贾第虫病毒影响贾第虫致病性的分子识别机制

Giardia duodenalis is an important zoonotic parasite that can cause human and animal diarrhea. The Giardiavirus was existed in some trophozoites of Giardia. There is still no report about what are the effects of Giardiavirus on the growth and pathogenicity of Giardia. TLRs plays an important role in host pattern recognition and innate resistance to parasite infection. In this study, Giardia trophozoites that without virus will be infected by Giardiavirus to construct the virus infection trophozoites strain in vitro, then the characteristics on the growth of Giardia and the pathogenicity to the host will be observed. TLRs and its pathway of host will be detected using Real-time PCR and immunoblot methods after Giardia that with/without virus infecting mice or immune cells. When TLRs and its pathway which related with Giardiavirus are clear, the pathogenicity to the host will be determined through blocking the target TLRs, to study the effect of pathogenicity resulted by the TLRs that related to Giardiavirus, and also to illuminate the mechanism of Giardia pathogenicity that affected by Giardiavirus. This study will provide the important theoretical basis and potential new drug or vaccine targets against Giardiasis.

贾第虫是一种重要的人兽共患原虫，可引起人和动物的腹泻。一些贾第虫体内存在贾第虫病毒，贾第虫病毒的存在是否影响虫体的生长发育和致病性尚无报导。在宿主模式识别和天然免疫体系中，TLRs 及其信号通路在机体抵抗寄生虫感染中起重要作用。本研究拟通过建立人工感染的贾第虫病毒株，观察贾第虫病毒对贾第虫生长发育特征及致病性的影响；利用荧光定量PCR和免疫印迹等技术，鉴定参与识别不同虫株的宿主TLRs分子及其信号通路；通过阻断贾第虫病毒相关的目标TLRs，探讨贾第虫病毒相关的TLRs在贾第虫致病过程中的作用，阐明贾第虫病毒影响贾第虫致病性的分子识别机制，为贾第虫病防治提供理论依据和潜在的的药物疫苗靶位。

贾第虫是一种重要的人兽共患原虫，可引起人和动物的腹泻。部分贾第虫虫株中存在贾第虫病毒，其是否会引起贾第虫致病性的改变？宿主如何识别不同虫株贾第虫？本研究以贾第虫病毒为切入点，分离贾第虫病毒感染无病毒株贾第虫建立人工感染贾第虫病毒VIG虫株，通过转录组测序分析及相关表型试验，发现贾第虫病毒能够通过调控贾第虫糖代谢信号通路影响贾第虫的增殖和成囊；以无病毒株、携病毒株及构建的VIG虫株分别感染野生型小鼠腹腔巨噬细胞，利用荧光定量PCR和western-blot等技术，鉴定出TLR2、TLR4、TLR9可识别无病毒株贾第虫，TLR2、TLR3、TLR4、TLR9可识别携病毒株贾第虫；利用TLRs缺失小鼠及相关通路抑制剂，通过体外筛选、体内验证的方式，在动物、细胞及分子水平分别进行病理指标检测、病原学检测、信号通路检测、细胞因子检测等，最终确定TLR3在携病毒株贾第虫致病过程中通过调控MAPK、NF-κB等信号通路介导IL-12、IL-6、TNF-α等细胞因子的产生，发挥抗感染作用；TLR9在无病毒和携病毒株贾第虫致病过程中通过协同调控AKT、MAPK和NF-κB等信号通路调控IL-12、IL-6、TNF-α等细胞因子的产生，主要发挥抗贾第虫感染的作用；而TLR2在贾第虫致病过程中通过调控AKT信号通路负调控IL-12、IL-6、TNF-α等细胞因子的产生，加剧贾第虫病的致病过程。综上，本项目的实施详细阐明了宿主TLRs受体在贾第虫感染中的天然免疫应答机制：贾第虫病毒可被宿主TLR3识别引起更强的宿主抗感染免疫反应，进而有利于宿主清除贾第虫感染；此外，也证明了TLR9抗贾第虫感染的作用。尤其值得注意的是，宿主TLR2在贾第虫感染中发挥免疫负调节作用，这是一种新的贾第虫免疫逃避机制。本项目的完成为贾第虫病防治提供理论依据和潜在的的药物疫苗靶位。