Reconstitution of Plasmodium Export in Toxoplasma

描述(由申请人提供)：Apicomplexan寄生虫，如恶性疟原虫和弓形虫，共享共同的专性细胞内生活方式，在这种生活方式下，寄生虫主动穿透宿主细胞，并驻留在宿主细胞质中独特的膜结合液泡中。虽然这些寄生虫被隔离在液泡内，但一个新兴的范例是，顶端复合体也将蛋白质输送到宿主细胞中，以调节宿主以实现最佳的细胞内生存。这一点在原虫的红内期尤为重要，因为红血球宿主在很大程度上代谢不活跃，缺乏许多可以在其他细胞中补充的膜运输系统和细胞器功能。为了弥补相对缺乏的宿主功能劫持，疟原虫有效地输出数百种分泌蛋白穿过液泡膜进入宿主细胞，这些蛋白极大地重塑红细胞，是寄生虫毒力的关键调节因子。蛋白质的输出是通过一种被称为PTEX易位子的特殊的液泡膜运输装置进行的，它是疟原虫所特有的，对寄生虫的生存是必不可少的。在这项研究中，我们利用顶端复合体中的分泌途径和寄生液泡的相似性质来重建弓形虫中的PTEX转位和出口途径。作为概念的初步验证，我们在弓形虫中表达了转位子的两个关键成分EXP2和HSP101，并表明它们分泌到寄生虫的液泡膜上。我们将在这些结果的基础上，表达剩下的三个转位子成分，并评估它们在液泡膜上的定位和形成复合体的能力。我们还将表达一个针对疟原虫宿主的报告蛋白，以确定我们是否可以在弓形虫中建立一个功能性的PTEX介导的出口途径。这项技术的发展将使剖析PTEX转运子的组装、结构和功能的新方法成为可能，并使专门针对这一关键蛋白质输出途径的新疗法的设计成为可能。

DESCRIPTION (provided by applicant): Apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii share a common obligate intracellular lifestyle in which the parasite actively penetrates the host cell and resides in a unique membrane-bound vacuole in the cytoplasm of the host. While these parasites are sequestered inside the vacuole, an emerging paradigm is that apicomplexans also deliver proteins into the host cell to modulate the host for optimal intracellular survival. This is particularly important in the intraerythrocytic stges of the Plasmodia, as the red blood cell host is largely metabolically inactive and devoid of many membrane transport systems and organellar functions that can be co-opted in other cells. To compensate for the relative lack of host functions to hijack, Plasmodium efficiently exports hundreds of secretory proteins across the vacuolar membrane and into the host cell that dramatically remodel of the erythrocyte and are key regulators of parasite virulence. Protein export occurs by a specialized vacuolar membrane transport apparatus known as the PTEX translocon, which is unique to Plasmodium and essential for parasite survival. In this proposal, we exploit the similar properties of the secretory pathway and parasitophorous vacuole in apicomplexans to reconstitute the PTEX translocon and export pathway in Toxoplasma. As an initial proof of concept, we have expressed two key components of the translocon, EXP2 and HSP101, in T. gondii and shown that they are secreted to the parasitophorous vacuole membrane. We will build on these results by expressing the remaining three translocon components and assessing their localization and ability to form a complex at the vacuolar membrane. We will also express a Plasmodium host-targeted reporter protein to determine if we can establish a functional PTEX-mediated export pathway in T. gondii. Development of this technology will enable new approaches to dissect the assembly, architecture and function of the PTEX translocon, and also enable the design of novel therapies that specifically target this critical protein export pathway.