Regulation of mitochondrial morphodynamics in Toxoplasma gondii

弓形虫线粒体形态动力学的调控

• 批准号: 9896491
• 负责人: Gustavo A Arrizabalaga
• 金额: $ 39.28万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-03 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9896491
• 关键词: Address; Affect; Amino Acids; Apical; Apicomplexa; Automobile Driving; Auxins; Biochemistry; Biological Assay; Biology; Biotinylation; Calcium; Cells; Cessation of life; Co-Immunoprecipitations; Complex; Coupling; Cytokinesis; Development; Disease; Drug Targeting; Economic Burden; Environment; Homeostasis; Image; Immunocompromised Host; In Vitro; Individual; Knock-out; Lasso; Lead; Life; Life Cycle Stages; Light; Lipids; Lytic Phase; Maps; Mechanics; Mediating; Membrane; Microscopy; Mitochondria; Molecular; Molecular Genetics; Monitor; Morphology; Mutation Analysis; Organelles; Parasites; Pharmaceutical Preparations; Phenotype; Phosphorylation Site; Physiology; Plasmodium falciparum; Play; Population; Positioning Attribute; Post-Translational Protein Processing; Process; Proteins; Proteomics; Regulation; Research; Resistance; Role; Shapes; Site; Societies; Stretching; Structure; System; Toxoplasma; Toxoplasma gondii; Toxoplasmosis; Transmembrane Domain; Tubular formation; Yeasts; base; combat; daughter cell; experimental study; extracellular; health economics; human pathogen; in vivo; mutant; novel; novel therapeutics; pathogen; protein complex; segregation; tissue culture; yeast two hybrid system

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.

顶复门寄生虫如刚地弓形虫的一个独特特征是存在一种 单管状囊泡，这是寄生虫生存所必需的，也是一个经过验证的药物靶点。大多数研究 的研究主要集中在其生物化学和生理学上。相比之下， 关于控制线粒体分裂和调节其结构的机制， 对于彻底探索线粒体作为药物靶点至关重要。弓形虫单一型 寄生虫是非常动态的，并在整个寄生虫的生命周期中经历形态变化 包括在从细胞内环境向细胞外环境转变的过程中。在宿主细胞内， 寄生虫维持在一个套索形状，围绕寄生虫周围伸展， 的耦合与寄生虫表膜，表明膜接触点的存在。退出后立即退出 从宿主细胞，这些接触点消失，和微粒子崩溃表明，动态 膜接触位点调节膜的定位。既没有功能意义， 弓形虫的寄生虫和表膜之间的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，它与蛋白质结合，当敲除正常蛋白质时， 微粒子的形态受到严重影响。在细胞内FIP1敲除的寄生虫中， 不像野生型寄生虫那样呈套索状，而是折叠的。此外，适当 在敲除的寄生虫中，线粒体分离被破坏，导致寄生虫没有线粒体。 以及寄生虫外的线粒体物质这些肉眼可见的形态学变化与 寄生虫繁殖的显著减少，并且可以通过重新引入Fip1的野生型拷贝来拯救。 因此，我们假设Fip 1介导了寄生虫和寄生虫表膜之间的接触， 一种可调节的方式，Fip1依赖的线粒体形态和动力学对于 寄生虫繁殖通过分子遗传学、显微镜和蛋白质组学的结合， 解决线粒体形态的功能相关性和机制。在目标一，我们将 对Fip1突变株进行彻底的体内和体外表型表征，以确定其作用 Fip1和线粒体形状在寄生虫生存能力。目标二侧重于识别和表征 Fip1复合物的组成部分，介导的协会与周边的 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， 当寄生虫离开其宿主细胞时形态发生变化。结合起来，这些实验将揭示 驱动和调节弓形虫形态动力学的分子机制。作为 这种重要的人类病原体的感染对其生存至关重要， 这些研究将为新疗法的开发发现新的靶点。