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

项目总结 恰加斯病的病原体克氏锥虫是一种专性的细胞内寄生虫，感染一种 据估计，美洲有1000万人，高危人口为7000万。尽管它被认为是 美洲影响最大的寄生虫病恰加斯病仍然没有得到充分的报道，也没有得到足够的研究 而且资金不足。克鲁兹毛滴虫生物学的基础研究此前因缺乏效率而受阻 基因工具，但CRISPR/Cas9基因编辑技术的问世为更深入的研究扫清了道路 分子分析。在人类感染寄生性锥虫中，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为感染形式，并传播给其哺乳动物 宿主通过其昆虫媒介的粪便直接生活在其哺乳动物宿主细胞的胞浆中，并利用 古老的细胞器摄食内吞胞外物质的方式很像它的食菌性自由物质- 活着的亲戚。这种内吞结构是由一个长的管状内陷(细胞咽部)组成的。 表面质膜小孔(细胞孔)，我们在这里称为细胞孔/细胞咽复合体或 SPC。SPC是一个高度动态的细胞器，它只以复制的形式存在并发挥作用 寄生虫在过渡到其感染阶段的过程中分解。直到最近，最高人民法院还只是 使用电子断层扫描技术进行检查，并抵制作为蛋白质的分子分析 组成它的组件仍然难以捉摸。我们最初发表的工作描述了已知的第一种蛋白质 以SPC为靶标，随后鉴定了一系列SPC靶向肌球蛋白马达，我们 显示对内吞过程有直接贡献。然而，最近我们发现了一种重要的肌球蛋白。 SPC的调节成分(MyAP1)，它的敲除导致寄生虫完全 缺乏可测量的内吞作用，且清除宿主脂类的能力有缺陷。虽然可行 在培养中，我们还通过感染昆虫媒介(R.prolixus)证明了内吞作用是 对于寄生虫来说，能够建立强大的感染并在昆虫的后肠定居是至关重要的，这是必要的 为有效传播到哺乳动物宿主而采取的步骤。这项研究的完成将使我们能够继续 阐明SPC的形成和功能的分子机制，并给出见解 为什么克氏锥虫保留了这种古老的营养吸收模式。我们的内吞零突变体也给了我们 最终检查这种寄生虫的“内细胞体”的独特机会。虽然不限于血脂，但我们 对初步数据感到兴奋，这使我们第一次能够区分哪些脂类是活跃的 通过内吞作用从寄生虫能够为自己内源性产生的物质中清除出来的。这个 这项提案的最终目标是更深入地了解寄生虫对媒介至关重要的途径 传递以及提供进入活跃存在的生物合成途径的窗口 是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.