恶性疟原虫K13基因M579I突变参与青蒿素抗性机制的研究

Malaria is one of the parasitic diseases which severely impairs humanity health. The discovery of artemisinin resistance in Plasmodium falciparum poses a great challenge to malaria control and elimination. It found that artemisinin-resistant P. falciparum was associated with the mutations of the K13 gene in Southeast area. However, no confirmed artemisinin resistance strain was found in Africa. Our previous study found that the resistance selective pressure in K13 gene in Equatorial Guinea is much higher than other countries in Africa, and the strain with M579I mutation in K13 gene displayed artemisinin resistance. Therefore, as the specific mutation M579I in Africa is possible associated with artemisinin resistance also. The project intends to further confirm the correlation of mutation M579I and artemisinin resistance of P. falciparum by CRISPR-Cas9 system; To confirm the targets of K13 and the affect of mutation M579I, the K13 will be over-expressed and silenced; Checking the development of the M579I mutation strain with artemisinin treatment, and the change of the related genes’ expression level, to verify the up-regulation of unfolded protein response (UPR) in artemisinin resistance, thereby provide new clues for the mechanism and monitoring of artemisinin resistance in P. falciparum.

疟疾是严重威胁人类健康的寄生虫病。恶性疟原虫青蒿素抗性株的发现给疟疾防控和消除工作带来巨大的挑战，研究发现东南亚地区的恶性疟原虫青蒿素抗性与其K13基因突变有关，但迄今尚未在非洲发现明确的青蒿素抗性虫株。本课题组前期研究发现，赤道几内亚恶性疟原虫K13基因的抗性选择压明显高于其他非洲国家，并发现了携带K13基因M579I突变的虫株对青蒿素具有抗性。因此，K13基因非洲特有的M579I突变很可能也与青蒿素抗性有关。本研究拟应用CRISPR-Cas9基因编辑技术进一步确认M579I突变与恶性疟原虫青蒿素抗性的相关性；通过对K13蛋白的过表达和抑制来确定K13作用的靶点及M579I突变后产生的影响；并通过观察青蒿素处理后M579I突变株发育状态的改变及其相关基因表达水平变化，验证未折叠蛋白反应（UPR）上调在疟原虫青蒿素抗性中的作用，从而为恶性疟原虫青蒿素抗性分子机理及监测提供新的线索。

疟疾是一种重要的虫媒传染病，严重威胁着人类健康。作为目前恶性疟和重症疟疾治疗的最后一线药物，青蒿素抗性株的发现给疟疾防控和消除工作带来巨大的挑战，尤其是疟疾重灾区非洲。研究发现恶性疟原虫青蒿素抗性与其K13的螺旋桨区域的氨基酸变异有关，C580Y进一步被证实是恶性疟原虫青蒿素抗性的重要分子标志。本课题组首次发现了非洲恶性疟对青蒿素出现了耐药性，尤其是一种新发现的K13突变类型M579I，紧邻抗性关键氨基酸位点580，二者共处于第4个螺旋桨的β-折叠。恶性疟原虫的抗性已被证实可以有不同的起源，这种非洲特有的K13基因M579I突变很可能也与青蒿素抗性有关。本研究应用CRISPR-Cas9基因编辑技术，将虫株CWXM579I进行定点突变，获得CWXI579M株，并对其进行环状体生存率分析，确认了M579I突变与恶性疟原虫青蒿素抗性的相关性；通过对K13蛋白的过表达，发现环状体期发育进程受到了影响；利用RT-PCR分析UPR相关基因的表达水平，发现UTR与环状体期疟原虫休眠的相关性，环状体期发育是后续研究的关键。本研究鉴定了非洲独立起源的K13基因M579I突变参与青蒿素抗性的机制，为疟原虫抗青蒿素分子机理的认识提供新的线索。