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

恶性疟原虫是一种单细胞微生物，具有复杂的生命周期，同时入侵宿主肝细胞和红细胞(RBC)。它生命周期的很大一部分时间是在红细胞(RBC)内度过的，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，这种寄生虫将大量的红细胞胞浆吸收到一个专门的酸性细胞器中，称为消化液泡(DV)。这种细胞器分解红细胞胞浆的主要成分--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(一种酸性的细胞内液泡)和哺乳动物细胞的溶酶体有相似之处。然而，典型的溶酶体酸性磷酸酶和糖苷酶的缺失表明，疟原虫的DV是一种特殊的细胞器，最有可能进化为有效地降解血红蛋白。因此，寄生虫的DV执行各种特殊和关键的功能来确保寄生虫的生存，包括血红蛋白降解、氧自由基的解毒、离子稳态以及营养物质和/或溶质的跨膜运输。位于DV膜上的两个最重要和最重要的转运蛋白(多药转运蛋白PfMDR1和氯喹耐药转运蛋白PfCRT)尚未完全确定。这些转运蛋白以及它们运输的底物(S)的功能作用仍然不清楚。然而，很明显，它们并不是简单地在寄生虫内进化来排出药物。*这种复杂细胞器的潜在生物学还不完整，也很难理解，这可能是因为过去的实验大多使用固定的样本。因此，这项研究计划的目标是利用我们实验室提供的针对恶性疟原虫量身定做的现代成像技术，深入了解寄生虫消化液泡的分子和细胞过程。*根据我们之前的NSERC建议，我们着手更好地表征PfMDR1转运蛋白。我们实现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活体寄生虫中PfMDR1转运体的动力学，一位期刊评论者将这项技术称为“巧妙的”。*本提议将继续关注DV中发生的各种重要过程的量化。我们将继续使用活细胞成像技术，以更好地了解寄生虫的原位动态。这是一种独特的方法，仅在世界范围内的有限数量的实验室中使用，将使我们能够更好地在活体寄生虫中实时了解这些过程。*

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.***