Dedkhad Watcharatip, Tran Tia, Fierro Manuel A, Brooks Carrie, Muralidharan Vasant
Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, GA 30602.
Department of Cellular Biology, University of Georgia, Athens, GA 30602.
bioRxiv. 2025 Aug 24:2025.08.20.671297. doi: 10.1101/2025.08.20.671297.
Malaria, caused by intracellular parasites, remains a major global health concern. These parasites reside and replicate within a vacuole in host red blood cells. Egress of daughter parasites out of the vacuolar and host membranes is tightly regulated via a complex mechanism. Prior studies have suggested that a cyclic-GMP driven calcium signaling pathway leads to the signal-dependent exocytosis of egress-specific vesicles that discharge several proteases into the parasitophorous vacuole. However, signal-dependent exocytosis during egress has not yet been observed in live parasitized RBCs. We targeted the exocytosis reporter, superecliptic pHlourin or SEP, to these egress-specific vesicles and utilized time-lapse imaging to observe exocytosis in live parasites. The spatiotemporal relationship between exocytosis and the breakdown of the parasitophorous vacuolar membrane (PVM) as well as parasite egress was also determined using a fluorescent reporter targeted to the PVM. Our data showed that exocytosis is triggered as early as 3 hours prior to merozoite egress. These data suggest that the PVM rupture occurs at a single site and rapidly expands from that initial site of rupture, releasing the merozoites into the RBC. This is followed by RBC membrane rupture and egress of merozoites. Using conditional mutants of the endoplasmic reticulum calcium-binding protein (PfERC), we demonstrate that knockdown of PfERC inhibits signal-dependent exocytosis of egress-specific vesicles. Together, these data demonstrate that signal-dependent exocytosis of egress-specific vesicles starts well before merozoites are formed via cytokinesis, PVM ruptures at a single site, and that PfERC is required for exocytosis of egress-specific vesicles.
由细胞内寄生虫引起的疟疾仍然是全球主要的健康问题。这些寄生虫在宿主红细胞的液泡内生存和繁殖。子代寄生虫从液泡膜和宿主细胞膜中逸出的过程受到复杂机制的严格调控。先前的研究表明，由环鸟苷酸驱动的钙信号通路会导致特定逸出囊泡的信号依赖性胞吐作用，这些囊泡会将几种蛋白酶释放到寄生泡中。然而，在活的被寄生红细胞中尚未观察到逸出过程中的信号依赖性胞吐作用。我们将胞吐作用报告分子超嗜碱pH荧光蛋白（SEP）靶向到这些特定逸出囊泡，并利用延时成像观察活寄生虫中的胞吐作用。还使用靶向寄生泡膜（PVM）的荧光报告分子确定了胞吐作用与寄生泡膜破裂以及寄生虫逸出之间的时空关系。我们的数据表明，胞吐作用早在裂殖子逸出前3小时就被触发。这些数据表明，PVM破裂发生在单个位点，并从该初始破裂位点迅速扩展，将裂殖子释放到红细胞中。随后是红细胞膜破裂和裂殖子逸出。利用内质网钙结合蛋白（PfERC）的条件突变体，我们证明敲低PfERC会抑制特定逸出囊泡的信号依赖性胞吐作用。总之，这些数据表明，特定逸出囊泡的信号依赖性胞吐作用在裂殖子通过胞质分裂形成之前就已经开始，PVM在单个位点破裂，并且PfERC是特定逸出囊泡胞吐作用所必需的。
