Spatiotemporal dynamics of signal dependent exocytosis and parasitophorous vacuolar membrane rupture during Plasmodium falciparum egress

Malaria, caused by intracellular Plasmodium falciparum 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 live imaging to observe exocytosis. 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 fused to EXP2. 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 Plasmodium endoplasmic reticulum calcium-binding protein (PfERC), we demonstrate that loss 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.