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（由申请人提供）：顶复合体寄生虫，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，寄生虫主动渗透宿主细胞，居住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，但一种新兴的范式是顶复合体也将蛋白质传递到宿主细胞中，以调节宿主细胞内的最佳生存。这在疟原虫的红细胞阶段尤其重要，因为红细胞宿主在很大程度上是代谢不活跃的，并且缺乏许多可以被其他细胞吸收的膜运输系统和细胞器功能。为了弥补宿主功能的相对缺乏，疟原虫有效地输出数百种分泌蛋白，穿过空泡膜进入宿主细胞，这些蛋白显著地重塑了红细胞，是寄生虫毒力的关键调节因子。蛋白质的输出是通过一种特殊的液泡膜运输装置进行的，这种装置被称为PTEX转位体，这是疟原虫所特有的，对寄生虫的生存至关重要。在本研究中，我们利用顶复合体分泌途径和寄生液泡的相似特性，重建了弓形虫PTEX的转运和输出途径。作为概念的初步证明，我们在弓形虫中表达了易位的两个关键成分EXP2和HSP101，并表明它们分泌到寄生物液泡膜上。我们将在这些结果的基础上表达剩余的三个易位成分，并评估它们的定位和在液泡膜上形成复合物的能力。我们还将表达一个疟原虫宿主靶向报告蛋白，以确定我们是否可以在弓形虫中建立一个功能性的ptex介导的输出途径。这项技术的发展将为剖析PTEX易位子的组装、结构和功能提供新的方法，也使设计专门针对这一关键蛋白质输出途径的新疗法成为可能。