弓形虫强毒株PPM3H蛋白的生物学功能研究

PPM3H of Toxoplasma gondii is a member of the protein phosphatase 2C (PP2C) family that plays important roles in regulation of immune response. Our previous study revealed that the virulence of PPM3H-KO strain is attenuated significantly, the amino acid sequences of T. gondii PPM3H possess the features of secretory proteins, variations in amino acid sequences are found between virulent and avirulent strains. Thus, we speculate that the PPM3H gene may be one of the reasons that result in variation in virulence among different T. gondii strains. PPM3H of virulent T. gondii strain may regulate phosphorylation status of specific target proteins and gene expression through its protein phosphatase activity, and then influence the biological features or virulence of T. gondii. To test this hypothesis, in this project, we will study whether PPM3H of virulent T. gondii contributes to virulence variation between virulent strain and avirulent strain by using gene replacement technology which is based on the CRISPR/Cas9 technology, will examine expression and subcellular location of PPM3H by using western blot and laser confocal technology, respectively, and will study the influences of PPM3H on protein dephosphorylation and gene regulation by using phosphorylation “omics” technology and high-throughput transcriptomic sequencing, respectively. The roles of PPM3H in host cell invasion, intracellular proliferation, phage formation and egress of T. gondii will also be studied. It is expected that the findings of this proposed project will elaborate the basic biological functions of PPM3H, and lay a theoretical foundation for elucidating the virulence mechanism of T. gondii.

弓形虫PPM3H蛋白属于蛋白磷酸酶2C家族成员，该家族成员在免疫反应调控中具有重要的作用。我们在前期研究中发现，弓形虫强毒株PPM3H基因被敲除后其毒力明显减弱，弓形虫PPM3H蛋白具有分泌蛋白的特征，且强毒株和弱毒株PPM3H蛋白磷酸酶结构域中的氨基酸序列存在较大差异。由此我们推测，PPM3H可能是造成不同弓形虫虫株毒力出现差异的原因之一，弓形虫强毒株的PPM3H可能会通过其特有的蛋白磷酸酶活性对某些蛋白的磷酸化状态、基因表达进行调节，进而影响弓形虫毒力。为验证此假说，本项目通过CRISPR/Cas9基因替换技术、激光共聚焦、western blot、蛋白磷酸化组、转录组、弓形虫入侵、胞内增殖、空斑形成等实验，对弓形虫强毒株PPM3H调控的蛋白去磷酸化修饰、基因表达及生物学特性等进行研究，预期研究结果将基本阐明弓形虫强毒株PPM3H的生物学功能，为阐明弓形虫的毒力形成机制奠定理论基础。

弓形虫是一种重要的人兽共患病病原，全世界曾有三分之一的人感染过弓形虫。蛋白磷酸酶是一种重要的蛋白翻译后修饰酶，其中蛋白磷酸酶2C家族成员在免疫反应调控中具有重要的作用，但弓形虫的蛋白磷酸酶2C蛋白——PPM3H的生物学功能尚未被研究和报道。本项目以弓形虫的PPM3H基因为研究对象，通过转基因、激光共聚焦、转录组学和磷酸化组学等技术对弓形虫PPM3H的亚细胞定位及生物学功能进行了解析。通过本项目，我们首次证实了弓形虫的PPM3H定位于弓形虫的棒状体，PPM3H基因敲除后被感染宿主的免疫、炎症相关基因的表达显著下调；转录相关的蛋白也发生了明显的去磷酸化修饰。强毒株RH株的PPM3H转入弱毒株PRU虫株后，转基因虫株在感染宿主中引起的免疫和炎症反应显著上调。我们不仅证实了弓形虫的PPM3H基因参与了小鼠免疫和炎症反应的调控，也证实了PPM3H基因并不影响弓形虫本身的生物学特性（例如：细胞入侵、逸出、胞内增殖、空斑形成等生物学特性）。通过加权基因共表达网络分析和基因差异表达分析，本研究共鉴定到21个受弓形虫PPM3H调控的小鼠关键核心基因，这些基因可能在弓形虫的小鼠致死、致病方面起着重要的作用。预期研究成果将为进一步揭示弓形虫的生物学特性、防治家畜的弓形虫病奠定坚实的基础。