TgMIC6：Chinese1型弓形虫毒力调控因子及其致病机制

Toxoplasma is an intracellular protozoa that can cause serious diseases to human and animals. Chinese1 of Toxoplasma is the predominant genotype prevalent in China, showing unique genetic and virulent characteristics that different from classic genotype of T. gondii. However, its virulence determinants and their modulating mechanisms are still remain elusive.TgMIC6 is a protein secreted by micronemes of Toxoplasma, and was found to play a negative regulatory role on the classic autophagy signaling pathway. Our previous study found that the expression level of TgMIC6 in TgCtwh3 (highly virulent) was significantly higher than that in TgCtwh6 (intermediately virulent). In the present study, CRISPR/Cas9 genome-editing technology is used to produce TgMIC6 knockout or complementation in TgCtwh3 or TgMIC6 overexpression in TgCtwh6. After infection of wild type mice or specific gene knockout mice with different strains of Toxoplasma, the survival rate of mice and related cytokines levels in mice are observed. Wild type or specific gene knockout or complementation of TgCtwh3, and wild type or specific gene overexpression of TgCtwh6 are used to infect different cell lines isolated from mouse or human, and the proliferation of the parasite, the autophagy level of host cell and the activation of key molecules involved in EGFR-Akt-mTOR classic autophagy pathway are evaluated. To further explain the molecular mechanism of how TgMIC6 modulates the virulence of Chinese1, cells are treated with different inhibitors or stimulators or gene specific siRNAs, and the proliferation of the parasite is determined. This study will reveal the virulence determinant and immune escape strategy of Toxoplasma Chinese1, the predominant genotype of Toxoplasma in China, as well as to formulate effective prevention and control strategies for toxoplasmosis.

Chinese1型弓形虫是我国人兽间流行的优势基因型，表现出与欧美流行的经典基因型弓形虫迥异的毒力特征，而目前对其毒力决定因子及其作用机制知之甚少。TgMIC6是对经典自噬信号通路具有负调节作用的微线体蛋白，前期转录组分析发现其在Chinese1强毒株TgCtwh3内的表达显著高于弱毒株TgCtwh6，强烈提示此蛋白与该基因型毒力密切相关。本研究拟利用CRISPR/Cas9技术构建TgMIC6基因敲除/回补的TgCtwh3株，TgMIC6过表达TgCtwh6株，观察不同虫株在野生型/基因敲除小鼠体内毒力；上述不同虫株分别感染各型人/鼠源细胞株，观察其胞内增殖、细胞自噬及EGFR-Akt-mTOR经典自噬信号通路中关键分子的激活，并运用药物/基因敲低信号通路不同环节加以干预，观察虫体的增殖水平。该研究对于揭示我国弓形虫优势基因型的毒力决定因子特征和致病机制、制定有效的防控策略具有重要的意义。

Chinese1型弓形虫是我国人兽间流行的优势基因型，表现出与欧美流行的经典基因型弓形虫迥异的毒力特征，而目前对其毒力相关因子及其作用机制知之甚少。本研究对我国人兽间流行的弓形虫优势基因型Chinese1的两个代表性虫株——TgCtwh3（强毒）和TgCtwh6（弱毒）进行了基因组和转录组分析，揭示了二者主要经典毒力因子的基因结构特征。转录组分析发现这两个代表性虫株在既往认为与经典基因型毒力相关的几个效应分子（ROP18、ROP5、ROP16、GRA15）的转录水平均没有明显的差异，但与弓形虫入侵/免疫调控有关的数个基因（MIC1、MIC4、MIC6、SAG1、ROP4、RON2、 RON4等）的转录水平在强毒株TgCtwh3 内显著升高，该结果也得到了实时荧光定量PCR 进一步验证。免疫印迹检测发现MIC6的相对表达在TgCtwh3内显著高于TgCtwh6。我们利用CRISPR/Cas9技术构建TgMIC6基因敲除/回补的TgCtwh3株，观察不同虫株在野生型/基因敲除小鼠体内毒力；上述不同虫株分别感染各型人/鼠源细胞株，观察其胞内增殖、细胞自噬及EGFR-Akt-mTOR经典自噬信号通路中关键分子的激活，并运用药物/基因敲低信号通路不同环节加以干预，观察虫体的增殖水平。结果提示TgMIC6在Chinese1基因型弓形虫强毒株TgCtwh3内高表达，一方面增强其细胞入侵能力，另一方增强其逃逸CD40-CD154诱导的经典自噬途径的清除。TgMIC6在Chinese1基因型不同毒力虫株中的差异表达，能部分解释二者致小鼠毒力差异。该研究对于揭示我国弓形虫优势基因型的毒力相关因子特征和致病机制、制定有效的防控策略具有重要的意义。