弓形虫硫氧还原蛋白家族抵抗巨噬细胞活性氧的作用研究

The resistance of Toxoplasma gondii to reactive oxygen species (ROS) from macrophages is an important to evade innate immunity of the host, the mechanism of which is related to the redox metabolism. By Library of Apicomplexan Metabolic Pathways (LAMP), 11 Trx family proteins were found to be involved in the redox pathway, but the key Trx family proteins were still uncertain. This project intends to compare the transcriptional level of Trx family genes of Toxoplasma gondii under oxidative stress by qPCR, and obtain significantly up-regulated Trx family recombinant proteins through prokaryotic expression and prepare polyclonal antibodies. The antioxidant ability of the recombinant proteins is tested by MCO assay.The differences of expression and subcellular localization of up-regulated Trx family proteins were compared by Western blot and IFA, and the key Trx family proteins were preliminarily screened. The gene knockout and complement strains were made by CRISPR/Cas9 technology.Then the in vitro anti-hydrogen peroxide ability, invasive efficiency, intracellular proliferation ability and pathogenicity to mice were compared. Finally, the role of Trx family key proteins in resisting reactive oxygen species (ROS) attack from macrophages was elucidated, which provided a theoretical basis for revealing the innate immunity of Toxoplasma gondii to escape host and drug molecular targets.

弓形虫抵抗巨噬细胞活性氧（ROS）是逃避宿主先天免疫的重要手段，其机制与虫体抗氧化系统有关。本课题组通过检索顶复门原虫代谢通路数据库（LAMP）发现11个硫氧还蛋白（Trx）家族成员参与了虫体抗氧化系统，但发挥关键作用Trx家族蛋白质仍未确定。本项目拟通过qPCR比较氧化应激条件下弓形虫Trx家族基因转录水平，通过原核表达获得显著上调的Trx家族重组蛋白质并制备多克隆抗体，利用MCO试验检测重组蛋白质的抗氧化能力，并利用Western blot和IFA方法检测转录中上调蛋白质的表达水平及亚细胞定位情况，初步筛选关键Trx家族蛋白；通过CRISPR/Cas9技术敲除拟关键蛋白质，构建基因缺失虫株和回补虫株，比较其体外抵抗H2O2能力、抵抗小鼠巨噬细胞ROS能力和致病力的差异，最终阐明Trx家族关键蛋白质抵抗巨噬细胞活性氧攻击的作用，为揭示弓形虫逃逸宿主先天免疫及药物分子靶点提供理论基础。

弓形虫（Toxoplasma gondii）是重要的人兽共患寄生虫原虫，宿主广泛，几乎能够入侵所有有核细胞。弓形虫能够感染宿主巨噬细胞并在其中增殖，其抵抗巨噬细胞活性氧（Reactive oxygen species）杀菌机制有待深入研究。已有研究表明，弓形虫具有较为完整的氧化还原系统，其中硫氧还原蛋白家族（Thioredoxin family）在虫体抗氧化过程中发挥重要作用，但发挥关键作用蛋白质尚未完全清楚。因此，本项通过绝对定量转录组测序技术（UID-RNAseq）分析弓形虫RH株速殖子体外氧化应激条件下基因转录水平特征，通过GO分析、KEEG分析以及差异表达基因（DEG）分析，发现虫体TGGT1_201800基因在强氧化应激条件下转录水平显著增加。通过生物信息学分析，该蛋白质具有硫氧还原蛋白结构域，预测定位于虫体的线粒体，维持虫体氧化还原稳态；进一步通过原核表达系统表达、纯化了TGGT1_201800重组蛋白质，并免疫小鼠获得多克隆抗体，并初步分析了该蛋白质在弓形虫RH速殖子中表达与定位情况。为了明确该蛋白质在虫体抗氧化应激中的作用，我们构建了该基因的敲除质粒，进一构建敲除虫株和回补虫株，分析基因敲除对虫体表型的影响，以确定其生物学功能。研究结果不但有助于揭示虫体逃避宿主细胞防御机制，还能够为确定新的抗弓形虫感染药物靶标和疫苗候选抗原奠定基础。