Reconstitution of Plasmodium Export in Toxoplasma

弓形虫中疟原虫输出的重建

• 批准号: 8463994
• 负责人: Peter John Bradley
• 金额: $ 7.32万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2015-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8463994
• 关键词: ATP phosphohydrolase; Architecture; Aspartic Endopeptidases; Binding; Biological Assay; Cells; Chimeric Proteins; Codon Nucleotides; Complex; Cytoplasm; Data; Development; Disease; Elements; Endoplasmic Reticulum; Engineering; Erythrocytes; Funding Mechanisms; Host Defense; Human; Immunocompromised Host; Invaded; Ion Channel; Licensing; Life Style; Malaria; Mediating; Medical; Membrane; Names; Nutrient; Parasites; Pathway interactions; Peptide Signal Sequences; Plasmodium; Plasmodium falciparum; Plasmodium malariae; Process; Property; Protein Export Pathway; Protein translocation; Proteins; Relative (related person); Reporter; System; Testing; Therapeutic Intervention; Toxin; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transmembrane Transport; Transport Process; Vacuole; Virulence; cell type; design; neonate; new technology; novel; novel strategies; obligate intracellular parasite; pathogen; plasmepsin; protein complex; protein transport; reconstitution; secretory protein; technology development; thioredoxin-like protein

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.

描述（由申请方提供）：顶复门寄生虫（如恶性疟原虫和刚地弓形虫）具有共同的专性细胞内生活方式，其中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空泡中。虽然这些寄生虫被隔离在液泡内，但一个新兴的范例是顶复门动物也将蛋白质递送到宿主细胞中以调节宿主以获得最佳的细胞内存活。这在疟原虫的红细胞内阶段中特别重要，因为红细胞宿主在很大程度上代谢不活跃，并且缺乏许多可以在其他细胞中增选的膜转运系统和细胞器功能。为了弥补宿主功能的相对缺乏，疟原虫有效地将数百种分泌蛋白穿过液泡膜并进入宿主细胞，这些蛋白显著地重塑红细胞，并且是寄生虫毒力的关键调节因子。蛋白质输出通过称为PTEX易位子的专门的液泡膜运输装置发生，PTEX易位子是疟原虫所特有的并且对于寄生虫存活是必需的。 在这个提议中，我们利用分泌途径和寄生虫空泡在顶复门的类似属性，重建弓形虫的PTEX易位和输出途径。作为初步的概念证明，我们表达了T. gondii，并显示它们分泌到寄生虫空泡膜。我们将建立在这些结果的基础上，通过表达其余三个translocon组件和评估其定位和能力，形成一个复合物在液泡膜。我们还将表达疟原虫宿主靶向报告蛋白，以确定我们是否可以在T。刚地。这项技术的发展将使新的方法来剖析PTEX translocon的组装，结构和功能，也使新的疗法，专门针对这一关键的蛋白质输出途径的设计。