Export of parasite proteins into the host erythrocyte is essential for survival of Plasmodium falciparum during its asexual life cycle. While several studies described key factors within the parasite that are involved in protein export, the mechanisms employed to traffic exported proteins within the host cell are currently unknown. Members of the Hsp70 family of chaperones, together with their Hsp40 cochaperones, facilitate protein trafficking in other organisms, and are thus likely used by P. falciparum in the trafficking of its exported proteins. A large group of Hsp40 proteins is encoded by the parasite and exported to the host cell, but only one Hsp70, P. falciparum Hsp70x (PfHsp70x), is exported with them. PfHsp70x is absent in most Plasmodium species and is found only in P. falciparum and closely related species that infect apes. Herein, we have utilized clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome editing in P. falciparum to investigate the essentiality of PfHsp70x. We show that parasitic growth was unaffected by knockdown of PfHsp70x using both the dihydrofolate reductase (DHFR)-based destabilization domain and the glmS ribozyme system. Similarly, a complete gene knockout of PfHsp70x did not affect the ability of P. falciparum to proceed through its intraerythrocytic life cycle. The effect of PfHsp70x knockdown/knockout on the export of proteins to the host red blood cell (RBC), including the critical virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1), was tested, and we found that this process was unaffected. These data show that although PfHsp70x is the sole exported Hsp70, it is not essential for the asexual development of P. falciparum. IMPORTANCE Half of the world's population lives at risk for malaria. The intraerythrocytic life cycle of Plasmodium spp. is responsible for clinical manifestations of malaria; therefore, knowledge of the parasite's ability to survive within the erythrocyte is needed to combat the deadliest agent of malaria, P. falciparum. An outstanding question in the field is how P. falciparum undertakes the essential process of trafficking its proteins within the host cell. In most organisms, chaperones such as Hsp70 are employed in protein trafficking. Of the Plasmodium species causing human disease, the chaperone PfHsp70x is unique to P. falciparum, and it is the only parasite protein of its kind exported to the host (S. Külzer et al., Cell Microbiol 14:1784-1795, 2012). This has placed PfHsp70x as an ideal target to inhibit protein trafficking and kill the parasite. However, we show that PfHsp70x is not required for export of parasite effectors and it is not essential for parasite survival inside the RBC.

To mediate its survival and virulence, the malaria parasite Plasmodium falciparum exports hundreds of proteins into the host erythrocyte. To enter the host cell, exported proteins must cross the parasitophorous vacuolar membrane (PVM) within which the parasite resides, but the mechanism remains unclear. A putative Plasmodium translocon of exported proteins (PTEX) has been suggested to be involved for at least one class of exported proteins; however, direct functional evidence for this has been elusive. Here we show that export across the PVM requires heat shock protein 101 (HSP101), a ClpB-like AAA+ ATPase component of PTEX. Using a chaperone auto-inhibition strategy, we achieved rapid, reversible ablation of HSP101 function, resulting in a nearly complete block in export with substrates accumulating in the vacuole in both asexual and sexual parasites. Surprisingly, this block extended to all classes of exported proteins, revealing HSP101-dependent translocation across the PVM as a convergent step in the multi-pathway export process. Under export-blocked conditions, association between HSP101 and other components of the PTEX complex was lost, indicating that the integrity of the complex is required for efficient protein export. Our results demonstrate an essential and universal role for HSP101 in protein export and provide strong evidence for PTEX function in protein translocation into the host cell.

The cis-polyisoprenoid lipids namely polyprenols, dolichols and their derivatives are linear polymers of several isoprene units. In eukaryotes, polyprenols and dolichols are synthesized as a mixture of four or more homologues of different length with one or two predominant species with sizes varying among organisms. Interestingly, co-occurrence of polyprenols and dolichols, i.e. detection of a dolichol along with significant levels of its precursor polyprenol, are unusual in eukaryotic cells. Our metabolomics studies revealed that cis-polyisoprenoids are more diverse in the malaria parasite Plasmodium falciparum than previously postulated as we uncovered active de novo biosynthesis and substantial levels of accumulation of polyprenols and dolichols of 15 to 19 isoprene units. A distinctive polyprenol and dolichol profile both within the intraerythrocytic asexual cycle and between asexual and gametocyte stages was observed suggesting that cis-polyisoprenoid biosynthesis changes throughout parasite's development. Moreover, we confirmed the presence of an active cis-prenyltransferase (PfCPT) and that dolichol biosynthesis occurs via reduction of the polyprenol to dolichol by an active polyprenol reductase (PfPPRD) in the malaria parasite.

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 falciparumendoplasmic 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.

The deadly malaria parasite Plasmodium falciparum contains a nonphotosynthetic plastid, known as the apicoplast, that functions to produce essential metabolites, and drugs that target the apicoplast are clinically effective. Several prokaryotic caseinolytic protease (Clp) genes have been identified in the Plasmodium genome. Using phylogenetic analysis, we focused on the Clp members that may form a regulated proteolytic complex in the apicoplast. We genetically targeted members of this complex and generated conditional mutants of the apicoplast-localized PfClpC chaperone and PfClpP protease. Conditional inhibition of the PfClpC chaperone resulted in growth arrest and apicoplast loss and was rescued by addition of the essential apicoplast-derived metabolite IPP. Using a double-conditional mutant parasite line, we discovered that the chaperone activity is required to stabilize the mature protease, revealing functional interactions. These data demonstrate the essential function of PfClpC in maintaining apicoplast integrity and its role in regulating the proteolytic activity of the Clp complex.

The vast majority of malaria mortality is attributed to one parasite species: Plasmodium falciparum. Asexual replication of the parasite within the red blood cell is responsible for the pathology of the disease. In Plasmodium, the endoplasmic reticulum (ER) is a central hub for protein folding and trafficking as well as stress response pathways. In this study, we tested the role of an uncharacterised ER protein, PfGRP170, in regulating these key functions by generating conditional mutants. Our data show that PfGRP170 localises to the ER and is essential for asexual growth, specifically required for proper development of schizonts. PfGRP170 is essential for surviving heat shock, suggesting a critical role in cellular stress response. The data demonstrate that PfGRP170 interacts with the Plasmodium orthologue of the ER chaperone, BiP. Finally, we found that loss of PfGRP170 function leads to the activation of the Plasmodium eIF2α kinase, PK4, suggesting a specific role for this protein in this parasite stress response pathway.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBC) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs, begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from two specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains seven transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only one rhoptry each. The single rhoptry in RON11 deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11 deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11 deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

Malaria remains a major global health problem, creating a constant need for research to identify druggable weaknesses in P. falciparum biology. As important components of cellular redox biology, members of the Thioredoxin (Trx) superfamily of proteins have received interest as potential drug targets in Apicomplexans. However, the function and essentiality of endoplasmic reticulum (ER)-localized Trx-domain proteins within P. falciparum has not been investigated. We generated conditional mutants of the protein PfJ2-an ER chaperone and member of the Trx superfamily-and show that it is essential for asexual parasite survival. Using a crosslinker specific for redox-active cysteines, we identified PfJ2 substrates as PfPDI8 and PfPDI11, both members of the Trx superfamily as well, which suggests a redox-regulatory role for PfJ2. Knockdown of these PDIs in PfJ2 conditional mutants show that PfPDI11 may not be essential. However, PfPDI8 is required for asexual growth and our data suggest it may work in a complex with PfJ2 and other ER chaperones. Finally, we show that the redox interactions between these Trx-domain proteins in the parasite ER and their substrates are sensitive to small molecule inhibition. Together these data build a model for how Trx-domain proteins in the P. falciparum ER work together to assist protein folding and demonstrate the suitability of ER-localized Trx-domain proteins for antimalarial drug development.

The malaria-causing parasite, Plasmodium falciparum completely remodels its host red blood cell (RBC) through the export of several hundred parasite proteins, including transmembrane proteins, across multiple membranes to the RBC. However, the process by which these exported membrane proteins are extracted from the parasite plasma membrane for export remains unknown. To address this question, we fused the exported membrane protein, skeleton binding protein 1 (SBP1), with TurboID, a rapid, efficient and promiscuous biotin ligase (SBP1TbID). Using time-resolved proximity biotinylation and label-free quantitative proteomics, we identified two groups of SBP1TbID interactors - early interactors (pre-export) and late interactors (post-export). Notably, two promising membrane-associated proteins were identified as pre-export interactors, one of which possesses a predicted translocon domain, that could facilitate the export of membrane proteins. Further investigation using conditional mutants of these candidate proteins showed that these proteins were essential for asexual growth and localize to the host-parasite interface during early stages of the intraerythrocytic cycle. These data suggest that they might play a role in ushering membrane proteins from the parasite plasma membrane for export to the host RBC.