Molecular mechanisms of dense granule trafficking in Toxoplasma gondii

 描述（由申请方提供）：弓形虫是一种专性细胞内寄生虫，是弓形虫病的病原体，如果不进行治疗，可导致免疫功能低下的个体和新生儿发生危及生命的疾病。该病的发病机制依赖于寄生虫在宿主体内的一个专门的细胞器--寄生虫空泡（PV）中生存和复制的能力。从分泌囊泡（dense granule，DG）中释放的蛋白质具有多种功能，包括调节PV结构和代谢活性、调节宿主细胞周期和调节宿主基因表达等，对细胞内的生存至关重要。这些致密的颗粒，即200 nm的小膜囊泡，如何在寄生虫体内运输并将其内容物释放到宿主细胞中是主要的悬而未决的问题。了解这一基本过程的机制水平将导致抗寄生虫药物开发的潜在目标的识别。的研究进展 迄今为止，由于不能对活寄生虫中的单个颗粒在其分泌路径上进行实时成像，该领域受到阻碍。R21的目标是开发先进的成像技术和生物试剂，以克服这一障碍，并开始阐明DG运输和分泌的分子机制。我已经用高空间（30 nm）和时间（10 fps）分辨率成像了活体寄生虫中的密集颗粒运动。我的初步数据表明，TgMyoF，一个未表征的XXVII类肌球蛋白马达，是必要的DG运输T。刚地。我推测DG是由TgMyoF沿着沿着肌动蛋白的轨道运输到目的地的。因此，在目标1中，我将使用最先进的单分子生物物理技术来定义TgMyoF在体外和寄生虫中转运货物的功能能力。然后，在目标2中，我将确定如何TgMyoF为基础的运输促进DG分泌。由于DG运输主要发生在寄生虫外周，我假设DG被运输，直到颗粒遇到一个特定的可及性位点，允许DG穿过内膜复合物（IMC）并到达质膜分泌（IMC;一系列位于寄生虫外周的扁平囊泡，可能充当分泌屏障）。为了检验这一假设，将在野生型和TgMyoF缺陷型寄生虫系中对DG分泌事件进行成像，以确定基于TgMyoF的转运的损失如何影响DG分泌。总的来说，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.