Regulation of mitochondrial morphodynamics in Toxoplasma gondii

弓形虫门的寄生虫，如弓形虫的一个独特特征是存在一种 单管线粒体，这是寄生虫生存所必需的，也是有效的药物靶点。大多数研究 对顶端复合体线粒体的研究主要集中在其生化和生理学上。相比之下，人们知之甚少。 关于控制线粒体分裂和调节其结构的机制，信息将 对于彻底探索线粒体作为药物靶点至关重要。弓形虫单纯性 线粒体非常动态，在寄生虫的整个生命周期中都会经历形态变化 包括在从细胞内环境到细胞外环境的转变期间。而在宿主细胞内， 线粒体保持套索形状，在寄生虫周围有区域延伸。 与寄生虫膜结合的可能性，表明存在膜接触部位。退场后立即退场 从宿主细胞中，这些接触点消失，线粒体崩溃，这表明动态 膜接触部位调节线粒体的位置。既没有功能意义，也没有 弓形虫线粒体与细胞膜接触所需的蛋白质是已知的。我们有 发现了一种新的蛋白质Fip1，它与线粒体相关，当敲除正常的 线粒体的形态受到严重影响。在细胞内的fip1基因敲除寄生虫中，线粒体 不是像野生型寄生虫那样处于套索形状，而是折叠起来的。此外，适当的 基因敲除寄生虫的线粒体分离被破坏，导致没有线粒体的寄生虫。 以及寄生虫外的线粒体物质。这些粗大的形态变化与 显著减少寄生虫的繁殖，并可以通过重新引入野生型Fip1来拯救。 因此，我们假设Fip1在线粒体和寄生虫膜之间的联系中起中介作用。 一种可调节的方式，依赖于Fip1的线粒体形态和动力学对 寄生虫的繁殖。通过分子遗传学、显微镜和蛋白质组学的结合，我们将 阐述线粒体形态的功能相关性和机制。在目标1中，我们将 对Fip1突变株进行彻底的体内和体外表型鉴定，以确定其作用 Fip1和线粒体形态对寄生虫存活率的影响。目标二侧重于识别和表征 Fip1复合体的组成部分，它介导线粒体与细胞外周的联系 寄生虫。最后，在目标三中，我们将确定驱动线粒体的调节机制。 当寄生虫离开宿主细胞时，形态会发生变化。同时，这些实验将揭示 驱动和调节弓形虫线粒体形态动力学的分子机制。AS 这种重要的人类病原体的线粒体对于它的生存和有效的药物靶点是必不可少的，我们的 研究将为新疗法的开发找到新的目标。

A unique feature of parasites of the phylum Apicomplexa, such as Toxoplasma gondii, is the presence of a single tubular mitochondrion, which is essential for parasite survival and a validated drug target. Most studies of the apicomplexan mitochondrion have focused on its biochemistry and physiology. By contrast little is known about the machinery that controls mitochondrial division and that regulate its structure, information that would be critical for a thorough exploration of the mitochondrion as a drug target. Toxoplasma's singular mitochondrion is very dynamic and undergoes morphological changes throughout the parasite's life cycle including during the transition from the intracellular to the extracellular environment. While inside a host cell the mitochondrion is maintained in a lasso shape that stretches around the parasite periphery where it has regions of coupling with the parasite pellicle, suggesting the presence of membrane contact sites. Promptly after exit from the host cell, these contact sites disappear, and the mitochondrion collapses indicating that dynamic membrane contact sites regulate the positioning of the mitochondrion. Neither the functional significance nor the proteins needed for the contact between Toxoplasma's mitochondrion and pellicle are known. We have discovered a novel protein, Fip1, that associates with the mitochondrion and that when knocked out the normal morphology of the mitochondrion is severely affected. In intracellular fip1 knockout parasites the mitochondrion is not in a lasso shape as seen in wildtype parasites, but instead it is collapsed. Additionally, proper mitochondrial segregation is disrupted in the knockout parasites, resulting in parasites with no mitochondrion and mitochondrial material outside of the parasites. These gross morphological changes are associated with a significant reduction of parasite propagation and can be rescued by reintroduction of a wildtype copy of Fip1. Accordingly, we hypothesize that Fip1 mediates contact between the mitochondrion and the parasite pellicle in a regulatable fashion, and that the Fip1 dependent mitochondrial morphology and dynamics are critical for parasite propagation. Through a combination of molecular genetics, microscopy and proteomics we will address the functional relevance and the mechanics of the mitochondrial morphology. In aim one we will conduct a thorough in vivo and in vitro phenotypic characterization of Fip1 mutant strains to determine the role of Fip1 and mitochondrial shape in parasite viability. Aim two focuses on identifying and characterizing components of the Fip1 complex that mediates the association of the mitochondrion with the periphery of the parasites. Finally, in aim three we will determine the regulatory mechanisms that drive the mitochondrial morphological changes as the parasite exits its host cell. In conjunction, these experiments will shed light onto the molecular mechanisms driving and regulating the morphodynamics of the Toxoplasma mitochondrion. As the mitochondrion of this important human pathogen is essential for its survival and a validated drug target, our studies will uncover novel targets for the development on new therapeutics.