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

项目摘要 查加斯病的病原体克氏锥虫是一种专性细胞内寄生虫， 据估计，美洲有1000万人，其中7000万人处于危险之中。尽管它被公认为 作为美洲影响最大的寄生虫感染，恰加斯病仍然没有得到充分报告和研究 资金不足对T. cruzi此前曾因缺乏有效的 基因工具，但CRISPR/Cas9基因编辑技术的出现为更深入的研究扫清了道路。 分子分析在人类感染的寄生锥虫中，T. cruzi是非常独特的， 原因有很多：它在昆虫的后肠中发展成感染形式，传播给哺乳动物 通过其昆虫载体的粪便感染宿主，直接生活在其哺乳动物宿主细胞的胞质溶胶中， 一种古老的摄食细胞器，以类似于其食菌性游离细胞的方式内吞细胞外物质， 活着的亲戚这种内吞结构由一个长管状内陷（细胞咽）组成， 表面质膜孔（cytostome），我们在这里称为cytostome/cytopharynx复合体，或 SPC。SPC是一种高度动态的细胞器，仅以细胞的复制形式存在并发挥作用。 寄生虫和解体的过渡期间，其传染性阶段。直到最近，SPC才被 使用电子断层扫描技术进行了检查，并抵制了分子分析，因为蛋白质 其组成部分仍然难以捉摸。我们最初发表的工作描述了已知的第一种蛋白质， 靶向SPC，随后鉴定了一个SPC靶向肌球蛋白马达家族， 显示直接参与内吞过程。然而，最近，我们发现了一种重要的肌球蛋白， SPC（MyAP 1）的调控成分，敲除该成分导致寄生虫完全 缺乏可测量的内吞作用，并且其吸收宿主脂质的能力有缺陷。虽然可行 在培养物中，我们还通过昆虫载体（R. prolixus），内吞作用是 关键的寄生虫能够建立一个强大的感染和定殖昆虫后肠，一个必要的 有效传播到哺乳动物宿主的步骤。这项研究的完成将使我们能够继续 阐明负责SPC形成和功能的分子机制，并提供见解 为什么是T cruzi保留了这种古老的营养吸收模式。我们的内吞无效突变体也给了我们 这是最终研究这种寄生虫的“胞内细胞组”的唯一机会。虽然不限于脂质，我们 我们对初步数据感到兴奋，这些数据使我们第一次能够区分哪些脂质正在积极地 通过内吞作用从寄生虫能够为自身内源性产生的那些物质中清除。的 该提案的目标是最终更深入地了解媒介所必需的寄生虫途径 传输以及提供一个窗口，进入生物合成途径，积极存在， 寄生虫生存所必需的。

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.