advanced fluorescence imaging: fluorescence resonance energy transfer (FRET) and deconvolution modules to study host-parasite ineractions during toxoplasma gondii infection

弓形虫病是由弓形虫引起的一种感染，弓形虫是一种单细胞寄生虫，可以在任何温血脊椎动物的任何细胞内生长，是一种主要的全球人畜共患病原体。这种寄生虫在加拿大不仅是具有巨大社会文化和经济影响的重要病原体（肉类工业食品污染的主要来源，水污染，子宫内儿童污染），而且是一种具有巨大潜力的生物系统，是细胞内寄生的几个方面的模型。我们的目标是了解弓形虫是如何刺激和干扰脊椎动物宿主的免疫反应的。利用细胞生物学、遗传学和免疫学的多学科方法，我们专注于分离相互作用最终影响感染结果的寄生虫和宿主因素。我们使用遗传方法来产生受损的寄生虫，这些寄生虫无法在宿主体内正常活动，然后找到导致缺陷的突变。这使我们能够确定感染所需的重要寄生虫因子。然后对这些分子进行表征：我们研究它们的性质，它们在寄生虫或宿主中的定位，并确定潜在的相互作用分子。“蛋白质-蛋白质相互作用”的研究涉及分子生物学、生物化学和显微镜技术。在活宿主细胞的生理条件下观察这些相互作用需要使用标记蛋白，以及称为“反褶积”和“荧光共振能量转移（FRET）”的特定显微镜技术；这两个模块（本提案所要求的）基本上是增加到现有的成像站。通过使用这些技术表征宿主和寄生虫蛋白的相互作用，我们可以(i)获得关于寄生虫如何操纵宿主以及宿主如何反应的知识；（ii）增加我们的创新机会，不仅在弓形虫和细胞内生物的研究，而且在许多生物学，生物化学和免疫学研究；（三）培养高素质的学生；（四）开发新技术和可申请专利的产品；(v)最终为加拿大和其他国家带来社会文化和经济效益。

Toxoplasmosis is an infection due to Toxoplasma gondii, which is a unicellular parasite that can grow within any cells of any warm-blooded vertebrates and is a major global zoonotic pathogen. This parasite is not only an important pathogen with enormous sociocultural and economical impact in Canada (major source of food contamination in meat industry, water contamination, children contaminated in utero), but also a biological system with great potential and a model for several aspects of intracellular parasitism. Our goal is to understand how Toxoplasma stimulates and interferes with the immune response in its vertebrate hosts. Using a multidisciplinary approach of cell biology, genetics and immunology, we focus on the isolation of parasite and host factors that interact to ultimately influence the outcome of the infection. We use genetic approaches to generate parasites that are impaired and unable to behave normally in their host, and then locate the mutation responsible for the defect. This allows us to identify important parasite factors that are required for an infection. These molecules are then characterized: we study their properties, their localization in the parasite or in the host, and we determine potential interacting molecules. The study of "protein-protein interactions" involves techniques of molecular biology, biochemistry and microscopy. Observing these interactions in physiological conditions in live host cells requires the use of labeled proteins, and specific technologies of microscopy termed "deconvolution" and "Fluorescence Resonance Energy Transfer (FRET)"; these two modules (requested in the present proposal) are basically added to an existing imaging station. By characterizing the interaction of host and parasite proteins using such technologies, we can (i) gain knowledge on how the parasite manipulates the host, and how the host reacts; (ii) increase our chances for innovation not only for research on Toxoplasma and intracellular organisms, but also for numerous biology, biochemistry and immunology studies; (iii) train high quality students; (iv) develop new technologies and patentable products; (v) ultimately bring sociocultural and economical benefits to Canada and other countries.