Structural basis of the polar tube invasion machinery from microsporidia parasites

项目摘要/摘要 微孢子虫是一种单细胞​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类条件病原体，并在 免疫受损的患者。​为了进入靶细胞，微孢子虫使用了一种非常独特的和 特殊的类似鱼叉的入侵机制称为极管，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，但新细胞的感染始于快速的 从孢子上快速挤出极管，将孢子固定在寄主细胞上。 在发射后，极管被认为是传染病传播的管道。 “孢子质”进入目标细胞，在那里可以开始复制。早期的工作已经产生了对这一点的全球洞察 入侵过程的分子和结构基础已经成熟，可以用 现代技术，如低温电子显微镜。这项工作旨在解决基本问题和 我们对微孢子虫极管机制及其如何驱动入侵宿主的理解中的悖论 细胞。我们将使用自下而上(结构生物学、生物化学等)相结合的​体外​技术 纯化蛋白质)和自上而下(​in vivo​光学显微镜，电子断层扫描)方法； 这些方法将使我们能够解开这一独特入侵过程的机制生物学。​在这里，我们 重点研究了三种人类病原体：​anncalia algerae，Encephalitoson cunruli​和​Encephalitoson hellem​。 具体目标是：1)描述极管发射和孢子质运动的动力学 通过试管使用高速光学显微镜，并对其成分进行全面界定 使用质谱学的极管​；2)对单个蛋白质进行生化和结构表征 用X射线结晶学、单粒子低温电子显微镜研究极管细胞器的组成 和蛋白质-蛋白质相互作用分析；3)阐明极管的整体结构和包装 孢子使用结构细胞生物学技术，如连续块面扫描电子显微镜 (SBFSEM)和低温聚焦离子束扫描电子显微镜(低温FIB-SEM)，然后是低温电子 体层摄影术(冷冻等)。

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).