Elucidating the Mechanistic Basis for Phagotrophy in the Protozoan Trypansoma cruzi

阐明原生动物克氏锥虫吞噬作用的机制基础

• 批准号: 10345248
• 负责人: RONALD DREW ETHERIDGE
• 金额: $ 30.2万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10345248
• 关键词: Actin-Binding Protein; Actins; Active Biological Transport; Address; Amino Acids; Animal Model; Area; Binding; Binding Proteins; Biogenesis; Biology; Carbon; Cell Survival; Cell membrane; Cells; Cholesterol; Cilia; Complement; Complex; Consumption; Digestion; Dimensions; Eating; Ecosystem; Endocytosis; Environment; Eukaryota; Family; Filament; Flagella; Food; Food Webs; Goals; Health; Human; In Vitro; Integral Membrane Protein; Knock-out; Life; Light; Lysosomes; Mass Spectrum Analysis; Mastigophora; Mechanics; Mediating; Membrane Microdomains; Methodology; Methods; Molecular; Molecular Analysis; Motor; Myosin ATPase; Nutrient; Oral; Organelles; Organism; Parasites; Parasitic Diseases; Phosphotransferases; Photons; Plants; Play; Positioning Attribute; Predatory Behavior; Process; Protein Isoforms; Proteins; Protozoa; Publishing; Receptor Protein-Tyrosine Kinases; Receptor Signaling; Reporting; Role; Route; Signal Pathway; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Source; Structure; Surface; Techniques; Trypanosoma cruzi; Tubular formation; Vesicle; Work; extracellular; feeding; inhibitor; insight; microbial; overexpression; palmitoylation; parasitism; polymerization; receptor; recruit; tool; trafficking; transmission process

PROJECT SUMMARY Whether its photons or fries eating is fundamental for life. From this basic principle, living organisms have evolved innumerable strategies to capture energy and nutrients from their environment, leading, in turn, to the incredible ecological diversity spanning the gamut from light eating photosynthetic autotrophs to predatory heterotrophs. As part of the world’s aquatic ecosystems, the expansive family of heterotrophic protozoan predators play a critical role in environmental carbon and nutrient cycling as they consume 75% of primary producing planktonic autotrophs daily. The vast majority of these flagellated phagotrophs use self-generated currents to funnel their prokaryotic prey into an ancient and highly enigmatic feeding apparatus prior to digestion. This feeding structure begins as a plasma membrane surface opening (cytostome), descends into an internal tubular invagination (cytopharynx) and ends with prey being enveloped within budding vesicles destined for lysosome fusion. Here we refer to this organelle as the cytostome/cytopharynx complex or SPC and, despite its near ubiquitous presence in protozoans, next to nothing is known mechanistically about how this structure is formed or functions. Intriguingly, a class of these phagotrophic predators known as the kinetoplastids, gave rise to a lineage of parasitic protozoa that can infect a wide variety of organisms ranging from plants to humans. Curiously, one species in particular, Trypanosoma cruzi, retained this ancestral organelle much like its free-living relatives (e.g. bodonids) and continues to use it as its primary route of endocytosis. Due to the fact that T. cruzi is easily culturable, genetically tractable and not reliant on SPC mediated endocytosis for viability in vitro, we have been able to conduct the first ever in-dept molecular analyses of this ubiquitous feeding organelle. Our initial published work on this structure described the first known proteins targeted to the SPC and was followed by a report on the identification of a family of SPC targeted myosin motors that we show contribute directly to the endocytic process. As a continuation of these studies, this proposal seeks to generate a holistic understanding of how SPC mediated endocytosis fundamentally functions. We will begin by dismantling the unified activity of endocytosis into its constituent processes; cargo capture through surface receptors (Aim1), receptor signal transduction and activation of endocytic machinery (Aim2) and finally active transport of phagocytosed cargo along the SPC for digestion (Aim3). Each of these aims will address important basic aspects of protozoan biology that continue to remain poorly understood. Critically, this proposal will combine both a broad approach to identify cytostomal surface receptors and SPC specific signaling components with a focused analysis of the role of the Act2 isoform in the endocytic process. By combining this model organism with a broad range of cutting-edge molecular tools and methodologies, we will be able to elucidate the mechanistic basis of this ancient protozoal feeding apparatus with the goal of providing insight into basic processes ranging from microbial food webs to parasitic diseases.

项目摘要 无论是光子还是薯条，吃都是生命的基础。从这个基本原理出发，生物体具有 进化出无数的策略来从环境中获取能量和营养，反过来， 令人难以置信的生态多样性，从光食光合自养生物到掠食性 异养生物作为世界水生生态系统的一部分， 捕食者在环境碳和营养循环中发挥着关键作用，因为它们消耗了75%的初级 每天都在生产营养自养生物。这些鞭毛噬菌体中的绝大多数利用自我生成的 它们将原核生物的猎物输送到一个古老而神秘的进食装置中， 消化.这种进食结构开始于质膜表面开口（细胞口）， 一种内部管状内陷（胞咽），以被包围在出芽囊泡内的猎物结束 用于溶酶体融合。在这里，我们将这种细胞器称为细胞口/细胞咽复合体或SPC 而且，尽管它在原生动物中几乎无处不在，但对于其机制几乎一无所知 该结构形成或起作用。有趣的是，一类被称为 动质体，产生了一个谱系的寄生原生动物，可以感染各种各样的生物， 从植物到人类。奇怪的是，有一种特别的物种，克氏锥虫， 细胞器很像它的自由生活的亲戚（例如bodonids），并继续使用它作为其主要途径 内吞作用由于T. cruzi很容易培养，遗传上容易控制，不依赖SPC 介导的内吞作用的体外生存能力，我们已经能够进行有史以来第一次的深入分子 分析这种无处不在的进食细胞器。我们最初发表的关于这种结构的工作描述了第一个 已知的蛋白质靶向的SPC，并随后由一个家庭的SPC的鉴定报告 靶向肌球蛋白马达，我们显示直接有助于内吞过程。作为这些活动的延续， 研究，该建议旨在产生一个整体的理解SPC如何介导的内吞作用 基本功能。我们将开始把统一的内吞活动分解成它的组成部分 过程;货物捕获通过表面受体（Aim 1），受体信号转导和激活 内吞机制（Aim 2）和最后吞噬货物的主动运输沿着SPC消化 （目标3）。这些目标中的每一个都将涉及原生动物生物学的重要基本方面， 不太了解。重要的是，该建议将联合收割机与识别细胞口表面的广泛方法相结合， 受体和SPC特异性信号传导组分，重点分析Act 2亚型在 内吞过程通过将这种模式生物与广泛的尖端分子工具相结合， 方法，我们将能够阐明这种古老的原生动物进食装置的机械基础 其目的是深入了解从微生物食物网到寄生虫病的基本过程。