A novel approach to characterize the Toxoplasma gondii secretome in vivo

描述（由申请人提供）：刚地弓形虫是一种重要的人类病原体，在美国感染了超过5000万人，在全世界感染了超过10亿人。这种病原体可在免疫功能低下的个体中引起严重疾病（例如，由于艾滋病毒/艾滋病），孕妇的原发性感染可导致胎儿死亡。弓形虫是一种专性细胞内病原体，能够向宿主细胞分泌效应蛋白，显著影响疾病的严重程度。迄今为止，标准的蛋白质组学方法还不能全面鉴定体内寄生虫分泌的弓形虫蛋白，因为它们只占宿主细胞蛋白含量的一小部分。利用一种新的方法来全面分析体内分泌的弓形虫蛋白，特别是那些分泌到宿主细胞中的蛋白，我们的目标是发现在弓形虫发病过程中起重要作用的新一类宿主相互作用蛋白。为此，我们对弓形虫进行了基因工程改造，使其表达一种突变的蛋氨酸基tRNA合成酶（MetRS），该酶能够用蛋氨酸类似物给内源性met -tRNA充能，这种蛋氨酸类似物可以通过点击化学环加成反应进行标记，从而允许从宿主细胞的复杂环境中进行后续纯化。初步数据表明，表达该突变型MetRS的弓形虫菌株很容易将蛋氨酸类似物整合到新生蛋白中，而野生型菌株则不会。该方法将用于1)定性分析寄生虫生长周期中分泌组的时间变化，2)使用质谱法选择性地纯化和鉴定体内分泌的蛋白质。这些研究将提供关于弓形虫在感染期间和何时分泌分泌物的复杂性的基本问题的答案。此外，考虑到分泌蛋白在包括弓形虫在内的所有细胞内病原体中发挥的重要作用，我们希望鉴定出以前未被发现的在寄生虫入侵、生长、细胞内存活和毒力中发挥重要作用的效应蛋白。这些效应物可能代表治疗干预的新潜在靶点。

DESCRIPTION (provided by applicant): Toxoplasma gondii is an important human pathogen that has infected over 50 million people in the U.S., and over a billion people worldwide. This pathogen can cause severe disease in immunocompromised individuals (e.g., due to HIV/AIDS) and primary infections in pregnant women can cause death of the fetus. Toxoplasma is an obligate intracellular pathogen, and is capable of secreting effector proteins into the host cell that dramatically affect the severity of disease. To date, standard proteomics approaches have not allowed for a comprehensive identification of Toxoplasma proteins that are secreted from the parasite in vivo since they represent only a small fraction of host cell protein content. Using a novel approach to comprehensively analyze Toxoplasma proteins secreted in vivo, particularly those proteins that are secreted into the host cell, we aim to identify new classes of host-interacting proteins that play significant roles in Toxoplasma pathogenesis. To do this we have genetically engineered Toxoplasma to express a mutant methionyl tRNA synthetase (MetRS) that is capable of charging endogenous Met-tRNAs with a methionine analog that can be tagged using click chemistry cycloaddition reactions, allowing for subsequent purification from the complex milieu of the host cell. Preliminary data show that Toxoplasma strains expressing this mutant MetRS readily incorporate the methionine analog into nascent proteins, while wild type strains do not. This approach will be used to 1) qualitatively analyze temporal changes in the secretome during the parasite growth cycle and 2) selectively purify and identify proteins secreted in vivo using mass spectrometry. These studies will provide answers to fundamental questions regarding the complexity of the Toxoplasma secretome during infection and when they are secreted. Moreover, given the important role that secreted proteins play in all intracellular pathogens, including Toxoplasma, we hope to identify previously uncharacterized effector proteins that play significant roles in parasite invasion, growth, intracellular survival, and virulence. Such effectors may represent new potential targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Toxoplasma gondii is a human pathogen that causes severe disease in immunocompromised individuals and in the developing fetus, and has infected over 50 million people in the U.S. The work described in this proposal aims to identify Toxoplasma gene products that may play a significant role in causing severe disease using a novel approach to comprehensively identify proteins that are secreted from the parasite into the host cell. These gene products may play important roles in Toxoplasma biology and represent new therapeutic targets for combating this important human pathogen.