A novel approach to characterize the Toxoplasma gondii secretome in vivo

描述(申请人提供)：弓形虫是一种重要的人类病原体，已感染美国超过5000万人，全球超过10亿人。这种病原体可导致免疫功能受损的人患上严重疾病(例如，由于艾滋病毒/艾滋病)，孕妇的原发感染可导致胎儿死亡。弓形虫是一种专有的细胞内病原体，能够分泌效应蛋白进入宿主细胞，极大地影响疾病的严重程度。到目前为止，标准的蛋白质组学方法还不能对体内弓形虫分泌的蛋白质进行全面鉴定，因为它们只占宿主细胞蛋白质含量的一小部分。利用一种新的方法来全面分析体内分泌的弓形虫蛋白，特别是分泌到宿主细胞中的蛋白，我们的目标是识别在弓形虫致病过程中发挥重要作用的新的宿主相互作用蛋白。为此，我们通过基因工程使弓形虫表达突变的甲硫基tRNA合成酶(MetRS)，该酶能够向内源性Met-tRNA充电，使用点击化学环加成反应可以标记甲硫氨酸类似物，从而能够从宿主细胞的复杂环境中进行后续纯化。初步数据显示，表达该突变MetRs的弓形虫菌株很容易将蛋氨酸类似物结合到新生蛋白中，而野生型菌株不能。这种方法将被用来1)定性分析寄生虫生长周期中分泌体的时间变化，以及2)利用质谱学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分泌体在感染过程中的复杂性以及它们何时分泌的基本问题的答案。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扮演的重要角色，我们希望找到以前未描述的效应蛋白，这些蛋白在寄生虫的入侵、生长、细胞内生存和毒力方面发挥重要作用。这样的效应器可能代表着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，可在免疫功能低下的个体和发育中的胎儿中引起严重疾病，并已在美国感染超过5000万人。该提案中描述的工作旨在通过一种新的方法来全面识别从寄生虫分泌到宿主细胞的蛋白质，以确定可能在导致严重疾病中发挥重要作用的弓形虫基因产物。这些基因产物可能在弓形虫生物学中发挥重要作用，并为抗击这种重要的人类病原体提供新的治疗靶点。

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.