The malaria digestive vacuole: elucidating its function in parasite physiology and development

疟疾消化液泡：阐明其在寄生虫生理和发育中的功能

• 批准号: RGPIN-2015-03952
• 负责人: Rohrbach, Petra
• 金额: $ 2.19万
• 依托单位: McGill University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2019
• 资助国家: 加拿大
• 起止时间: 2019-01-01 至 2020-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=683670
• 关键词: malaria; digestive; vacuole; elucidating; its

The malaria parasite Plasmodium falciparum is a single-celled microorganism with a complex life cycle that invades both host hepatocytes and red blood cells (RBCs). It spends a great proportion of its life cycle within the red blood cell (RBC), a cell comprised mostly of hemoglobin and lacking in nuclei and organelles. The invasion and subsequent modification of the RBC is a crucial step for the survival of this deadly parasite.***To support the dynamic multiplication of malaria parasites, the parasite takes up large amounts of RBC cytosol into a specialized acidic organelle called the digestive vacuole (DV). This organelle breaks down the RBC cytosol's primary constituent - hemoglobin - to provide itself with nutrients (i.e. amino acids) that are crucial for its growth.***The DV has been thought to have similarities to both tonoplasts, an acidic intracellular vacuole of plant cells, and lysosomes of mammalian cells. However, the absence of the typical lysosomal acid phosphatase and glycosidase indicates that the DV of Plasmodium is a specialized organelle that most likely evolved to efficiently degrade hemoglobin. Consequently, the parasite's DV carries out a variety of specialized and critical functions to ensure the survival of the parasite, including hemoglobin degradation, detoxification of oxygen radicals, ion homeostasis, and nutrient and/or solute transport across its membrane. The two most prominent and essential transporters (the multi-drug transporter PfMDR1 and the chloroquine resistance transporter PfCRT) situated on the DV membrane are yet to be fully characterized. The functional role of these transporters, as well as the substrate(s) they transport, remains obscure. It is clear, however, that they did not evolve within the parasite simply to efflux drugs.***The underlying biology of this complex organelle is incomplete and poorly understood, possibly due to past experiments that mostly used fixed samples. Therefore, the objective of this research program is the development of an in-depth understanding of the molecular and cellular processes of the parasite's digestive vacuole using modern imaging techniques tailored to P. falciparum and available in our lab.****With our previous NSERC proposal, we set out to better characterize the PfMDR1 transporter. We accomplished an important objective of this proposal in that we were able to quantify the kinetics of the PfMDR1 transporter in live parasites using a reverse imaging assay, a technique that one of the journal reviewers termed "ingenious".***The present proposal will keep our focus on the quantification of various important processes occurring in the DV. We will continue to use live cell imaging techniques to get a better understanding of parasite dynamics in situ. This is a unique approach that is used in only a limited number of labs world-wide and will allow us to better understand these processes in real time within the live parasite.***

疟疾寄生虫恶性疟原虫是一种单细胞微生物，具有复杂的生命周期，侵入宿主肝细胞和红细胞（RBC）。它的生命周期中有很大一部分是在红细胞（RBC）中度过的，红细胞是一种主要由血红蛋白组成的细胞，缺乏细胞核和细胞器。红细胞的入侵和随后的修饰是这种致命寄生虫生存的关键一步。为了支持疟疾寄生虫的动态繁殖，寄生虫将大量的RBC胞质溶胶吸收到称为消化泡（DV）的专门酸性细胞器中。这种细胞器分解红细胞胞质溶胶的主要成分-血红蛋白-为自身提供对其生长至关重要的营养物质（即氨基酸）。DV被认为与植物细胞的液泡膜（一种酸性细胞内液泡）和哺乳动物细胞的溶酶体具有相似性。然而，典型的溶酶体酸性磷酸酶和糖苷酶的缺乏表明，疟原虫DV是一个专门的细胞器，最有可能进化到有效地降解血红蛋白。因此，寄生虫的DV执行各种专门的和关键的功能，以确保寄生虫的存活，包括血红蛋白降解，氧自由基解毒，离子稳态，以及营养和/或溶质跨膜转运。位于DV膜上的两个最突出和最重要的转运蛋白（多药转运蛋白PfMDR 1和氯喹抗性转运蛋白PfCRT）尚未完全表征。这些转运蛋白的功能作用以及它们转运的底物仍然不清楚。然而，很明显，它们在寄生虫体内的进化并不仅仅是为了排出药物。这种复杂细胞器的基础生物学是不完整的，也很难理解，可能是由于过去的实验主要使用固定的样品。因此，本研究计划的目标是使用针对恶性疟原虫定制的现代成像技术深入了解寄生虫消化泡的分子和细胞过程，并在我们的实验室中提供。在我们之前的NSERC提案中，我们着手更好地表征PfMDR 1转运蛋白。我们完成了这项提议的一个重要目标，因为我们能够使用反向成像分析来量化PfMDR 1转运蛋白在活寄生虫中的动力学，该技术被一位期刊评审员称为“巧妙的”。目前的建议将保持我们的重点是量化的各种重要过程中发生的DV。我们将继续使用活细胞成像技术，以更好地了解寄生虫的动态原位。这是一种独特的方法，仅在全球有限数量的实验室中使用，并将使我们能够更好地了解活寄生虫内真实的这些过程。