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 它的照片还是薯条饮食是生命的基础。从这个基本原则，生物有 进化的无数策略，从其环境中捕获能量和营养 令人难以置信的生态多样性，跨越范围从光饮光合自动噬菌到掠食性 异育。作为世界水生生态系统的一部分，异营原生动物的多余家族 捕食者在环境碳和营养循环中起着至关重要的作用，因为它们消耗了75％的初级 每天产生浮游生体。这些鞭毛的相位营养中的绝大多数都使用自我生成 将其原核生物猎物汇入古老而高度神秘的喂养装置之前的电流 消化。这种喂养结构以质膜表面开口（细胞抑制剂）的形式开始 内部结节内陷（细胞咽部），结束，猎物被包裹在萌芽的蔬菜中 注定要进行溶酶体融合。在这里，我们将此细胞器称为细胞击和细胞咽部复合物或SPC 而且，使其在原生动物中几乎无处不在的地传教，几乎没有任何知名度 这种结构是形成或功能的。有趣的是，一类这些水性捕食者称为 动质塑料产生了寄生原生动物的谱系，可以感染各种各样的生物 从植物到人类。奇怪的是，尤其是一种物种，锥虫cruzi，保留了这个祖先 Organelle很像它的自由生活亲戚（例如Bostonids），并继续将其用作其主要途径 内吞作用。由于克鲁兹很容易具有文化，遗传性的疗法并且不依赖于SPC，因此 介导的内吞作用是在体外生存力的 这种无处不在的喂养细胞器的分析。我们关于此结构的最初发表的工作描述了第一个 针对SPC的已知蛋白质，随后是关于鉴定SPC家族的报告 我们显示的靶向肌球蛋白电动机直接对内吞过程做出了贡献。作为这些的延续 研究，该建议旨在对SPC介导的内吞作用产生整体理解 从根本上起作用。我们将首先将内吞作用的统一活动拆除为其成分 过程；货物通过表面受体捕获（AIM1），受体信号传递和激活 内吞机械（AIM2），最后是沿SPC吞噬货物的主动运输以消化 （AIM3）。这些目标中的每一个都将解决原生动物生物学的重要基本方面，这些方面继续保持 理解不佳。至关重要的是，该提案将结合两种识别细胞表面的广泛方法 接收器和SPC特定信号传导组件，重点分析ACT2同工型在 内吞过程。通过将这种模型有机体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喂养设备的机械基础 目的是洞悉从微生物食物网到寄生疾病等基本过程。