Structural basis of the polar tube invasion machinery from microsporidia parasites

微孢子虫寄生虫极管入侵机制的结构基础

• 批准号: 10563182
• 负责人: Gira Bhabha
• 金额: $ 68.34万
• 依托单位: NEW YORK UNIVERSITY SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10563182
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Address; Animals; Architecture; Biochemical; Biochemistry; Biological Assay; Biology; Biophysics; Bombyx; Categories; Cells; Cellular biology; Cilia; Classification; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Data; Disease; Encephalitozoon cuniculi; Encephalitozoon hellem; Environment; Face; Farm; Fishes; Fluorescent Dyes; Freezing; Growth; Human; Image; Immunocompromised Host; In Situ; In Vitro; Individual; Infection; Insecta; Invaded; Ions; Light; Mass Spectrum Analysis; Microscopy; Microsporidia; Microsporidiosis; Microtubules; Modernization; Molecular; Molecular Conformation; Movement; Names; National Institute of Allergy and Infectious Disease; Optics; Organelles; Parasites; Patients; Process; Protein Subunits; Proteins; Proteome; Reproduction spores; Resolution; Sampling; Scanning Electron Microscopy; Sequence Homology; Side; Speed; Structure; Techniques; Thinness; Tube; Visualization; Work; X-Ray Crystallography; burden of illness; electron tomography; emerging pathogen; experimental study; genetic manipulation; human pathogen; in vivo; insight; light microscopy; lightspeed; mortality; opportunistic pathogen; organ transplant recipient; particle; pathogen; protein complex; protein protein interaction; protein purification; reconstruction; structural biology

Project Summary/Abstract Microsporidia are unicellular, ​fungal​ parasites with a wide host-range, from insects to humans. ​They are emerging pathogens, classified as NIAID Category B opportunistic pathogens, and cause microsporidiosis in immunocompromised patients.​ To gain entry into a target cell, microsporidia employ a remarkably unique and specialized harpoon-like invasion machinery called the polar tube, which is conserved among microsporidial species. While initially coiled neatly within the spore of the parasite, infection of a new cell begins with the rapid extrusion of the polar tube from the spore on a fast timescale (< 2s), which anchors the spore to the host cell. After it has been fired, the polar tube is thought to act as a conduit for the transfer of the infectious “sporoplasm” into the target cell, where replication can begin. Early work has yielded global insights into this process, and the molecular and structural underpinnings of the invasion process are ripe for exploration with modern techniques, such as cryo electron microscopy. This work aims to address fundamental questions and paradoxes in our understanding of the microsporidial polar tube machinery and how it drives invasion into host cells. We will use a combined bottom-up (structural biology, biochemistry and other ​in vitro​ techniques on purified proteins) and top-down (​in vivo​ light microscopy, electron tomography) approach; the intersection of these approaches will allow us to unravel the mechanistic biology of this unique invasion process. ​Here we focus on three human pathogens: ​Anncaliia algerae, Encephalitozoon cuniculi​ and ​Encephalitozoon hellem​. The specific aims are 1) To characterize the dynamics of polar tube firing and movement of sporoplasm through the tube using high-speed optical microscopy, and to comprehensively define the composition of the polar tube​ using mass spectrometry; 2) To biochemically and structurally characterize the individual protein components of the polar tube organelle using X-ray crystallography, single particle cryo electron microscopy and protein-protein interaction assays; 3) To elucidate the overall architecture and packing of the polar tube in the spore using structural cell biology techniques such as serial block face scanning electron microscopy (SBFSEM) and cryo focused ion beam scanning electron microscopy (cryo FIB-SEM) followed by cryo electron tomography (cryo ET).

项目摘要/摘要 微孢子虫是单细胞的真菌寄生虫，具有宽阔的宿主范围，从昆虫到人类。他们是 新兴的病原体，被归类为NIAID类别B的机会病原体，并引起微孢子虫病 免疫功能低下的患者。为了进入目标细胞，Microsporidia员工是一个非常独特的，并且 专门的鱼叉状入侵机械称为极地管，在微孢子虫中是保守的 虽然最初在寄生虫的酱汁中整齐地盘绕，但新细胞的感染始于快速 在快速时间尺度（<2s）上从酱汁中挤出极性管，将酱汁固定在宿主细胞上。 解雇后，极地管被认为是传染性转移的管道 可以开始复制的目标细胞中的“孢子质”。早期工作已经对此产生了全球见解 过程，以及入侵过程的分子和结构基础，可以与 现代技术，例如冷冻电子显微镜。这项工作旨在解决基本问题和 悖论在我们对微孢子极性管机械的理解中以及它如何将入侵驱动到宿主中 细胞。我们将使用合并的自下而上（结构生物学，生物化学和其他体外技术 纯化的蛋白质）和自上而下（体内光显微镜，电子断层扫描）方法；交集 这些方法将使我们能够揭示这一独特入侵过程的机械生物学。我们在这里 专注于三种人类病原体：Anncalia Algerae，脑静脉曲张和脑病。 具体目的是1）表征极性管的动力学和孢子质的运动 使用高速光学显微镜通过管，并全面定义 使用质谱法的极性管； 2）在生化和结构上表征单个蛋白 使用X射线晶体学，单个颗粒冷冻电子显微镜，极性管细胞器的组件 和蛋白质 - 蛋白质相互作用测定； 3）阐明极地管的整体结构和包装 使用结构细胞生物学技术（例如串行块面扫描电子显微镜）的孢子 （SBFSEM）和浓缩的离子光束扫描电子显微镜（冷冻FIB-SEM），然后是冷冻电子 断层扫描（Cryo ET）。