Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

Sequence elements within the PEXEL motif and its downstream region modulate PTEX-dependent protein export in Plasmodium falciparum.

The parasite Plasmodium falciparum causes the most severe form of malaria and to invade and replicate in red blood cells (RBCs), it exports hundreds of proteins across the encasing parasitophorous vacuole membrane (PVM) into this host cell. The exported proteins help modify the RBC to support rapid parasite growth and avoidance of the human immune system. Most exported proteins possess a conserved Plasmodium export element (PEXEL) motif with the consensus RxLxE/D/Q amino acid sequence, which acts as a proteolytic cleavage recognition site within the parasite's endoplasmic reticulum (ER). Cleavage occurs after the P1 L residue and is thought to help release the protein from the ER so it can be putatively escorted by the HSP101 chaperone to the parasitophorous vacuole space surrounding the intraerythrocytic parasite. HSP101 and its cargo are then thought to assemble with the rest of a Plasmodium translocon for exported proteins (PTEX) complex, that then recognises the xE/D/Q capped N-terminus of the exported protein and translocates it across the vacuole membrane into the RBC compartment. Here, we present evidence that supports a dual role for the PEXEL's conserved P2 ' position E/Q/D residue, first, for plasmepsin V cleavage in the ER, and second, for efficient PTEX mediated export across the PVM into the RBC. We also present evidence that the downstream 'spacer' region separating the PEXEL motif from the folded functional region of the exported protein controls cargo interaction with PTEX as well. The spacer must be of a sufficient length and permissive amino acid composition to engage the HSP101 unfoldase component of PTEX to be efficiently translocated into the RBC compartment.

Bottoms up! Malaria parasite invasion the right way around.

A critical part of the malaria parasite's life cycle is invasion of red blood cells (RBCs) by merozoites. Inside RBCs, the parasite forms a schizont, which undergoes segmentation to produce daughter merozoites. These cells are released, establishing cycles of invasion. Traditionally, merozoites are represented as nonmotile, egg-shaped cells that invade RBCs 'narrower end' first and pack within schizonts with this narrower end facing outwards. Here, we discuss recent evidence and re-evaluate previous data which suggest that merozoites are capable of motility and have spherical or elongated-teardrop shapes. Furthermore, merozoites invade RBCs 'wider end' first and pack within schizonts with this wider end facing outwards. We encourage the field to review this revised model and consider its implications for future studies.

The dual action of human antibodies specific to Plasmodium falciparum PfRH5 and PfCyRPA: Blocking invasion and inactivating extracellular merozoites.

The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) is the current leading blood-stage malaria vaccine candidate. PfRH5 functions as part of the pentameric PCRCR complex containing PTRAMP, CSS, PfCyRPA and PfRIPR, all of which are essential for infection of human red blood cells (RBCs). To trigger RBC invasion, PfRH5 engages with RBC protein basigin in a step termed the RH5-basigin binding stage. Although we know increasingly more about how antibodies specific for PfRH5 can block invasion, much less is known about how antibodies recognizing other members of the PCRCR complex can inhibit invasion. To address this, we performed live cell imaging using monoclonal antibodies (mAbs) which bind PfRH5 and PfCyRPA. We measured the degree and timing of the invasion inhibition, the stage at which it occurred, as well as subsequent events. We show that parasite invasion is blocked by individual mAbs, and the degree of inhibition is enhanced when combining a mAb specific for PfRH5 with one binding PfCyRPA. In addition to directly establishing the invasion-blocking capacity of the mAbs, we identified a secondary action of certain mAbs on extracellular parasites that had not yet invaded where the mAbs appeared to inactivate the parasites by triggering a developmental pathway normally only seen after successful invasion. These findings suggest that epitopes within the PfCyRPA-PfRH5 sub-complex that elicit these dual responses may be more effective immunogens than neighboring epitopes by both blocking parasites from invading and rapidly inactivating extracellular parasites. These two protective mechanisms, prevention of invasion and inactivation of uninvaded parasites, resulting from antibody to a single epitope indicate a possible route to the development of more effective vaccines.

Conditional expression of NanoLuc luciferase through a multimodular system offers rapid detection of antimalarial drug activity.

Conditional gene expression is a powerful tool to investigate putative vaccine and drug targets, especially in a haploid organism such as Plasmodium falciparum. Inducible systems based on regulation of either transcription, translation, protein or mRNA stability, among others, allow switching on an off the expression of any desired gene causing specific gain or loss of function phenotypes. However, those systems can be cumbersome involving the construction of large plasmids and generation of multiple transgenic parasite lines. In addition, the dynamic range of regulation achieved is not predictable for each individual gene and can be insufficient to generate detectable phenotypes when the genes of interest are silenced. Here, we combined up to three distinct inducible systems to regulate the expression of a single gene. Expression of the reporter NanoLuc luciferase was regulated over 40-fold, which correlates to the regulation achieved by each individual system multiplied by each other. We applied the conditionally expressed NanoLuc to evaluate the effect of fast-acting antimalarials such as chloroquine and artesunate as well as of slower-acting ones such as atovaquone. The conditionally expressed reporter allowed faster and more reliable detection of toxicity to the parasite, which correlated to the expected action of each compound. Bioluminescence achieved by the expression of this inducible highly sensitive reporter is therefore a promising tool to investigate the temporal effect of potential new antimalarials. This single plasmid combination system might also prove useful to achieve sufficient regulation of genes of interest to produce loss-of-function phenotypes.

A Pyridyl-Furan Series Developed from the Open Global Health Library Block Red Blood Cell Invasion and Protein Trafficking in Plasmodium falciparum through Potential Inhibition of the Parasite's PI4KIIIB Enzyme.

With the resistance increasing to current antimalarial medicines, there is an urgent need to discover new drug targets and to develop new medicines against these targets. We therefore screened the Open Global Health Library of Merck KGaA, Darmstadt, Germany, of 250 compounds against the asexual blood stage of the deadliest malarial parasite Plasmodium falciparum, from which eight inhibitors with low micromolar potency were found. Due to its combined potencies against parasite growth and inhibition of red blood cell invasion, the pyridyl-furan compound OGHL250 was prioritized for further optimization. The potency of the series lead compound (WEHI-518) was improved 250-fold to low nanomolar levels against parasite blood-stage growth. Parasites selected for resistance to a related compound, MMV396797, were also resistant to WEHI-518 as well as KDU731, an inhibitor of the phosphatidylinositol kinase PfPI4KIIIB, suggesting that this kinase is the target of the pyridyl-furan series. Inhibition of PfPI4KIIIB blocks multiple stages of the parasite's life cycle and other potent inhibitors are currently under preclinical development. MMV396797-resistant parasites possess an E1316D mutation in PfPKI4IIIB that clusters with known resistance mutations of other inhibitors of the kinase. Building upon earlier studies that showed that PfPI4KIIIB inhibitors block the development of the invasive merozoite parasite stage, we show that members of the pyridyl-furan series also block invasion and/or the conversion of merozoites into ring-stage intracellular parasites through inhibition of protein secretion and export into red blood cells.

The delayed bloodstream clearance of Plasmodium falciparum parasites after M5717 treatment is attributable to the inability to modify their red blood cell hosts.

M5717 is a promising antimalarial drug under development that acts against multiple stages of the life cycle of Plasmodium parasites by inhibiting the translation elongation factor 2 (PfeEF2), thereby preventing protein synthesis. The parasite clearance profile after drug treatment in preclinical studies in mice, and clinical trials in humans showed a notable delayed clearance phenotype whereby parasite infected red blood cells (iRBCs) persisted in the bloodstream for a significant period before eventual clearance. In a normal P. falciparum infection iRBCs sequester in the deep circulation by cytoadherence, allowing them to avoid surveillance and clearance in the spleen. We found that M5717 blocks parasite modification of their host red blood cells (RBCs) by preventing synthesis of new exported proteins, rather than by directly blocking the export of these proteins into the RBC compartment. Using in vitro models, we demonstrated that M5717 treated ring/trophozoite stage iRBCs became less rigid, and cytoadhered less well compared to untreated iRBCs. This indicates that in vivo persistence of M5717 treated iRBCs in the bloodstream is likely due to reduced cytoadherence and splenic clearance.

PTEX helps efficiently traffic haemoglobinases to the food vacuole in Plasmodium falciparum.

A key element of Plasmodium biology and pathogenesis is the trafficking of ~10% of the parasite proteome into the host red blood cell (RBC) it infects. To cross the parasite-encasing parasitophorous vacuole membrane, exported proteins utilise a channel-forming protein complex termed the Plasmodium translocon of exported proteins (PTEX). PTEX is obligatory for parasite survival, both in vitro and in vivo, suggesting that at least some exported proteins have essential metabolic functions. However, to date only one essential PTEX-dependent process, the new permeability pathways, has been described. To identify other essential PTEX-dependant proteins/processes, we conditionally knocked down the expression of one of its core components, PTEX150, and examined which pathways were affected. Surprisingly, the food vacuole mediated process of haemoglobin (Hb) digestion was substantially perturbed by PTEX150 knockdown. Using a range of transgenic parasite lines and approaches, we show that two major Hb proteases; falcipain 2a and plasmepsin II, interact with PTEX core components, implicating the translocon in the trafficking of Hb proteases. We propose a model where these proteases are translocated into the PV via PTEX in order to reach the cytostome, located at the parasite periphery, prior to food vacuole entry. This work offers a second mechanistic explanation for why PTEX function is essential for growth of the parasite within its host RBC.

Sulfonylpiperazine compounds prevent Plasmodium falciparum invasion of red blood cells through interference with actin-1/profilin dynamics.

With emerging resistance to frontline treatments, it is vital that new antimalarial drugs are identified to target Plasmodium falciparum. We have recently described a compound, MMV020291, as a specific inhibitor of red blood cell (RBC) invasion, and have generated analogues with improved potency. Here, we generated resistance to MMV020291 and performed whole genome sequencing of 3 MMV020291-resistant populations. This revealed 3 nonsynonymous single nucleotide polymorphisms in 2 genes; 2 in profilin (N154Y, K124N) and a third one in actin-1 (M356L). Using CRISPR-Cas9, we engineered these mutations into wild-type parasites, which rendered them resistant to MMV020291. We demonstrate that MMV020291 reduces actin polymerisation that is required by the merozoite stage parasites to invade RBCs. Additionally, the series inhibits the actin-1-dependent process of apicoplast segregation, leading to a delayed death phenotype. In vitro cosedimentation experiments using recombinant P. falciparum proteins indicate that potent MMV020291 analogues disrupt the formation of filamentous actin in the presence of profilin. Altogether, this study identifies the first compound series interfering with the actin-1/profilin interaction in P. falciparum and paves the way for future antimalarial development against the highly dynamic process of actin polymerisation.

Defining species-specific and conserved interactions of apical membrane protein 1 during erythrocyte invasion in malaria to inform multi-species vaccines.

Plasmodium falciparum and P. vivax are the major causes of human malaria, and P. knowlesi is an important additional cause in SE Asia. Binding of apical membrane antigen 1 (AMA1) to rhoptry neck protein 2 (RON2) was thought to be essential for merozoite invasion of erythrocytes by Plasmodium spp. Our findings reveal that P. falciparum and P. vivax have diverged and show species-specific binding of AMA1 to RON2, determined by a ß-hairpin loop in RON2 and specific residues in AMA1 Loop1E. In contrast, cross-species binding of AMA1 to RON2 is retained between P. vivax and P. knowlesi. Mutation of specific amino acids in AMA1 Loop1E in P. falciparum or P. vivax ablated RON2 binding without impacting erythrocyte invasion. This indicates that the AMA1-RON2-loop interaction is not essential for invasion and additional AMA1 interactions are involved. Mutations in AMA1 that disrupt RON2 binding also enable escape of invasion inhibitory antibodies. Therefore, vaccines and therapeutics will need to be broader than targeting only the AMA1-RON2 interaction. Antibodies targeting AMA1 domain 3 had greater invasion-inhibitory activity when RON2-loop binding was ablated, suggesting this domain is a promising additional target for vaccine development. Targeting multiple AMA1 interactions involved in invasion may enable vaccines that generate more potent inhibitory antibodies and address the capacity for immune evasion. Findings on specific residues for invasion function and species divergence and conservation can inform novel vaccines and therapeutics against malaria caused by three species, including the potential for cross-species vaccines.

Characterisation of PfCZIF1 and PfCZIF2 in Plasmodium falciparum asexual stages.

Plasmodium falciparum exerts strong temporal control of gene expression across its lifecycle. Proteins expressed exclusively during late schizogony of blood stages, for example, often have a role in facilitating merozoite invasion of the host red blood cell (RBC), through merozoite development, egress, invasion or early establishment of infection in the RBC. Here, we characterise P. falciparum C3H1 zinc finger 1 (PfCZIF1, Pf3D7_1468400) and P. falciparum C3H1 zinc finger 2 (PfCZIF2, Pf3D7_0818100) which we identified as the only C3H1-type zinc finger proteins with peak expression at schizogony. Previous studies reported that antibodies against PfCZIF1 inhibit merozoite invasion, suggesting this protein may have a potential role during RBC invasion. We show using C-terminal truncations and gene knockouts of each of Pfczif1 and Pfczif2 that neither are essential for blood stage growth. However, they could not both be knocked out simultaneously, suggesting that at least one is needed for parasite growth in vitro. Immunofluorescence localisation of PfCZIF1 and PfCZIF2 indicated that both proteins occur in discrete foci on the periphery of the parasite's cytosol and biochemical assays suggest they are peripherally associated to a membrane. Transcriptomic analyses for the C-terminal truncation mutants reveal no significant expression perturbations with PfCZIF1 truncation. However, modification of PfCZIF2 appears to modify the expression for some exported proteins including PfKAHRP. This study does not support a role for PfCZIF1 or PfCZIF2 in merozoite invasion of the RBC and suggests that these proteins may help regulate the expression of proteins exported into the RBC cytosol after merozoite invasion.

Dissecting EXP2 sequence requirements for protein export in malaria parasites.

Apicomplexan parasites that reside within a parasitophorous vacuole harbor a conserved pore-forming protein that enables small-molecule transfer across the parasitophorous vacuole membrane (PVM). In Plasmodium parasites that cause malaria, this nutrient pore is formed by EXP2 which can complement the function of GRA17, an orthologous protein in Toxoplasma gondii. EXP2, however, has an additional function in Plasmodium parasites, serving also as the pore-forming component of the protein export machinery PTEX. To examine how EXP2 can play this additional role, transgenes that encoded truncations of EXP2, GRA17, hybrid GRA17-EXP2, or EXP2 under the transcriptional control of different promoters were expressed in EXP2 knockdown parasites to determine which could complement EXP2 function. This revealed that EXP2 is a unique pore-forming protein, and its protein export role in P. falciparum cannot be complemented by T. gondii GRA17. This was despite the addition of the EXP2 assembly strand and part of the linker helix to GRA17, which are regions necessary for the interaction of EXP2 with the other core PTEX components. This indicates that the body region of EXP2 plays a critical role in PTEX assembly and/or that the absence of other T. gondii GRA proteins in P. falciparum leads to its reduced efficiency of insertion into the PVM and complementation potential. Altering the timing and abundance of EXP2 expression did not affect protein export but affected parasite viability, indicating that the unique transcriptional profile of EXP2 when compared to other PTEX components enables it to serve an additional role in nutrient exchange.

PfATP4 inhibitors in the Medicines for Malaria Venture Malaria Box and Pathogen Box block the schizont-to-ring transition by inhibiting egress rather than invasion.

The cation efflux pump Plasmodium falciparum ATPase 4 (PfATP4) maintains Na+ homeostasis in malaria parasites and has been implicated in the mechanism of action of many structurally diverse antimalarial agents, including >7% of the antimalarial compounds in the Medicines for Malaria Venture's 'Malaria Box' and 'Pathogen Box'. Recent screens of the 'Malaria Box' and 'Pathogen Box' revealed that many PfATP4 inhibitors prevent parasites from exiting their host red blood cell (egress) or entering new host cells (invasion), suggesting that these compounds may have additional molecular targets involved in egress or invasion. Here, we demonstrate that five PfATP4 inhibitors reduce egress but not invasion. These compounds appear to inhibit egress by blocking the activation of protein kinase G, an enzyme that, once stimulated, rapidly activates parasite egress. We establish a direct link between egress and PfATP4 function by showing that the inhibition of egress is attenuated in a Na+-depleted environment and in parasites with a mutation in pfatp4. Finally, we show that PfATP4 inhibitors induce host cell lysis when administered prior to the completion of parasite replication. Since host cell lysis mimics egress but is not followed by invasion, this phenomenon likely explains why several PfATP4 inhibitors were previously classified as invasion inhibitors. Collectively, our results confirm that PfATP4-mediated Na+ efflux is critical to the regulation of parasite egress.

The Medicines for Malaria Venture Malaria Box contains inhibitors of protein secretion in Plasmodium falciparum blood stage parasites.

Plasmodium falciparum parasites which cause malaria, traffic hundreds of proteins into the red blood cells (RBCs) they infect. These exported proteins remodel their RBCs enabling host immune evasion through processes such as cytoadherence that greatly assist parasite survival. As resistance to all current antimalarial compounds is rising new compounds need to be identified and those that could inhibit parasite protein secretion and export would both rapidly reduce parasite virulence and ultimately lead to parasite death. To identify compounds that inhibit protein export we used transgenic parasites expressing an exported nanoluciferase reporter to screen the Medicines for Malaria Venture Malaria Box of 400 antimalarial compounds with mostly unknown targets. The most potent inhibitor identified in this screen was MMV396797 whose application led to export inhibition of both the reporter and endogenous exported proteins. MMV396797 mediated blockage of protein export and slowed the rigidification and cytoadherence of infected RBCs-modifications which are both mediated by parasite-derived exported proteins. Overall, we have identified a new protein export inhibitor in P. falciparum whose target though unknown, could be developed into a future antimalarial that rapidly inhibits parasite virulence before eliminating parasites from the host.

The Plasmodium falciparum parasitophorous vacuole protein P113 interacts with the parasite protein export machinery and maintains normal vacuole architecture.

Infection with Plasmodium falciparum parasites results in approximately 627,000 deaths from malaria annually. Key to the parasite's success is their ability to invade and subsequently grow within human erythrocytes. Parasite proteins involved in parasite invasion and proliferation are therefore intrinsically of great interest, as targeting these proteins could provide novel means of therapeutic intervention. One such protein is P113 which has been reported to be both an invasion protein and an intracellular protein located within the parasitophorous vacuole (PV). The PV is delimited by a membrane (PVM) across which a plethora of parasite-specific proteins are exported via the Plasmodium Translocon of Exported proteins (PTEX) into the erythrocyte to enact various immune evasion functions. To better understand the role of P113 we isolated its binding partners from in vitro cultures of P. falciparum. We detected interactions with the protein export machinery (PTEX and exported protein-interacting complex) and a variety of proteins that either transit through the PV or reside on the parasite plasma membrane. Genetic knockdown or partial deletion of P113 did not significantly reduce parasite growth or protein export but did disrupt the morphology of the PVM, suggesting that P113 may play a role in maintaining normal PVM architecture.

A revised mechanism for how Plasmodium falciparum recruits and exports proteins into its erythrocytic host cell.

Plasmodium falciparum exports ~10% of its proteome into its host erythrocyte to modify the host cell's physiology. The Plasmodium export element (PEXEL) motif contained within the N-terminus of most exported proteins directs the trafficking of those proteins into the erythrocyte. To reach the host cell, the PEXEL motif of exported proteins is processed by the endoplasmic reticulum (ER) resident aspartyl protease plasmepsin V. Then, following secretion into the parasite-encasing parasitophorous vacuole, the mature exported protein must be unfolded and translocated across the parasitophorous vacuole membrane by the Plasmodium translocon of exported proteins (PTEX). PTEX is a protein-conducting channel consisting of the pore-forming protein EXP2, the protein unfoldase HSP101, and structural component PTEX150. The mechanism of how exported proteins are specifically trafficked from the parasite's ER following PEXEL cleavage to PTEX complexes on the parasitophorous vacuole membrane is currently not understood. Here, we present evidence that EXP2 and PTEX150 form a stable subcomplex that facilitates HSP101 docking. We also demonstrate that HSP101 localises both within the parasitophorous vacuole and within the parasite's ER throughout the ring and trophozoite stage of the parasite, coinciding with the timeframe of protein export. Interestingly, we found that HSP101 can form specific interactions with model PEXEL proteins in the parasite's ER, irrespective of their PEXEL processing status. Collectively, our data suggest that HSP101 recognises and chaperones PEXEL proteins from the ER to the parasitophorous vacuole and given HSP101's specificity for the EXP2-PTEX150 subcomplex, this provides a mechanism for how exported proteins are specifically targeted to PTEX for translocation into the erythrocyte.

Property activity refinement of 2-anilino 4-amino substituted quinazolines as antimalarials with fast acting asexual parasite activity.

Malaria is a devastating disease caused by Plasmodium parasites. Emerging resistance against current antimalarial therapeutics has engendered the need to develop antimalarials with novel structural classes. We recently described the identification and initial optimization of the 2-anilino quinazoline antimalarial class. Here, we refine the physicochemical properties of this antimalarial class with the aim to improve aqueous solubility and metabolism and to reduce adverse promiscuity. We show the physicochemical properties of this class are intricately balanced with asexual parasite activity and human cell cytotoxicity. Structural modifications we have implemented improved LipE, aqueous solubility and in vitro metabolism while preserving fast acting P. falciparum asexual stage activity. The lead compounds demonstrated equipotent activity against P. knowlesi parasites and were not predisposed to resistance mechanisms of clinically used antimalarials. The optimized compounds exhibited modest activity against early-stage gametocytes, but no activity against pre-erythrocytic liver parasites. Confoundingly, the refined physicochemical properties installed in the compounds did not engender improved oral efficacy in a P. berghei mouse model of malaria compared to earlier studies on the 2-anilino quinazoline class. This study provides the framework for further development of this antimalarial class.

The Role of Malaria Parasite Heat Shock Proteins in Protein Trafficking and Remodelling of Red Blood Cells.

The genus Plasmodium comprises intracellular eukaryotic parasites that infect many vertebrate groups and cause deadly malaria disease in humans. The parasites employ a suite of heat shock proteins to help traffic other proteins to different compartments within their own cells and that of the host cells they parasitise. This review will cover the role of these chaperones in protein export and host cell modification in the asexual blood stage of the human parasite P. falciparum which is the most deadly and well-studied parasite species. We will examine the role chaperones play in the import of proteins into the secretory pathway from where they are escorted to the vacuole space surrounding the intraerythrocytic parasite. Here, other heat shock proteins unfold protein cargoes and extrude them into the red blood cell (RBC) cytosol from where additional chaperones of parasite and possibly host origin refold the cargo proteins and guide them to their final functional destinations within their RBC host cells. The secretory pathway also serves as a launch pad for proteins targeted to the non-photosynthetic apicoplast organelle of endosymbiotic origin, and the role of heat shock proteins in trafficking proteins here will be reviewed. Finally, the function of chaperones in protein trafficking into the mitochondrion, the remaining organelle of endosymbiotic origin, will be discussed.

Defining the Essential Exportome of the Malaria Parasite.

To survive inside red blood cells (RBCs), malaria parasites export many proteins to alter their host cell's physiological properties. Although most proteins of this exportome are involved in immune avoidance or in the trafficking of exported proteins to the host membrane, about 20% are essential for parasite survival in culture but little is known about their biological functions. Here, we have combined information from large-scale genetic screens and targeted gene-disruption studies to tabulate all currently known Plasmodium falciparum exported proteins according to their likelihood of being essential. We also discuss the essential functional pathways that exported proteins might be involved in to help direct research efforts towards a more comprehensive understanding of host-cell remodelling.

Characterisation of complexes formed by parasite proteins exported into the host cell compartment of Plasmodium falciparum infected red blood cells.

During its intraerythrocytic life cycle, the human malaria parasite Plasmodium falciparum supplements its nutritional requirements by scavenging substrates from the plasma through the new permeability pathways (NPPs) installed in the red blood cell (RBC) membrane. Parasite proteins of the RhopH complex: CLAG3, RhopH2, RhopH3, have been implicated in NPP activity. Here, we studied 13 exported proteins previously hypothesised to interact with RhopH2, to study their potential contribution to the function of NPPs. NPP activity assays revealed that the 13 proteins do not appear to be individually important for NPP function, as conditional knockdown of these proteins had no effect on sorbitol uptake. Intriguingly, reciprocal immunoprecipitation assays showed that five of the 13 proteins interact with all members of the RhopH complex, with PF3D7_1401200 showing the strongest association. Mass spectrometry-based proteomics further identified new protein complexes; a cytoskeletal complex and a Maurer's clefts/J-dot complex, which overall helps clarify protein-protein interactions within the infected RBC (iRBC) and is suggestive of the potential trafficking route of the RhopH complex itself to the RBC membrane.