Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.
PLoS One. 2013 Aug 5;8(8):e70637. doi: 10.1371/journal.pone.0070637. Print 2013.
Toxoplasma gondii is an obligate intracellular parasite of the phylum Apicomplexa. The interaction of two well-studied proteins, Apical Membrane Antigen 1 (AMA1) and Rhoptry Neck protein 2 (RON2), has been shown to be critical for invasion by the asexual tachyzoite stage. Recently, two paralogues of these proteins, dubbed sporoAMA1 and sporoRON2 (or RON2L2), respectively, have been identified but not further characterized in proteomic and transcriptomic analyses of Toxoplasma sporozoites. Here, we show that sporoAMA1 and sporoRON2 localize to the apical region of sporozoites and that, in vitro, they interact specifically and exclusively, with no detectable interaction of sporoAMA1 with generic RON2 or sporoRON2 with generic AMA1. Structural studies of the interacting domains of sporoRON2 and sporoAMA1 indicate a novel pairing that is similar in overall form but distinct in detail from the previously described interaction of the generic pairing. Most notably, binding of sporoRON2 domain 3 to domains I/II of sporoAMA1 results in major alterations in the latter protein at the site of binding and allosterically in the membrane-proximal domain III of sporoAMA1 suggesting a possible role in signaling. Lastly, pretreatment of sporozoites with domain 3 of sporoRON2 substantially impedes their invasion into host cells while having no effect on tachyzoites, and vice versa for domain 3 of generic RON2 (which inhibits tachyzoite but not sporozoite invasion). These data indicate that sporozoites and tachyzoites each use a distinct pair of paralogous AMA1 and RON2 proteins for invasion into host cells, possibly due to the very different environment in which they each must function.
刚地弓形虫是顶复门的一种专性细胞内寄生虫。两个研究较为深入的蛋白，顶膜抗原 1（AMA1）和裂殖体颈蛋白 2（RON2）的相互作用，被证明对无性速殖子阶段的入侵至关重要。最近，这两种蛋白的两个同源物，分别被称为spor AMA1 和 spor RON2（或 RON2L2），已在刚地弓形虫孢子虫的蛋白质组学和转录组学分析中被鉴定出来，但尚未进一步表征。在这里，我们表明 spor AMA1 和 spor RON2 定位于孢子虫的顶端区域，并且在体外，它们特异性地相互作用，spor AMA1 与通用的 RON2 或 spor RON2 与通用的 AMA1 没有可检测到的相互作用。对 spor RON2 和 spor AMA1 的相互作用域的结构研究表明，一种新的配对方式在整体形式上相似，但在细节上与先前描述的通用配对的相互作用不同。最值得注意的是，spor RON2 结构域 3 与 spor AMA1 的结构域 I/II 的结合导致后者蛋白在结合部位发生重大改变，并在 spor AMA1 的膜近端结构域 III 上产生变构效应，提示其可能在信号转导中发挥作用。最后，用 spor RON2 的结构域 3 预处理孢子虫，可显著阻止其侵入宿主细胞，而对速殖子没有影响；而通用的 RON2 的结构域 3 则相反（其抑制速殖子但不抑制孢子虫的入侵）。这些数据表明，孢子虫和速殖子分别使用一对独特的同源 AMA1 和 RON2 蛋白来侵入宿主细胞，这可能是由于它们各自所处的环境非常不同。
