Ca²⁺-mediated exocytosis of subtilisin-like protease 1: a key step in egress ofPlasmodium falciparummerozoites

Abstract

SummaryEgress of Plasmodium falciparum merozoites from host erythrocytes is a critical step in multiplication of blood-stage parasites. A cascade of proteolytic events plays a major role in degradation of membranes leading to egress of merozoites. However, the signals that regulate the temporal activation and/or secretion of proteases upon maturation of merozoites in intra-erythrocytic schizonts remain unclear. Here, we have tested the role of intracellular Ca 2+ in regulation of egress of P. falciparum merozoites from schizonts. A sharp rise in intracellular Ca 2+ just before egress, observed by timelapse video microscopy, suggested a role for intracellular Ca 2+ in this process. Chelation of intracellular Ca 2+ with chelators such as BAPTA-AM or inhibition of Ca 2+ release from intracellular stores with a phospholipase C (PLC) inhibitor blocks merozoite egress. Interestingly, chelation of intracellular Ca 2+ in schizonts was also found to block the discharge of a key protease PfSUB1 (subtilisin-like protease 1) from exonemes of P. falciparum merozoites to parasitophorous vacuole (PV). This leads to inhibition of processing of PfSERA5 (serine repeat antigen 5) and a block in parasitophorous vacuolar membrane (PVM) rupture and merozoite egress. A complete understanding of the steps regulating egress of P. falciparum merozoites may provide novel targets for development of drugs that block egress and limit parasite growth.

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…Calcium ions are ubiquitous and versatile signalling molecules that play pleiotropic roles in the complex cellular organization and life cycle [38][39][40][41][42] , including during the egress from and the subsequent invasion of host cells by Apicomplexan parasites 25,[43][44][45] . The secretion route of eukaryotic cells has a gradient of Ca 2 þ concentration ([Ca 2 þ ]), being high in the ER (up to 400 mM), decreasing through the Golgi apparatus (250-130 mM) to reach values lower than 100 mM within secretory vesicles 38 .…”

“…SUB1 activation needs a shift in [Ca 2 þ ] to dissociate the stable non-covalent complex formed between the pro-region and the catalytic domain. Such a cGMP-mediated drastic shift in [Ca 2 þ ] has recently been shown to immediately precede the egress of Plasmodium Mz and to trigger SUB1 secretion into the lumen of the PV 25,48 . It is tempting to propose a model (Fig.…”

“…It is tempting to propose a model (Fig. 6) in which the sharp rise in [Ca 2 þ ] not only promotes SUB1 secretion from the exonemes into the PV 25 , but also triggers SUB1 prodomain release and degradation via the cleavage of the belt loop at the a/b-sites, promoting full activation of the secreted catalytic domain. Thus, compared with other known subtilases, the SUB1 belt domain adds an additional level of spatio-temporal regulation to this critical Plasmodium protease.…”

“…Egress of Plasmodium Mz from infected erythrocytes is a precisely regulated proteasedependent multi-step process that requires rupture of the parasitophorous vacuole (PV) membrane followed by rupture of the red cell membrane [22][23][24] . Mz egress is preceded by a sharp rise of intracellular Ca 2 þ that triggers the discharge of the exoneme content in the PV 25 , including an active form of SUB1. Subsequently, SUB1 matures the serine-rich repeat antigens (SERAs) 11 , a serine and papain-like family of proteases that participates in the rupture of the PV and infected RBC membranes 26 .…”

A Key Role for Plasmodium Subtilisin-like SUB1 Protease in Egress of Malaria Parasites from Host Hepatocytes

“…active PfSUB1 from merozoite exonemes into the PV (41,42). It is tempting to propose that, together with SUB1/SERA, the parasite PKG composes a multipartner-regulated pathway that plays equivalent roles in Plasmodium egress from hepatocytes and red blood cells.…”