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: 9913209
• 负责人: Gira Bhabha
• 金额: $ 64.85万
• 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/9913209
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Address; Animals; Architecture; Bees; Biochemical; Biochemistry; Biological Assay; Biology; Biophysics; Bombyx; Categories; Cells; Cellular Structures; Cellular biology; Cilia; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Data; Disease; Encephalitozoon cuniculi; Encephalitozoon hellem; Environment; Face; Farming environment; Fishes; Fluorescent Dyes; Freezing; Growth; Honey; Human; Image; Immunocompromised Host; In Situ; In Vitro; Individual; Infection; Insecta; Ions; Light; Mass Spectrum Analysis; Microscopy; Microsporidia; Microsporidiosis; Microtubules; Modernization; Molecular Conformation; Molecular Structure; Movement; Names; National Institute of Allergy and Infectious Disease; Optics; Organ Transplantation; Organelles; Parasites; Parasitic infection; Patients; Process; Protein Subunits; Proteins; Proteome; Reproduction spores; Resolution; Sampling; Scanning Electron Microscopy; Sequence Homology; Side; Speed; Structure; Techniques; Thinness; Transplant Recipients; Tube; Work; X-Ray Crystallography; base; burden of illness; electron tomography; experimental study; genetic manipulation; human pathogen; in vivo; insight; light microscopy; lightspeed; mortality; particle; pathogen; protein complex; protein protein interaction; 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).

项目概要/摘要 微孢子虫是单细胞真菌寄生虫，宿主范围广泛，从昆虫到人类。​他们是 新出现的病原体，被归类为 NIAID B 类机会病原体，并引起微孢子虫病 免疫功能低下的患者。为了进入靶细胞，微孢子虫采用了一种非常独特的、 称为极管的特殊鱼叉状入侵机制，在微孢子虫中保守存在 物种。虽然最初在寄生虫的孢子内整齐地盘绕，但新细胞的感染开始于快速 在快速时间尺度（< 2s）内从孢子中挤出极管，将孢子锚定到宿主细胞上。 发射后，极管被认为充当传染性物质传播的管道 “孢子质”进入靶细胞，在那里复制可以开始。早期的工作已经产生了对此的全球见解 过程，入侵过程的分子和结构基础已经成熟，可以探索 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 我们对微孢子极管机制及其如何驱动宿主入侵的理解中的悖论 细胞。我们将使用自下而上的组合（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（体内光学显微镜、电子断层扫描）方法；的交集 这些方法将使我们能够解开这种独特入侵过程的生物学机制。​在这里我们 重点关注三种人类病原体：​藻类环孢菌、兔脑炎原虫​和​地狱脑炎原虫​。 具体目标是 1) 表征极管发射和孢子质运动的动力学 使用高速光学显微镜通过管子，并全面确定其成分 使用质谱分析的极管； 2) 对单个蛋白质进行生化和结构表征 使用 X 射线晶体学、单粒子冷冻电子显微镜观察极管细胞器的组成 和蛋白质-蛋白质相互作用测定； 3) 阐明极管的整体结构和封装 使用结构细胞生物学技术（例如串行块面扫描电子显微镜）观察孢子 (SBFSEM) 和冷冻聚焦离子束扫描电子显微镜 (cryo FIB-SEM)，然后是冷冻电子显微镜 断层扫描（冷冻 ET）。