代谢调节来维持毒力及在吞噬细胞中生存。其缺失后不利于ADP转化ATP，但可通过基因调控网络调节线粒体能量代谢。. 研究结果揭示，ATP合酶参与了白念珠菌致死性感染的关键环节，且与耐药性直接相关。这为寻找抗白念珠菌药物的新靶标提供了直接的科学依据。. 如期完成了合同书内容。发表论文11篇（SCI收录7篇）。正在投稿2篇。