Elucidating the Role of Endocytosis Via the Cytostome in the Life Cycle of Trypanosoma cruzi

阐明细胞口内吞作用在克氏锥虫生命周期中的作用

• 批准号: 10414106
• 负责人: RONALD DREW ETHERIDGE
• 金额: $ 47.98万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-06-01 至 2026-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10414106
• 关键词: Address; Adult; Americas; Arthropod Vectors; Basic Science; Biological; Biology; CRISPR/Cas technology; Cell membrane; Cells; Chagas Disease; Clinical; Complex; Cytosol; Data; Defect; Digestion; Disease; Elements; Endocytosis; Environment; Etiology; Family; Feces; Gastrointestinal tract structure; Gene Deletion; Genes; Genetic; Goals; Growth; Hindgut; Human; Individual; Infection; Ingestion; Insect Vectors; Insecta; Invaded; Knock-out; Leishmania; Life; Life Cycle Stages; Light; Lipids; Measurable; Mediating; Molecular; Molecular Analysis; Molecular Motors; Molecular Target; Motor; Myosin ATPase; Nutrient; Oral; Organelles; Organism; Orphan; Parasites; Parasitic infection; Pathway interactions; Persons; Play; Populations at Risk; Process; Proteins; Proteomics; Publishing; Rectum; Resources; Role; Structure; Suggestion; Surface; Techniques; Technology; Time; Trypanosoma brucei brucei; Trypanosoma cruzi; Tubular formation; Validation; Vesicle; Work; burden of illness; combat; comparative; electron tomography; extracellular; feeding; gastrointestinal; insight; knockout gene; lipidomics; mutant; neglect; novel; obligate intracellular parasite; pathogen; protein complex; subcellular targeting; tool; transmission process; uptake; vector; vector transmission; virtual

PROJECT SUMMARY The etiological agent of Chagas disease, Trypanosoma cruzi, is an obligate intracellular parasite that infects an estimated 10 million people in the Americas, with an at-risk population of 70 million. Despite its recognition as the highest impact parasitic infection of the Americas, Chagas disease remains underreported, understudied and underfunded. Basic research into the biology of T. cruzi has previously been hindered by a lack of efficient genetic tools, but the advent of CRISPR/Cas9 gene editing technology has cleared the way for more in-depth molecular analyses. Among the human infecting parasitic trypanosomatids, T. cruzi is extremely unique for a number of reasons: it develops into its infectious form in the hindgut of insects, is transmitted to its mammalian host via the feces of its insect vector, lives directly in the cytosol of its mammalian host cell and utilizes an ancient feeding organelle to endocytose extracellular material in a manner much like its bacterivorous free- living relatives. This endocytic structure is composed of a long tubular invagination (cytopharynx) starting at a surface plasma membrane pore (cytostome) which we refer to here as the cytostome/cytopharynx complex or SPC. The SPC is a highly dynamic organelle that is present and functional only in the replicating forms of the parasite and disassembles during the transition to its infectious stages. Until recently the SPC had only been examined using electron tomography techniques and had resisted molecular analysis as the protein components comprising it remained elusive. Our initial published work described the first proteins known to be targeted to the SPC and was followed by the identification of a family of SPC targeted myosin motors that we show contribute directly to the endocytic process. Recently, however, we have identified an essential myosin regulatory component of the SPC (MyAP1), a knockout of which resulted in parasites that are completely devoid of measurable endocytosis and who are defective in their ability to scavenge host lipids. Although viable in culture, we have also demonstrated through infections of the insect vector (R. prolixus) that endocytosis is critical for the parasite to be able to establish a robust infection and colonize the insect hindgut, a necessary step for effective transmission to its mammalian host. Completion of this study will allow us to continue to elucidate the molecular machinery responsible for the formation and function of the SPC as well as give insight into why it is that T. cruzi retained this ancient mode of nutrient uptake. Our endocytic-null mutant also gives us the unique opportunity to finally examine the “endocytome” of this parasite. Although not limited to lipids, we are excited by preliminary data allowing us for the first time to now distinguish which lipids are actively being scavenged through endocytosis from those which the parasite is able to endogenously produce for itself. The goal for this proposal is ultimately to give greater insight into parasite pathways essential for vector transmission as well as provide a window into the biosynthetic pathways that are actively present and necessary for parasite viability.

项目概要 恰加斯病的病原体克氏锥虫是一种专性细胞内寄生虫，可感染 据估计，美洲有 1000 万人，其中高危人群为 7000 万。 作为美洲影响最大的寄生虫感染，南美锥虫病的报告和研究仍然不足 克氏锥虫生物学的基础研究此前一直因缺乏有效的研究而受到阻碍。 基因工具，但 CRISPR/Cas9 基因编辑技术的出现为更深入的基因编辑扫清了道路 在人类感染的寄生锥虫中，克氏锥虫是极其独特的。 原因有很多：它在昆虫的后肠中发展成传染性形式，并传播给哺乳动物 通过其昆虫载体的粪便传播宿主，直接生活在其哺乳动物宿主细胞的细胞质中，并利用 古老的摄食细胞器以类似于其食菌自由的方式内吞细胞外物质 这种内吞结构由一个长管状内陷（细胞咽）组成。 表面质膜孔（细胞口），我们在此称为细胞口/细胞咽复合体或 SPC 是一种高度动态的细胞器，仅以复制形式存在并发挥功能。 直到最近，SPC 才在向感染阶段过渡期间被寄生和分解。 使用电子断层扫描技术进行检查，并拒绝对蛋白质进行分子分析 我们最初发表的工作描述了第一个已知的蛋白质。 靶向 SPC，随后鉴定了一系列 SPC 靶向肌球蛋白马达，我们 然而，最近我们发现了一种重要的肌球蛋白。 SPC (MyAP1) 的调节成分，敲除该成分会导致寄生虫完全死亡 缺乏可测量的内吞作用，并且清除宿主脂质的能力有缺陷，尽管有活力。 在培养中，我们还通过昆虫载体（R. prolixus）的感染证明了内吞作用 对于寄生虫能够建立强大的感染并在昆虫后肠定殖至关重要，这是必要的 有效传播到哺乳动物宿主的步骤将使我们能够继续研究。 阐明负责 SPC 形成和功能的分子机制并提供见解 我们的内吞无效突变体也为我们提供了为什么克氏锥虫保留了这种古老的营养吸收模式。 最终检查这种寄生虫的“内细胞组”的独特机会虽然不限于脂质，但我们。 初步数据使我们感到兴奋，这使我们现在第一次能够区分哪些脂质正在被积极地利用 通过内吞作用从寄生虫自身能够内源产生的物质中清除。 该提案的最终目标是更深入地了解媒介所必需的寄生虫途径 传输以及提供一个了解活跃存在和的生物合成途径的窗口 寄生虫生存所必需的。