Vesicle dynamics during the egress of malaria gametocytes from the red blood cell

疟疾寄生虫是专性细胞内病原体，在寄生液泡内的人红细胞中复制。虽然细胞内的区隔为寄生虫提供了庇护所，但它们需要离开红细胞，以继续生命周期进程，并确保人-蚊子传播。在蚊子中肠中，红细胞的分裂需要两个阶段，即红细胞分裂时形成的分裂子和配子体发生时形成的红细胞配子体。这两个阶段经过严格和精心安排的分子过程退出程序，最终导致包膜红细胞的裂解。退出程序遵循由内到外的模式，在此过程中，寄生物液泡膜在红细胞膜之前破裂，两种膜的破坏需要寄生物的专门分泌囊泡的胞吐。配子细胞表现出至少两种类型的出口囊泡，一种是参与破坏寄生液泡膜的亲渗小体，另一种是含有红细胞膜裂解所需的穿孔蛋白PPLP2的出口囊泡。在SPP 2225中，我们的目标是了解两种类型的出口囊泡在激活配子细胞的红细胞退出过程中的动力学。在spp2225的第一个资助期，我们重点研究了出口囊泡的特征，并表明这两种囊泡类型相互独立地作用，在pplp2阳性囊泡流出之前，嗜渗透体会分泌胞吐。我们进一步确定了这两种囊泡类型的蛋白质组，并鉴定了143种配子细胞出口囊泡蛋白，包括亲渗体的新标记物以及各种蛋白酶及其假定的底物。在SPP 2225的第二个资助期，我们的目标是通过表达分析和功能丧失表型结合超分辨率和透射电子显微镜对新鉴定的出口囊泡蛋白进行功能表征。一个特别的重点将在于潜在的蛋白水解激活这些蛋白通过出口相关的蛋白酶。此外，我们旨在揭示控制出口囊泡胞吐的分子机制。为此，我们将对在第一个资助期确定的推定的疟原虫v-SNARE进行功能表征，特别关注v-SNARE候选物Vti1。在第二步中，选定的v-SNAREs的相互作用组将通过BioID-MS进行分析，以确定囊泡融合机制的进一步组成部分。在该项目中获得的数据将提供疟疾配子体红细胞裂解机制的深入知识，并可能导致识别传播阻断干预的新靶点。

Malaria parasites are obligate intracellular pEathogens that replicate in human red blood cells harbored by a parasitophorous vacuole. While the intracellular compartmentalization provides shelter to the parasites, they need to exit from the red blood cell to continue life-cycle progression and ensure human-to-mosquito transmission. Two stages are required to exit the red blood cell, the merozoites that are formed during erythrocytic schizogony, and the intraerythrocytic gametocytes at the onset of gametogenesis in the mosquito midgut. The two stages pass a strict and orchestrated exit programme of molecular processes, which eventually results in lysis of the enveloping red blood cell. The exit programme follows an inside-out mode, during which the parasitophorous vacuole membrane ruptures prior to the red blood cell membrane, and destruction of both membranes requires the exocytosis of specialized secretory vesicles of the parasite. Gametocytes exhibit at least two types of egress vesicles, the osmiophilic bodies involved in rupturing the parasitophorous vacuole membrane, and egress vesicles that harbor the perforin-like protein PPLP2 required for lysis of the red blood cell membrane. Within the SPP 2225, it is our goal to understand the dynamics of the two types of egress vesicles during red blood cell exit by activated gametocytes. During the first funding period of the SPP 2225, we focused on the characterization of the egress vesicles and showed that the two vesicle types act independently from each other with exocytosis of the osmiophilic bodies preceding discharge of the PPLP2-positive vesicles. We further determined the proteomes of both vesicle types and identified 143 gametocyte egress vesicle proteins, including novel markers of osmiophilic bodies as well as various proteases and their putative substrates. During the second funding period of the SPP 2225, we aim at functionally characterizing the newly identified egress vesicle proteins by expression analysis and loss-of-function phenotyping in combination with super resolution and transmission electron microscopy. A special focus will lie on potential proteolytical activation of these proteins by egress-related proteases. Further, we aim at unveiling the molecular machinery governing exocytosis of the egress vesicles. For this, we will functionally characterize putative plasmodial v-SNAREs that have been identified during the first funding period with a special focus on the v-SNARE candidate Vti1. In a second step, the interactomes of selected v-SNAREs will be analyzed by BioID-MS to identify further components of the vesicle fusion machinery. Data gained during the project will provide in-depth knowledge of the mechanisms of red blood cell lysis by malaria gametocytes and may lead to the identification of novel targets for transmission-blocking interventions.