顶膜抗原1在柔嫩艾美耳球虫入侵宿主细胞中作用与机制研究

Most Apicomplexa, including Toxoplasma, Plasmodium, and Eimeria, are obligate intracellar parasites. Many invade host cells by a conserved mechanism involving the formation of a zone of tight attachment between the parasite apex and the host cell, called Moving junction (MJ). MJ is primarily a molecular bridge between the parasite sub-membrane actin-myosin motor and a stable and stationary anchor associated with the host cell surface/cortex..The transmembrane protein apical membrane antigen 1 (AMA1) is thought to shape the MJ on the parasite side. The cytoplasmic tail of AMA1 was reported to bind aldolase in vitro, considered a signature of proteins that bind parasite actin and the motor. The ectodomain of AMA1 tightly binds in parasite extracts to the rhoptry neck 2 (RON2) protein, a protein secreted from the parasite rhoptries that specifically localizes at the MJ, where it inserts in the host cell membrane and is presumably linked to the cell cortical cytoskeleton via other RON proteins, like RON4. Moreover, antibodies or peptides that inhibit the AMA1-RON2 interaction drastically reduce host cell invasion by Plasmodium merozoites and Toxoplasma tachyzoites indicating that AMA1 is essential for apicomplexan invasion..Eimeria parasites have complex developmental life cycles with an exogenous phase in the environment and an endogenous phase in the intestine. Sporozoites and merozoites invasion are the most important and obligatory steps of the Eimeria life cycle. Our previous studies showed that Eimeria tenella Apical Membrane Antigen-1 (EtAMA1) was expressed at the highest levels in sporozoites among the 4 development stages. Its antibodies had a potent inhibitory effect on parasite invasion, decreasing it by approximately 70% and the localization of EtAMA1 was mainly on the apical end of intracellular sporozoites. These results indicate EtAMA1 might play an important role in sporozoite invasion and development. But its mechanism has not been studied..The purpose of this project is to investigate the potential role and molecular mechanism of EtAMA1 in Eimeria tenella host cell invasion with the following works: 1) Effects of different EtAMA1domains on the invasion ability and process (including of gliding motility, adhesion, moving junction formation and parasitophorous vacuole formation) of Eimeria tenella sporozoites and merozoites were studied to identify the EtAMA1 functional domain and the inhibiting invasive stage; 2) EtAMA1 interacting proteins from sporozoite, merozoite and host cell were screened and verified to identify the EtAMA1-associated molecules. .Theses studies not only could help to understand the role and molecular mechanism of EtAMA1 in Eimeria tenella host cell invasion, but also provided important rationale to understand how to invasion processes and find new targets for studying new vaccines and drugs against coccidiosis.

子孢子和裂殖子入侵宿主细胞是球虫建立感染的前提，但机制不清。已证实顶膜抗原1（AMA1）是顶复器门原虫入侵关键结构——运动连接环（moving junction，MJ）重要组成成分，是疟原虫和弓形虫入侵宿主细胞的关键蛋白。迄今，AMA1在球虫中功能尚不清楚。前期研究发现AMA1在柔嫩艾美耳球虫子孢子和裂殖子中转录和蛋白水平以及蛋白分布均明显不同，其抗体能明显抑制子孢子入侵。本项目拟在此基础上，利用体外抑制、免疫电镜、免疫荧光、CoIP、BiFC等技术，研究AMA1不同结构域对球虫子孢子和裂殖子入侵细胞能力和过程的影响，筛选与AMA1相互作用的子孢子、裂殖子和宿主细胞蛋白，并对作用蛋白在球虫入侵中功能进行研究，以确定AMA1发挥功能的结构域、影响的入侵阶段以及作用的分子基础，弄清楚AMA1在子孢子和裂殖子入侵中扮演角色的异同。结果对阐明球虫入侵的分子机制及筛选防治球虫病关键分子具有重要意义。

子孢子和裂殖子入侵宿主细胞是球虫建立感染的前提，但机制不清。为了阐明AMA1在球虫子孢子和裂殖子入侵宿主细胞中扮演角色的异同，本项目研究了顶膜抗原1不同结构域对柔嫩艾美耳球虫入侵宿主细胞能力的影响，发现对子孢子入侵能力起主要抑制作用是EtAMA1结构域I（DI），但对裂殖子入侵能力无显著性影响，表明EtAMA1是子孢子入侵关键蛋白，其发挥主要功能的是结构域I。研究了顶膜抗原1对柔嫩艾美耳球虫入侵宿主细胞过程的影响，发现EtAMA1抗体能明显降低子孢子的滑行和粘附能力，但对胞内增殖无显著性影响，表明EtAMA1抗体是通过降低子孢子的滑行和粘附能力而抑制了子孢子的入侵。对3个柔嫩艾美耳球虫EtAMA1潜在互作蛋白——Et0440、EtMIC2、EtEsp进行克隆、表达，制备相应的抗体，体外抑制试验结果表明，该三个蛋白均参与了子孢子入侵宿主细胞。利用免疫共沉淀技术、双分子荧光互补技术（BiFC）和GST pull-down 技术验证了4个潜在互作蛋白与EtAMA1之间的互作关系，发现EtEsp和EtRON2能与EtAMA1发生互作，而Et0440和EtMIC2不能，EtAMA1- EtRON2互作表明EtAMA1具有与其他顶复门原虫相同的特性，EtAMA1-EtEsp互作表明球虫EtAMA1具有自身的特性。对EtAMA1及其互作蛋白EtEsp、EtRON2在球虫入侵宿主细胞中功能进行了研究，发现EtAMA1及其互作蛋白抗体单独或联合使用，均可明显抑制子孢子的入侵能力，表明EtAMA1与EtEsp、EtRON2互作在子孢子入侵中发挥重要的功能。对EtAMA1同源分子在球虫入侵宿主细胞中功能进行了研究，发现球虫AMAs均是微线体分泌蛋白，EtAMA1和EtAMA3是子孢子阶段调控蛋白，是子孢子入侵的关键蛋白；EtAMA2是裂殖子阶段调控蛋白，是裂殖子入侵的关键蛋白，表明在子孢子和裂殖子入侵细胞中扮演重要角色的AMAs明显不同。以上研究结果为弄清AMAs在球虫入侵宿主细胞的机制和寻找防治鸡球虫病重要“关键”分子奠定基础。.

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