The C-Terminus of Toxoplasma RON2 Provides the Crucial Link between AMA1 and the Host-Associated Invasion Complex

Host cell invasion by apicomplexan parasites requires formation of the moving junction (MJ), a ring-like apposition between the parasite and host plasma membranes that the parasite migrates through during entry. The Toxoplasma MJ is a secreted complex including TgAMA1, a transmembrane protein on the parasite surface, and a complex of rhoptry neck proteins (TgRON2/4/5/8) described as host cell-associated. How these proteins connect the parasite and host cell has not previously been described. Here we show that TgRON2 localizes to the MJ and that two short segments flanking a hydrophobic stretch near its C-terminus (D3 and D4) independently associate with the ectodomain of TgAMA1. Pre-incubation of parasites with D3 (fused to glutathione S-transferase) dramatically reduces invasion but does not prevent injection of rhoptry bulb proteins. Hence, the entire C-terminal region of TgRON2 forms the crucial bridge between TgAMA1 and the rest of the MJ complex but this association is not required for rhoptry protein injection.顶复门寄生虫侵染宿主细胞的过程，依赖于移动连接体（moving junction, MJ）的形成：该结构为寄生虫与宿主细胞膜之间形成的环状贴合界面，寄生虫在侵入宿主时需通过该结构完成迁移。弓形虫（Toxoplasma）的移动连接体属于分泌型复合物，包含寄生虫表面的跨膜蛋白TgAMA1，以及被报道定位于宿主细胞侧的棒状体颈部蛋白复合物（TgRON2/4/5/8）。此前学界尚未明确这些蛋白如何介导寄生虫与宿主细胞的连接。本研究证实，TgRON2定位于移动连接体，且其C端疏水序列两侧的两段短片段（D3与D4）可分别独立结合TgAMA1的胞外域。将寄生虫与融合了谷胱甘肽S-转移酶（glutathione S-transferase, GST）的D3片段预孵育后，寄生虫的侵染能力显著下降，但并未阻断棒状体球部蛋白的注入过程。综上，TgRON2的完整C端区域构成了TgAMA1与移动连接体其余复合物之间的关键桥梁，但该结合并非棒状体蛋白注入所必需的环节。