The plant-like vacuole of Toxoplasma gondii

描述(申请人提供)：弓形虫属于尖端复合门，是免疫受损患者先天性疾病和感染的重要原因。该寄生虫有一个复杂的分泌系统，对寄主细胞入侵和寄生液泡的建立是必不可少的。这些分泌细胞器的生物发生仍然知之甚少，但有几个小组假设，在高尔基体和成熟的微丝和棒状体之间有一个隔室，在那里进行分类和加工。我们最近发现了一个新的弓形虫后高尔基体区，我们将其命名为类植物液泡(PLV)，本项目的目的是研究它在寄生虫生物学中的功能。这种细胞器是一个大的、多泡的结构，与植物细胞中发现的中央液泡有相似之处。这种细胞器可以被针对与液泡植物泵和通道非常相似的蛋白质的抗体标记，例如K敏感的V-H PPase(TgVP1)和液泡膜样水通道蛋白(TgAQP1)。生理学研究进一步揭示了与植物液泡的相似之处，如存在V-H ATPase、Na？那钙呢？抗转运蛋白和钙储存。此外，作为植物液泡，PLV还含有组织蛋白L(TgCPL)等蛋白酶。我们和合作者的结果表明，PLV是胞外速殖子的一个显著特征，作为植物的液泡，具有多种潜在功能，包括在蛋白质分选和抵抗环境胁迫方面的作用。例如，这个液泡在至少两个微线体前体蛋白proTgM2AP和proTgMIC3的蛋白质降解成熟过程中所起的作用表明，这个细胞器可能代表一个内胞/胞外枢纽，在那里可以对针对不同细胞器的蛋白质进行分类。在PLV中高表达VP1的弓形虫速殖子对钠胁迫具有更强的抵抗力，表明PLV在抵抗环境离子胁迫中发挥着重要作用。离子胁迫是弓形虫在离开宿主细胞进入细胞外基质时所面临的主要挑战。在细胞内和细胞外环境之间，钠、钾、钙等离子的浓度发生了巨大的变化。应对这些应激变化对寄生虫的生存非常重要，寄生虫需要主动入侵其他宿主细胞才能继续其裂解周期。我们将使用遗传学和细胞生物学实验相结合的方法，检验基于植物液泡的功能和对寄生虫的初步观察而开发的两个假说。这种新的细胞器起到调节离子和/或作为高尔基体后分选室的作用，用于定位微线体、棒状体和酸性钙体的分泌蛋白。如果我们考虑到植物液泡具有一系列复杂的功能(储存、分类、胁迫、植物生长等)，这些模型并不是相互排斥的。我们认为，在寄生虫的胞外期，PLV不仅在抵抗环境压力方面发挥着核心作用，而且在它为入侵下一个宿主细胞做准备的过程中也发挥着核心作用。 公共卫生相关性：这项工作旨在检验弓形虫类植物液泡(PLV)参与调节钙和pH动态平衡和对盐胁迫的反应，以及参与蛋白质到细胞器(如棒状体和酸性钙体)的分选和加工的假设。我们的工作可能为化疗提供潜在的靶点。

DESCRIPTION (provided by applicant): Toxoplasma gondii belongs to the phylum Apicomplexa and is an important cause of congenital disease and infection in immunocompromised patients. The parasite has a complex secretory system essential for host cell invasion and establishment of the parasitophorous vacuole. Biogenesis of these secretory organeles remains poorly understood but several groups have postulated a compartment situated between Golgi and mature micronemes and rhoptries in which sorting and processing occurs. We have recently discovered a novel post-Golgi compartment of T. gondii that we named the plant-like vacuole (PLV) and the goal of this project is to investigate its function in the biology of the parasite. This organelle is a large multi-vesicular structure that possesses similarities to the central vacuole found in plant cells. This organelle can be labeled with antibodies against proteins with great similarity to vacuolar plant pumps and channels, such as a K+sensitive V-H+PPase (TgVP1), and a tonoplast-like aquaporin (TgAQP1). Physiological studies further revealed similarities to plant vacuoles such as the presence of a V-H+ATPase, Na? and Ca2? antiporters, and calcium storage. Furthermore, as plant vacuoles, the PLV also contains proteases such as cathepsin L (TgCPL) and others. Our results and those of our collaborators showed that the PLV is a prominent feature of extracellular tachyzoites and, as the plant vacuole, has multiple potential functions including roles in protein sorting and resistance to environmental stresses. For example, the role of this vacuole in the proteolytic maturation of at least two microneme precursor proteins, proTgM2AP and proTgMIC3, suggests that this organelle could represent an endocytic/exocytic hub where sorting of proteins targeted to various organelles occurs. T. gondii tachyzoites that overexpress VP1 in the PLV, become more resistant to sodium stress pointing to an important role of the PLV in resistance to environmental ionic stress. Ionic stress is a major challenge that T. gondii confronts when exiting the host cell into the extracellular media. There is a dramatic change in the concentration of sodium, potassium, calcium and other ions between the intracellular and the extracellular environment. Dealing with these stressful changes is important for the survival of the parasite, which needs to actively invade other host cells to continue with its lytic cycle. We will test two hypotheses, which have been developed based on the functions of the plant vacuole and preliminary observations in the parasite, using a combination of genetic and cell biological experiments. This novel organelle acts to regulate ions and/or as a post Golgi sorting compartment for secretory proteins destined to the micronemes, rhoptries, and acidocalcisomes. These models are not mutually exclusive if we take into account the fact that the plant vacuole is home to a complex set of functions (storage, sorting, stress, plant growth, etc.). We think that the PLV plays a central role during the extracellular phase of the parasite not only in resisting environmental stress but also as it prepares itself for invading the next host cell. PUBLIC HEALTH RELEVANCE: This work is designed to test the hypotheses that the plant-like vacuole (PLV) of T. gondii is involved in the regulation of calcium and pH homeostasis and response to salt stress, as well as in sorting and processing of proteins to organelles such as the rhoptries and acidocalcisomes. Our work could provide potential targets for chemotherapy.