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，在人体红细胞中复制，并由寄生虫空泡窝藏。虽然细胞内的区室化为寄生虫提供了庇护，但它们需要离开红细胞以继续生命周期的进展并确保人-蚊传播。蚊子中肠的红细胞需要经过两个阶段才能从红细胞中排出，即红细胞内配子发生时形成的裂殖子和红细胞内配子母细胞。这两个阶段通过一个严格和精心策划的退出程序的分子过程，最终导致溶解的包膜红细胞。退出程序如下内-外模式，在此期间，寄生虫空泡膜破裂之前的红细胞膜，和破坏两个膜需要专门的分泌囊泡的寄生虫胞吐作用。配子体表现出至少两种类型的出口囊泡，嗜锇小体参与破坏寄生虫空泡膜，和出口囊泡，港口穿孔素样蛋白PPLP2所需的红细胞膜裂解。在SPP 2225中，我们的目标是了解激活的配子母细胞在红细胞退出期间两种类型的出口囊泡的动力学。在SPP 2225的第一个资助期内，我们重点研究了外出囊泡的特征，并表明两种囊泡类型相互独立地起作用，在PPLP2阳性囊泡排出之前嗜锇小体的胞吐作用。我们进一步确定了这两种囊泡类型的蛋白质组，并确定了143配子体出口囊泡蛋白，包括新的嗜锇小体标记以及各种蛋白酶及其推定的底物。在SPP 2225的第二个资助期内，我们的目标是通过表达分析和功能丧失表型分析，结合超分辨率和透射电子显微镜，对新发现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质的出口相关的蛋白酶。此外，我们的目标是揭示分子机制的出口囊泡胞吐。为此，我们将在功能上表征在第一个资助期内已经确定的假定疟原虫v-SNARE，特别关注v-SNARE候选Vti1。在第二步中，将通过BioID-MS分析选定的v-SNARE的相互作用组，以鉴定囊泡融合机制的其他组分。项目期间获得的数据将提供关于疟疾配子体溶解红细胞机制的深入知识，并可能导致确定阻断传播干预措施的新目标。

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.