嘌呤作为核酸和辅酶的组成成分，在能量供应和代谢调节等方面起着重要的作用，是弓形虫生长繁殖所必需的物质。然而弓形虫缺失从头合成嘌呤的能力，必须依赖于转运和补救机制从宿主窃取嘌呤，来维持自身的生命活动。研究发现弓形虫的基因组中含有4个嘌呤转运蛋白。前期我们利用CRISPR-Cas9技术发现仅NBT1不能被完全敲除，由此可见NBT1为弓形虫主要的嘌呤转运蛋白。基于前期的研究基础，本项目将对NBT1进行过表达和条件性敲除，鉴定其在弓形虫入侵、逸出、胞内增殖及在小鼠中的功能，并明确NBT1缺失株是否为嘌呤营养缺陷株。通过生物素标记技术进一步筛选与NBT1相互作用的蛋白并阐明它们之间的内在联系。通过GC-MS技术寻找NBT1的下游蛋白并系统阐明弓形虫NBT1转运嘌呤的作用机制及转运通路。预期成果将基本阐明NBT1在弓形虫中生长繁殖及转运嘌呤中的作用机制，从而为防控弓形虫病的药物开发奠定理论基础。

Purine nucleosides and nucleobases are essential for T. gondii, because they are fundamental to the constitution of nucleic acids and coenzymes, and they play an important role in the modulation of enzymatic reactions and energy metabolism. However, T. gondii lacks the machinery to synthesize the purine ring de novo and they rely entirely on essential capture, transport and salvage machinery to steal purines from their hosts for incorporation into the parasite nucleotide pools. Purines captured from the host cell and transported into the parasite cytosol are interconverted by nucleoside and nucleobase transporters. T. gondii genome contains four nucleoside and nucleobase transporters designated as AT1, AT2, AT3 and NBT1. Using CRISPR-Cas9, AT1, AT2, AT3 genes were successfully disrupted in Toxoplasma and the three ATs are not essential for T. gondii lytic cycle and also not virulence factors for mice. However, numerous efforts failed to disrupt NBT1 by CRISPR-Cas9 indicating that NBT1 is essential for T. gondii. Based on our previous study, in this project, we will ablate NBT1 in a conditional null mutant and generate a NBT1 overexpression mutant to study the effects of NBT1 on the parasite’s ability to infect cells in vitro and mice in vivo. Using BioID technique, we will screen the interaction proteins with NBT1 and study the specific relationship between them. Using GC-MS technique, the pathways for purine uptake will be also investigated. Whether transgenic parasites lacking the NBT1 are auxotrophic for purine nucleoside and nucleobase are also worth studying. Our expected results will elaborate the basic functions of NBT1 and the mechanism of purine uptake, which will lay the theoretical foundation for the research and development of T. gondii drugs.