A novel approach to characterize the Toxoplasma gondii secretome in vivo

描述（由申请人提供）：弓形虫是一种重要的人类病原体，在美国已感染超过5000万人，全世界超过十亿人。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）和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，能够分泌效应蛋白进入宿主细胞，显著影响疾病的严重程度。迄今为止，标准蛋白质组学方法还不允许全面鉴定体内从寄生虫分泌的弓形虫蛋白，因为它们仅代表宿主细胞蛋白质含量的一小部分。使用一种新的方法来全面分析弓形虫体内分泌的蛋白质，特别是那些分泌到宿主细胞中的蛋白质，我们的目标是确定新的类的主机相互作用的蛋白质，在弓形虫的发病机制中发挥重要作用。为此，我们对弓形虫进行了基因工程改造，使其表达突变型甲硫氨酰tRNA合成酶（MetRS），该合成酶能够用甲硫氨酸类似物装载内源性Met-tRNA，该甲硫氨酸类似物可以使用点击化学环加成反应进行标记，从而允许随后从宿主细胞的复杂环境中纯化。初步数据显示，表达该突变MetRS的弓形虫菌株容易将甲硫氨酸类似物掺入新生蛋白质中，而野生型菌株则不然。该方法将用于1）定性分析寄生虫生长周期期间分泌组的时间变化，2）使用质谱法选择性纯化和鉴定体内分泌的蛋白质。这些研究将为弓形虫分泌组在感染过程中的复杂性以及何时分泌提供基本问题的答案。此外，由于分泌蛋白在所有细胞内病原体（包括弓形虫）中发挥的重要作用，我们希望确定以前未表征的效应蛋白，这些效应蛋白在寄生虫入侵、生长、细胞内存活和毒力中发挥重要作用。这种效应器可能代表治疗干预的新的潜在靶点。 公共卫生相关性：弓形虫是一种人类病原体，在免疫功能低下的个体和发育中的胎儿中引起严重的疾病，并且在美国已经感染了超过5000万人。本提案中描述的工作旨在鉴定弓形虫基因产物，这些基因产物可能在使用一种新方法引起严重疾病中发挥重要作用，以全面鉴定从寄生虫分泌到宿主细胞中的蛋白质。这些基因产物可能在弓形虫生物学中发挥重要作用，并代表了对抗这种重要的人类病原体的新的治疗靶点。

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.