利用iNOS基因敲除大鼠研究宿主NO对弓形虫增殖与分化的调控

Toxoplasma gondii is a key and global distributed zoonotic parasitic protozoan, which causes a great threat to human health and significant economic loss in livestock husbandry. T. gondii infection in humans or in domestic animals usually causes chronic signs with tissue cysts. The cyst formation is a key step for the parasite in its life cycle and pathogenesis. However, the mechanism for the cyst-formation remains far to well understand. Based on our previous studies, the natural resistance of rats to T. gondii infection is strongly linked to the high expression level of inducible nitric oxide synthase (iNOS) and large amount of nitric oxide (NO). We are asking if NO deficiency in rat will cause the loss of natural resistance to this parasite infection. To address this question, we generated iNOS knock-out rat for the first time in the world and carried out related experiments. Very surprisingly, pioneer experiments showed that neither parasitemia nor brain cysts were found in the iNOS-/- rats infected with T. gondii. Two key scientific points are raised. 1) Rats launched a back-up and more effective defensing machinery against T. gondii after deficiency of NO; 2) Differentiation from tachyzoites to cyst (bradyzoites) of T. gondii requires sufficient NO. Therefore, we hypothese that NO molecule plays a key role in interaction between T. gondii and host, and may also be a key molecule for T. gondii to sense host’s immune status and to regulate its differentiation. To test this hypothese, we will carry out on the analysis on transcriptomic, proteomic approaches and genetic manipulating techniques to identify the potential elements of NO-independent T. gondii-killing machinery, and to identify the potential sensors and signaling networks for T. gondii to monitor the host’s immune status and checkpoints to the differentiation. The findings from this project will explain why T. gondii cysts does not exist in iNOS knock-out rats and would provide essential molecular evidences to better understand the co-evolution of T. gondii and its hosts.

弓形虫是重要的人兽共患寄生原虫，对人类健康和畜牧业发展造成严重威胁。弓形虫主要以慢性感染的包囊形式存在。但包囊形成机理至今没有明确解释。我们前期的研究发现，大鼠对弓形虫的敏感性与一氧化碳（NO）浓度呈负相关。那么减少NO是否导致大鼠对弓形虫易感？为此，我们构建了世界上首个iNOS基因敲除大鼠，但实验结果令人震惊，弓形虫在敲除大鼠中既不出现虫血症也不形成包囊。这可能涉及两个重要的科学问题：1，NO缺失后大鼠启动了更有效的新抑虫机制；2、弓形虫分化成囊需要足够NO参与。因此我们提出新的假说：NO是弓形虫与宿主相互作用的关键分子，也是其识别宿主免疫状态以调控自身分化的关键分子。为验证此假说，本项目拟利用转录组、蛋白组和基因操作等技术，鉴定iNOS缺失后大鼠的抑虫因子，阐明弓形虫监测宿主NO及调控通路，以揭示弓形虫在iNOS敲除大鼠中不成囊的原因，为解释弓形虫与宿主协同进化提供重要分子证据。

弓形虫是重要的人兽共患寄生原虫，对人类健康和畜牧业发展造成严重威胁。感染弓形虫后的发病情况与宿主免疫状况，尤其是上调的一氧化氮（NO）浓度高度相关。我们构建了世界上首个iNOS基因敲除大鼠，但实验结果令人震惊，弓形虫在敲除大鼠中既不出现虫血症也不形成包囊。这可能涉及两个重要的科学问题： NO缺失后大鼠启动了更有效的新抑虫机制；弓形虫分化成囊需要足够NO参与。我们提出新的假说：NO是弓形虫与宿主相互作用的关键分子，也是其识别宿主免疫状态以调控自身分化的关键分子。本研究主要鉴定iNOS及产物NO对宿主抗弓形虫的免疫作用，以及iNOS缺失后大鼠的抑虫因子和弓形虫的应对机制。研究发现，1、宿主免疫状况变化时，iNOS表达受影响，若降低NO的浓度，可加重宿主弓形虫病的发病症状。2、但在iNOS敲除的大鼠模型中，尽管缺少了NO杀灭弓形虫，巨噬细胞却改为显著上调活性氧（ROS），进而启动了焦亡通路。这一通路是依赖于弓形虫的GRA43等致密颗粒蛋白的分泌。3、弓形虫TgIST蛋白可拮抗宿主IDO1为主的免疫应答。4、弓形虫的棒状体蛋白可磷酸化宿主RTN1-C，进而通过GPR78/HDAC通路引起宿主细胞凋亡。上述四条与免疫系统的互作通路充分揭示了宿主具有完善且复杂的免疫抑虫网络（iNOS、ROS、IDO1），而弓形虫也具有GRA43、TgIST等重要互作蛋白。另一方面，我们也开展了宿主NO对弓形虫分化成囊作用的研究。成果发现，NO对弓形虫分化成囊起着重要作用，鉴定出关键的调控因子NOF1，并证实其为NO刺激相关的分化抑制因子。总的来说，本课题提供了可靠的分子证据，以iNOS为代表的宿主免疫蛋白与NOF1为代表的弓形虫蛋白互作。研究结果以弓形虫感染的动物及细胞模型，更好地了解弓形虫的致病机理与分化成囊机制。