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: 10630908
• 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/10630908
• 关键词: Actin-Binding Protein; Actins; Active Biological Transport; Amino Acids; Area; Binding; Binding Proteins; Biogenesis; Biology; Carbon; Cell Survival; Cell membrane; Cells; Cholesterol; Cilia; Complement; Complex; Consumption; Curiosities; Cytoplasm; 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; Polymers; 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; glycosylation; induced pluripotent stem cell; inhibitor; insight; microbial; model organism; 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.

项目概要 无论是光子还是薯条的吃都是生命的基础，从这个基本原理来看，生物体都有。 进化出无数的策略来从环境中获取能量和营养，进而导致 令人难以置信的生态多样性，涵盖从光食光合自养生物到掠食性生物的各个领域 异养生物是世界水生生态系统的一部分，是一个庞大的异养原生动物家族。 捕食者在环境碳和养分循环中发挥着关键作用，因为它们消耗了 75% 的初级资源 每天产生浮游自养生物，这些有鞭毛的吞噬生物中的绝大多数使用自生生物。 电流将它们的原核猎物输送到一个古老且高度神秘的进食装置中 这种进食结构从质膜表面开口（细胞口）开始，下降到消化。 内部管状内陷（细胞咽），最终猎物被包裹在出芽的囊泡内 此处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，但我们对它是如何发生的却几乎一无所知。 有趣的是，这种结构的形成或功能是一类被称为吞噬性捕食者。 动质体产生了一系列寄生原生动物，可以感染多种生物体 奇怪的是，有一个物种，克氏锥虫，保留了这一祖先。 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 由于 T. cruzi 易于培养、遗传易驯化且不依赖于 SPC。 介导的内吞作用在体外的活力，我们已经能够进行有史以来第一个深入的分子 我们最初发表的关于这种结构的研究描述了第一个。 已知针对 SPC 的蛋白质，随后发布了关于 SPC 家族鉴定的报告 我们展示的靶向肌球蛋白马达直接有助于内吞过程。 研究中，该提案旨在全面了解 SPC 如何介导内吞作用 我们首先将内吞作用的统一活动分解为其组成部分。 过程；通过表面受体（Aim1）捕获货物、受体信号转导和激活 内吞机制 (Aim2) 以及最终沿着 SPC 主动运输吞噬的货物进行消化 （目标 3）。每个目标都将解决原生动物生物学中仍然存在的重要基本方面。 至关重要的是，该提案将结合两种广泛的方法来识别细胞造口表面。 受体和 SPC 特异性信号传导成分，重点分析 Act2 同工型在 通过将这种模型生物与广泛的尖端分子工具相结合， 方法论，我们将能够阐明这种古老的原生动物进食装置的机制基础 目标是深入了解从微生物食物网到寄生虫病的基本过程。