Exocytosis of Plasmodium egress and invasion organelles

疟原虫出口和入侵细胞器的胞吐作用

• 批准号: 10888455
• 负责人: Vasant Muralidharan
• 金额: $ 54.02万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-22 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10888455
• 关键词: Antimalarials; Binding; Bioinformatics; Biological; Biological Assay; Calcium; Calcium Binding; Calcium Oscillations; Calcium Signaling; Calcium-Binding Domain; Calcium-Binding Proteins; Cells; Cessation of life; Clinical; Coupled; Cytoplasm; Cytoprotection; Data; Daughter; Development; Disease; EF-Hand Domain; Endoplasmic Reticulum; Ensure; Erythrocytes; Exocytosis; Extracellular Space; Fluorescence Microscopy; Genetic; Growth; Hand; Hepatocyte; Human; Immune system; Invaded; Lead; Life Cycle Stages; Malaria; Mediating; Membrane; Microscopy; Neck; Organelles; Organism; Outcome; Output; Parasites; Pathway interactions; Plasmodium; Plasmodium falciparum; Play; Proteins; Proteomics; Publishing; Reporter; Research; Resistance development; Role; Rupture; Second Messenger Systems; Secretory Vesicles; Series; Set protein; Shapes; Signal Pathway; Signal Transduction; Symptoms; Testing; Translating; Vacuole; Vesicle; asexual; calcium indicator; conditional mutant; drug development; innovation; knock-down; novel; obligate intracellular parasite; prevent; protein function; protein protein interaction; response; rhoptry; vector mosquito

Project Summary Human malaria caused by the intracellular parasite Plasmodium falciparum is responsible for nearly 600,000 deaths every year. The clinical symptoms of malaria are caused by the exponential asexual growth of parasites within human red blood cells. This cycle begins with the invasion of P. falciparum into the erythrocyte, where it hides within a vacuole (parasitophorous vacuole or PV) to divide into daughter merozoites and ends with the rapid release of merozoites that invade a red blood cell (RBC) to start the cycle anew. The egress and invasion of merozoites requires the signal-dependent exocytosis of specialized vesicles known as exonemes into the PV during egress and organelles known as rhoptries into the RBC during invasion. Data show that intracellular calcium oscillations lead to exocytosis of both exonemes and rhoptries within minutes of each other. But it is not known how the exocytic machinery on exonemes and rhoptries differentiate between the two intracellular calcium oscillations. It is likely that the proteins responsible for exocytosis are differentially sensitive to calcium and hence, respond differentially to the varied calcium signals during egress and invasion. However, the proteins localized in the secretory pathway required for signal dependent exocytosis remain mostly unknown. Therefore, we took a bioinformatic approach to identify several proteins in the secretory pathway with calcium binding domains that we hypothesized to function in organelle discharge during Plasmodium egress and invasion. This approach led to the identification of a calcium binding protein (PfERC) with an essential role in P. falciparum egress. Based on these published data, we wanted to test if PfERC functions in exoneme exocytosis but organelle discharge during egress or invasion has never been observed in live parasites. In the first aim, we developed fluorescence microscopy-based assays to investigate signal-dependent exoneme exocytosis and we will develop assays to assess calcium oscillations during egress of live P. falciparum. In PfERC conditional mutants, these live microscopy assays show that PfERC knockdown inhibits exoneme exocytosis. Using a quantitative proteomic approach, we have identified PfERC interactors and generated conditional mutants for prioritized candidates. Another candidate, rhoptry neck protein 11 (RON11), is essential for merozoite invasion. Therefore, in the second aim, we will focus on P. falciparum invasion and the proposed research will lead to the development of reporters to study rhoptry discharge as well as calcium oscillations in live merozoites during invasion. Using these assays, we will test the function of RON11 in rhoptry discharge, calcium oscillations as well as merozoite invasion into the RBC. The proposed research will identify several putative targets for antimalarial drug development. Since organelle secretion is required for egress and invasion of Plasmodium parasites during all stages of their lifecycle, this research may lead to the identification of pan-active antimalarials.

项目摘要 由恶性疟原虫疟原虫引起的人类疟疾几乎是 每年有60万人死亡。疟疾的临床症状是由指数无性恋引起的 人类红细胞中寄生虫的生长。这个周期始于恶性疟原虫的入侵 红细胞，它藏在液泡（寄生虫或PV）中，以分为女儿 梅龙夫人和以侵略红细胞（RBC）开始的蛋白酶的快速释放开始 重新循环。梅罗寄生虫的出口和入侵需要信号依赖性胞吐作用 在出口期间被称为外显子囊泡的专门囊泡，被称为Rhoptries的细胞器 入侵期间进入RBC。数据表明，细胞内钙振荡导致两者的胞吐作用 彼此几分钟之内的外部和犀牛。但尚不知道外囊肿机械如何 除外和体性分化了两个细胞内钙振荡。很可能 负责胞吐作用的蛋白质对钙差异敏感，因此反应差异 在出口和入侵期间的各种钙信号。但是，蛋白质本地分泌中 信号依赖性胞吐作用所需的途径主要未知。因此，我们接受了 生物信息学方法可以在分泌途径中识别具有钙结合结构域的分泌途径中的几种蛋白质 我们假设在疟原虫出口和侵袭过程中在细胞器排出中发挥作用。这 方法导致鉴定钙结合蛋白（PFERC）在P中至关重要的作用。 恶性阵容。基于这些已发布的数据，我们想测试PFERC是否在Exoneme中功能 在活寄生虫中从未观察到胞吞作用，但在出口或入侵期间的细胞器排出。 在第一个目的中，我们开发了基于荧光显微镜的测定以研究信号依赖性 外生胞吐作用，我们将开发测定法以评估活体的出口期间的钙振荡。 恶意。在PFERC条件突变体中，这些实时显微镜测定表明PFERC敲低 抑制外生胞吐作用。使用定量蛋白质组学方法，我们已经确定了PFERC 相互作用的人和生成有条件的突变体，用于优先级的候选者。另一个候选人，罗波特脖子 蛋白质11（RON11），对于梅洛祖铁虫入侵至关重要。因此，在第二个目标中，我们将重点关注 恶性疟原虫的入侵和拟议的研究将导致记者的发展 在入侵期间，实时泌尿生组中的Rhoptry放电以及钙振荡。使用这些测定， 我们将测试RON11在Rhoptry放电，钙振荡和梅罗龙米特入侵中的功能 进入RBC。拟议的研究将确定抗疟药的几个假定靶标 发展。由于疟原虫的出口和入侵需要细胞器分泌 在其生命周期的所有阶段，这项研究可能会导致池活性抗疟药的鉴定。