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: 10349551
• 负责人: Gira Bhabha
• 金额: $ 70.3万
• 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/10349551
• 关键词: 3-Dimensional; Acquired Immunodeficiency Syndrome; Address; Animals; Architecture; Bees; Biochemical; Biochemistry; Biological Assay; Biology; Biophysics; Bombyx; Categories; Cells; Cellular biology; Cilia; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Data; Disease; Encephalitozoon cuniculi; Encephalitozoon hellem; Environment; Face; Farm; 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; Molecular Conformation; Movement; Names; National Institute of Allergy and Infectious Disease; Optics; Organelles; Parasites; Parasitic infection; Patients; Process; Protein Subunits; Proteins; Proteome; Reproduction spores; Resolution; Sampling; Scanning Electron Microscopy; Sequence Homology; Side; Speed; Structure; Techniques; Thinness; Tube; Work; X-Ray Crystallography; base; 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; 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).

项目总结/摘要 微孢子虫是单细胞真菌寄生虫，具有广泛的宿主范围，从昆虫到人类。 他们是 新出现的病原体，被归类为NIAID B类条件致病菌，并在 为了进入靶细胞，微孢子虫采用非常独特的， 一种特殊的鱼叉状入侵机器，称为极管，它在微孢子虫中是保守的。 物种虽然最初整齐地盘绕在寄生虫的孢子内，但新细胞的感染开始于快速的 在短时间内（< 2s）从孢子中挤出极管，将孢子固定在宿主细胞上。 在它被发射后，极管被认为是传染性疾病转移的管道。 将“孢子质”导入靶细胞，在那里复制可以开始。早期的工作已经产生了全球性的见解 过程，入侵过程的分子和结构基础已经成熟，可以进行探索， 现代技术，如冷冻电子显微镜。这项工作旨在解决基本问题， 我们对微孢子虫极管机制及其如何驱动侵入宿主的理解存在矛盾 细胞我们将使用自下而上（结构生物学，生物化学和其他体外技术）的组合， 纯化蛋白质）和自上而下（体内光学显微镜、电子断层扫描）方法;两者的交叉 这些方法将使我们能够解开这种独特的入侵过程的生物学机制。 这里我们 重点研究了三种人类病原体：藻类Anncalia algerae、兔脑孢子虫和赫莱姆脑孢子虫。 其具体目的是：1）研究极管放电和孢子质运动的动态 通过管使用高速光学显微镜，并全面定义的组成， 使用质谱的极性管; 2）对单个蛋白质进行生物化学和结构表征 用X射线晶体学、单粒子低温电子显微镜和透射电镜观察了极管细胞器的组成 蛋白质-蛋白质相互作用分析; 3）阐明了极管的整体结构和包装， 使用结构细胞生物学技术如连续块面扫描电子显微镜 低温聚焦离子束扫描电子显微镜（SBFSEM）和低温聚焦离子束扫描电子显微镜（cryo FIB-SEM），随后进行低温电子显微镜（cryo electron microscopy）。 断层扫描（cryo ET）。