An Endoplasmic Reticulum CREC Family Protein Regulates the Egress Proteolytic Cascade in Malaria Parasites Manuel A. Fierro, a,b Beejan Asady, a Carrie F. Brooks, a David W. Cobb, a,b Alejandra Villegas, a,b Silvia N. J. Moreno, a,b Vasant Muralidharan a,b a Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA b Department of Cellular Biology, University of Georgia, Athens, Georgia, USA ABSTRACT The endoplasmic reticulum (ER) is thought to play an essential role dur- ing egress of malaria parasites because the ER is assumed to be required for biogen- esis 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 es- sential for parasite egress but is not required for protein trafficking or calcium stor- age. We found that PfERC knockdown prevents the rupture of the parasitophorous vacuole membrane. This is because PfERC knockdown inhibited the proteolytic mat- uration of the subtilisin-like serine protease SUB1. Using double mutant parasites, we showed that PfERC is required for the proteolytic maturation of the essential as- partic protease plasmepsin X, which is required for SUB1 cleavage. Further, we showed that processing of substrates downstream of the proteolytic cascade is in- hibited 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 ma- laria parasites. IMPORTANCE The divergent eukaryotic parasites that cause malaria grow and di- vide 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 pro- teolytic 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 proteo- lytic 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 re- lease of daughter parasites. KEYWORDS calcium-binding protein, malaria, Plasmodium, egress, endoplasmic reticulum M embers of the phylum Apicomplexa are responsible for severe human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. Together, the members of this group of obligate intracellular parasites cause several hundred million infections every year and remain among the major drivers of infant mortality (1–4). In fact, malaria results in nearly half a million deaths each year and most of the mortality is attributed to one species, Plasmodium falciparum. All the clinical symptoms of malaria are directly Citation Fierro MA, Asady B, Brooks CF, Cobb DW, Villegas A, Moreno SNJ, Muralidharan V. 2020. An endoplasmic reticulum CREC family protein regulates the egress proteolytic cascade in malaria parasites. mBio 11:e03078-19. https:// doi.org/10.1128/mBio.03078-19. Invited Editor Vern B. Carruthers, University of Michigan Medical School Editor L. David Sibley, Washington University School of Medicine Copyright © 2020 Fierro et al. This is an open- access article distributed under the terms of the Creative Commons Attribution 4.0 International license. Address correspondence to Vasant Muralidharan, vasant@uga.edu. Received 26 November 2019 Accepted 17 January 2020 Published RESEARCH ARTICLE Host-Microbe Biology crossm January/February 2020 Volume 11 Issue 1 e03078-19 ® mbio.asm.org 1 25 February 2020 on July 24, 2020 by guest http://mbio.asm.org/ Downloaded from