Elucidating the Mechanistic Basis for Phagotrophy in the Protozoan Trypansoma cruzi

项目总结 无论是吃光子还是吃薯条，都是生命的根本。根据这一基本原理，有生命的有机体 进化出无数的策略来从他们的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，从捕食光合作用的自养生物到捕食性的 异养生物。作为世界水生生态系统的一部分，异养原生动物的庞大家族 捕食者在环境碳和养分循环中扮演着关键角色，因为它们消耗了75%的初级 每天生产浮游自养生物。这些有鞭毛的吞噬生物中的绝大多数使用自我生成的 洋流将它们的原核生物猎物输送到一个古老而高度神秘的摄食装置中 消化。这种摄食结构开始于质膜表面的开口(细胞气孔)，下降到 一种内部管状内陷(细胞咽喉)，末端猎物被包裹在萌芽的小泡中 注定要进行溶酶体融合。在这里，我们将这种细胞器称为细胞气孔/细胞咽复合体或SPC 而且，尽管它几乎无处不在地存在于原生动物中，但从机制上来说，几乎没有人知道它是如何 这种结构是形成的或起作用的。有趣的是，一类这样的食性捕食者被称为 动质体，产生了寄生原生动物的谱系，可以感染范围广泛的各种生物 从植物到人类。奇怪的是，有一种特别的物种，克氏锥虫，保留了这个祖先 细胞器很像它的自由生活的近亲(如狗形目动物)，并继续使用它作为它的主要途径 内吞作用。由于克鲁兹毛滴虫很容易培养，遗传上容易驯化，而且不依赖SPC 介导的内吞作用在体外存活，我们已经能够进行有史以来第一个深入的分子 对这种无处不在的摄食细胞器的分析。我们最初发表的关于这种结构的工作描述了第一个 以SPC为靶点的已知蛋白质，随后是一份关于SPC家族鉴定的报告 我们展示的靶向肌球蛋白马达直接参与内吞过程。作为这些的延续 研究，这项建议试图对SPC如何介导内吞作用产生一个整体的理解 从根本上说是有效的。我们将首先将内吞作用的统一活动分解为其组成部分 过程；通过表面受体捕获货物(Aim1)，受体信号转导和激活 吞噬机械(AIM2)和最终吞噬的货物沿着SPC主动运输以进行消化 (目标3)。这些目标中的每一个都将涉及原生动物生物学的重要基本方面，这些方面继续存在 人们对此知之甚少。关键的是，这项建议将结合两种广泛的方法来识别细胞造口表面 受体和SPC特异性信号成分，重点分析Act2异构体在SPC中的作用 内吞过程。通过将这种模式生物与广泛的尖端分子工具和 方法论，我们将能够阐明这种古老的原生动物喂养装置的机制基础。 目的是洞察从微生物食物网到寄生虫病的基本过程。

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.