Regulation of mitochondrial morphodynamics in Toxoplasma gondii

弓形虫线粒体形态动力学的调控

基本信息

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
项目类别：
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

项目摘要

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（例如弓形虫）的一个独特特征是存在单管状线粒体，对于寄生虫的生存和经过验证的药物靶点至关重要。大多数研究顶端复合体线粒体的研究重点是其生物化学和生理学。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，这些信息对于彻底探索线粒体作为药物靶点至关重要。弓形虫的独特之处线粒体非常活跃，在寄生虫的整个生命周期中经历形态变化包括从细胞内环境到细胞外环境的转变过程。当在宿主细胞内时线粒体保持套索形状，围绕寄生虫外围延伸，其中有区域与寄生虫薄膜的耦合，表明存在膜接触位点。退出后立即从宿主细胞中，这些接触位点消失，线粒体崩溃，表明动态膜接触位点调节线粒体的定位。既没有功能意义，也没有弓形虫线粒体和表膜之间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时，线粒体的形态受到严重影响。细胞内 fip1 敲除寄生虫线粒体不像野生型寄生虫那样呈套索形状，而是折叠的。另外，适当敲除寄生虫中的线粒体分离被破坏，导致寄生虫没有线粒体以及寄生虫外部的线粒体物质。这些总体形态变化与显着减少寄生虫繁殖，并且可以通过重新引入 Fip1 野生型副本来挽救。因此，我们假设 Fip1 介导线粒体和寄生虫表膜之间的接触一种可调节的方式，并且 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、显微镜学和蛋白质组学的结合，我们将解决线粒体形态的功能相关性和机制。在目标一中，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 和线粒体形状对寄生虫活力的影响。目标二侧重于识别和表征 Fip1 复合体的组成部分，介导线粒体与线粒体外周的关联寄生虫。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发生变化。结合起来，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。作为这种重要的人类病原体的线粒体对其生存至关重要，并且是经过验证的药物靶标，我们的研究将发现新疗法开发的新靶点。