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

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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.