The plant-like vacuole of Toxoplasma gondii

描述（申请人提供）：刚地弓形虫属于顶复合体门，是免疫功能低下患者先天性疾病和感染的重要原因。寄生物具有复杂的分泌系统，这是寄主细胞入侵和寄主液泡形成所必需的。这些分泌细胞器的生物发生尚不清楚，但有几个研究小组假设在高尔基体和成熟微元体之间有一个隔间，在那里分选和加工发生。我们最近发现了一种新的弓形虫后高尔基区室，我们将其命名为植物样液泡（PLV），本项目的目标是研究其在弓形虫生物学中的功能。这种细胞器是一个大的多泡结构，与植物细胞中的中央液泡相似。这种细胞器可以用抗体标记与液泡植物泵和通道非常相似的蛋白质，如K+敏感的V-H+PPase （TgVP1）和tono质体样水通道蛋白（TgAQP1）。生理学研究进一步揭示了与植物液泡的相似之处，如V-H+ atp酶的存在，Na？和游离钙吗?反向转运蛋白和钙储存。此外，作为植物液泡，PLV还含有组织蛋白酶L （TgCPL）等蛋白酶。我们和合作者的研究结果表明，PLV是细胞外速殖子的一个突出特征，作为植物液泡，具有多种潜在功能，包括蛋白质分选和抵抗环境胁迫的作用。例如，该液泡在至少两种微粒前体蛋白（proTgM2AP和proTgMIC3）的蛋白水解成熟过程中所起的作用表明，该细胞器可能代表一个胞内/胞外中心，在这里发生针对各种细胞器的蛋白质分选。在PLV中过表达VP1的弓形虫速殖子对钠胁迫的抗性更强，这表明PLV在抵抗环境离子胁迫中起着重要作用。离子胁迫是弓形虫离开宿主细胞进入细胞外介质时面临的主要挑战。在细胞内和细胞外环境之间，钠、钾、钙和其他离子的浓度有很大的变化。处理这些压力变化对寄生虫的生存很重要，因为寄生虫需要主动侵入其他宿主细胞以继续其分解周期。我们将采用遗传和细胞生物学实验相结合的方法，根据植物液泡的功能和对寄生虫的初步观察，验证两个假设。这种新型细胞器的作用是调节离子和/或作为分泌蛋白的后高尔基分选室，这些蛋白被送往微组、异构体和酸细胞体。如果我们考虑到植物液泡是一组复杂功能（储存、分选、胁迫、植物生长等）的家园，这些模型并不相互排斥。我们认为，在寄生虫的细胞外阶段，PLV不仅在抵抗环境压力方面起着核心作用，而且在准备入侵下一个宿主细胞时也起着核心作用。

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.