Molecular mechanisms of dense granule trafficking in Toxoplasma gondii

 描述(申请人提供)：弓形虫是一种专有的细胞内寄生虫，是弓形虫病的病原体，如果不治疗，可能会导致免疫功能受损的个人和新生儿患上危及生命的疾病。该病的发病机制依赖于寄生虫在寄主中的一个特殊细胞器内生存和复制的能力，即寄生性液泡(PV)。分泌小泡释放的致密颗粒(DGs)具有多种功能，对细胞内的生存至关重要，包括改变PV的结构和代谢活性，调节宿主细胞周期和改变宿主基因的表达。如何在寄生虫体内转运200 nm的膜包裹的致密颗粒，并将其内容物释放到宿主细胞中，是目前亟待解决的主要问题。在机制层面上了解这一基本过程将有助于确定抗寄生虫药物开发的潜在目标。在中国的研究进展 到目前为止，由于无法在活体寄生虫中实时成像单个颗粒分泌过程中的颗粒，这一领域一直受到阻碍。R21建议的目标是开发先进的成像技术和生物试剂来克服这一障碍，并开始阐明DG运输和分泌的分子机制。我已经以高空间(30纳米)和时间(10fps)分辨率成像了活体寄生虫的致密颗粒运动。我的初步数据显示，TgMyoF是弓形虫DG运输所必需的，它是一种未鉴定的XXVII类肌球蛋白马达。我假设DG是由TgMyoF沿着肌动蛋白轨道连续运输到目的地的。因此，在目标1中，我将使用尖端的单分子生物物理技术来定义TgMyoF在体外和寄生虫中运输货物的功能能力。然后，在目标2中，我将确定基于TgMyoF的转运如何促进DG的分泌。由于DG的运输主要发生在寄生虫的外围，我假设DG被运输，直到颗粒遇到一个特定的可接近的位置，使DG能够穿过内膜复合体(IMC)并到达质膜进行分泌(IMC：位于寄生虫外围的一系列扁平的小泡，可能起到分泌障碍的作用)。为了验证这一假设，将在野生型和TgMyoF缺陷寄生虫系中对DG分泌事件进行成像，以确定基于TgMyoF的运输丢失如何影响DG分泌。总而言之，这些研究将提供有关DG运输和分泌过程的基本新信息。

 DESCRIPTION (provided by applicant): Toxoplama gondii is an obligate intracellular parasite and the causative agent of Toxoplasmosis which, if left untreated, can cause life-threatening disease in immunocompromised individuals and neonates. Pathogenesis of the disease relies on the parasites ability to survive and replicate within a specialized organelle in the host, the parasitophorous vacuole, (PV). Proteins released from secretory vesicles, the dense granules (DGs), have diverse functions which are critical for intracellular survival including modification f PV structure and metabolic activity, regulation of host cell cycle and modification of host gene expression. How the dense granules, small 200nm membrane enclosed vesicles, are transported within the parasite and their contents released into the host cell are major outstanding questions. Understanding this essential process on a mechanistic level would lead to the identification of potential targets of anti-parasitic drug development. Research progress in this area has been hampered to date by the inability to image individual granules on their path to secretion in real-time, in live parasites. The goal of this R21 proposal is to develop the advanced imaging techniques and biological reagents to overcome this hurdle and to begin elucidating the molecular mechanisms underlying DG transport and secretion. I have already imaged dense granule motions in live parasites with high spatial (30nm) and temporal (10fps) resolution. My preliminary data shows that TgMyoF, an uncharacterized class XXVII myosin motor, is necessary for DG transport in T. gondii. I hypothesize that DGs are transported processively by TgMyoF along actin tracks to their destination. Thus, in Aim 1 I will define TgMyoF's functional capacity to transport cargo in vitro and in parasites using cutting edge single molecule biophysical techniques. Then, in Aim 2 I will determine how TgMyoF-based transport facilitates DG secretion. Since DG transport occurs predominately at the parasite periphery I hypothesize that the DGs are transported until the granule encounters a specific site of accessibility that allow DGs to traverse the inner membrane complex (IMC) and reach the plasma membrane for secretion (IMC; a series of flattened vesicles that are located at the parasite periphery, which likely act as a barrier to secretion). To test this hypothesis DG secretion events will be imaged in both wildtype and TgMyoF deficient parasite lines to determine how loss of TgMyoF-based transport affects DG secretion. Collectively, these studies will provide fundamental new information about the process of DG transport and secretion.