An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites

Abstract

The endoplasmic reticulum (ER) is thought to play an essential role during egress of malaria parasites because the ER is assumed to be required for biogenesis and secretion of egress-related organelles. However, no proteins localized to the parasite ER have been shown to play a role in egress of malaria parasites. In this study, we generated conditional mutants of the Plasmodium falciparum endoplasmic reticulum-resident calcium-binding protein (PfERC), a member of the CREC family. Knockdown of the PfERC gene showed that this gene is essential for asexual growth of P. falciparum. Analysis of the intraerythrocytic life cycle revealed that PfERC is essential for parasite egress but is not required for protein trafficking or calcium storage. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic maturation of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential aspartic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is inhibited by PfERC knockdown. Thus, these data establish that the ER-resident CREC family protein PfERC is a key early regulator of the egress proteolytic cascade of malaria parasites. IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and divide within a vacuole inside a host cell, which they have to break open once they finish cell division. The egress of daughter parasites requires the activation of a proteolytic cascade, and a subtilisin-like protease initiates a proteolytic cascade to break down the membranes blocking egress. It is assumed that the parasite endoplasmic reticulum plays a role in this process, but the proteins in this organelle required for egress remain unknown. We have identified an early ER-resident regulator essential for the maturation of the recently discovered aspartic protease in the egress proteolytic cascade, plasmepsin X, which is required for maturation of the subtilisin-like protease. Conditional loss of PfERC results in the formation of immature and inactive egress proteases that are unable to breakdown the vacuolar membrane barring release of daughter parasites.

A redox-active crosslinker reveals an essential and inhibitable oxidative folding network in the endoplasmic reticulum of malaria parasites

“…Anti-HA immunoprecipitation (IP) assays were performed as previously described, using anti-HA magnetic beads (Pierce) [ 56 ]. Anti-V5 IP assays were performed in the same manner as with anti-HA, but anti-V5 magnetic beads were used according to manufacturer instructions (MBL International Corporation).…”

Malaria parasite plasmepsins: More than just plain old degradative pepsins

“…PM X is autocatalytic, but in parasites not all of the PM X gets processed; they retain some as proenzyme (66 kDa) or as active 51-and 44-kDa versions (156 -158). An ER calciumbinding protein called PfERC is required for PM X maturation in an unknown fashion (161).…”

Stochastic Expression of Invasion Genes in Plasmodium falciparum Schizonts

“…In addition to invasion genes, we also discovered variation in a wide range of other (non-invasion) genes, such as, Pferc, cept, aquaglyceroporin, AP2 transcription factor and thioredoxin reductase involved in a variety of functions such as, parasite egress 96 , nutrient/protein channel, lipid metabolism, glycerol transport, transcription regulation and redox homeostasis respectively. This suggests the presence of unique 'transient' transcriptional states of individual parasites in a population, some of which may be suitable in defence to antagonists or coping to uctuating environments; a strategy known as bet hedging.…”