The plant-like vacuole of Toxoplasma gondii

描述（申请人提供）：弓形虫属于顶复门，是免疫功能低下患者先天性疾病和感染的重要原因。该寄生虫具有复杂的分泌系统，对于宿主细胞的入侵和寄生液泡的建立至关重要。这些分泌细胞器的生物发生仍然知之甚少，但一些研究小组推测，高尔基体和成熟的微线体和菱形体之间有一个隔室，在其中进行分类和加工。 我们最近发现了弓形虫的一种新型后高尔基体区室，我们将其命名为植物样液泡（PLV），该项目的目标是研究其在寄生虫生物学中的功能。这种细胞器是一个大型的多囊泡结构，与植物细胞中发现的中央液泡相似。这种细胞器可以用针对与液泡植物泵和通道非常相似的蛋白质的抗体进行标记，例如 K 敏感性 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，例如 V-H ATP 酶、Na？和钙？逆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。我们和我们合作者的结果表明，PLV 是细胞外速殖子的一个显着特征，并且作为植物液泡，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，该液泡在至少两种微线体前体蛋白 proTgM2AP 和 proTgMIC3 的蛋白水解成熟中的作用表明，该细胞器可能代表一个内吞/胞吐中心，其中发生针对各种细胞器的蛋白质分选。在 PLV 中过度表达 VP1 的弓形虫速殖子对钠胁迫具有更强的抵抗力，这表明 PLV 在抵抗环境离子胁迫方面发挥着重要作用。离子应激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。细胞内和细胞外环境之间钠、钾、钙等离子的浓度发生巨大变化。应对这些压力变化对于寄生虫的生存非常重要，寄生虫需要主动侵入其他宿主细胞以继续其裂解周期。 我们将结合遗传和细胞生物学实验来检验两个假设，这两个假设是根据植物液泡的功能和寄生虫的初步观察而提出的。这种新型细胞器的作用是调节离子和/或作为高尔基体后分选室，用于分泌蛋白，将其输送至微线体、棒状体和酸钙体。如果我们考虑到植物液泡是一组复杂功能（存储、分类、应激、植物生长等）的所在地，那么这些模型并不相互排斥。我们认为，PLV 在寄生虫的细胞外阶段发挥着核心作用，不仅在抵抗环境压力方面，而且在它为入侵下一个宿主细胞做好准备方面。

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.