Exocytosis of Plasmodium egress and invasion organelles

项目摘要 由细胞内寄生虫恶性疟原虫（Plasmodium falciparum）引起的人类疟疾， 每年有60万人死亡。疟疾的临床症状是由指数无性生殖引起的 寄生虫在人体红细胞内的生长。这个循环开始于恶性疟原虫侵入 红细胞，在那里它隐藏在一个空泡（寄生虫空泡或PV）分裂成子细胞 裂殖子，并以裂殖子的快速释放结束，裂殖子侵入红细胞（RBC）以开始 循环往复。裂殖子的排出和侵入需要信号依赖的胞吐作用， 在出口过程中，被称为外丝的专门囊泡进入PV， 进入红细胞数据表明，细胞内钙振荡导致胞吐作用， 外丝体和棒状体的距离只有几分钟但目前尚不清楚， 外丝和棒状体区分两种细胞内钙振荡。很可能 负责胞吐作用的蛋白质对钙的敏感性不同，因此反应不同 在流出和侵入过程中变化的钙信号。然而，蛋白质定位于分泌型 信号依赖性胞吐作用所需的途径仍然是未知的。因此，我们采取了 生物信息学方法鉴定分泌途径中具有钙结合结构域的几种蛋白质 我们假设它在疟原虫外出和入侵期间的细胞器放电中起作用。这 的方法导致的钙结合蛋白（PfERC）的鉴定与一个重要的作用，在P。 恶性疟原虫出口基于这些已发表的数据，我们想测试PfERC是否在外显子中起作用 在活寄生虫中从未观察到胞吐作用，但在外出或侵入期间细胞器排出。 在第一个目标中，我们开发了基于荧光显微镜的测定来研究信号依赖性 外切丝胞吐作用，我们将开发分析，以评估钙振荡在出口的活P。 恶性疟原虫。在PfERC条件突变体中，这些活的显微镜测定显示PfERC敲低 抑制外切丝胞吐作用。使用定量蛋白质组学方法，我们鉴定了PfERC 相互作用，并为优先候选人生成条件突变体。另一个候选人， 蛋白11（RON 11）是裂殖子侵入所必需的。因此，在第二个目标中，我们将重点关注 P.并提出了恶性疟原虫入侵研究的发展方向，以供记者研究 棒状体放电以及活裂殖子在侵入过程中的钙振荡。使用这些测定， 我们将测试RON 11在棒状体放电、钙振荡以及裂殖子侵入中的功能 进入RBC。这项拟议中的研究将确定抗疟药物的几个假定靶点 发展由于疟原虫寄生虫的外出和入侵需要细胞器分泌 在其生命周期的所有阶段，这项研究可能导致确定泛活性抗疟药。

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.