Within the human host, the symptoms of malaria are caused by the replication of malaria parasites within erythrocytes. Growth inside the erythrocyte exposes the parasites to the normal surveillance of erythrocytes by the host organism, in particular the clearance of erythrocytes in the spleen. Here we show that the malaria parasite Plasmodium falciparum undergoes a rapid, multi-step metamorphosis that transforms the invasive merozoite into an amoeboid-shaped cell within minutes after invading erythrocytes. This transformation involves an increase in the parasite surface area and is mediated by factors already present in the merozoite, including the parasite phospholipid transfer protein PV6. Parasites lacking PV6 do not assume an amoeboid form and instead are spherical and have a smaller surface area than amoeboid forms. Furthermore, erythrocytes infected with P. falciparum parasites lacking PV6 undergo a higher loss of surface area upon infection, which affects the traversal of infected erythrocytes through the spleen. This is the first evidence that after invasion, the parasite undergoes a rapid, complex metamorphosis within the host erythrocyte that promotes survival in the host.

Plasmodium falciparum plasmepsin X (PMX) has become a target of choice for the development of new antimalarial drugs due to its essential role across the parasite life cycle. Here we describe the 1.7 Å crystallographic structure of PMX noncovalently bound to a potent macrocyclic peptidomimetic inhibitor (7k) possessing a hydroxyethylamine (HEA) scaffold. Upon 7k binding, the enzyme adopts a novel conformation, with significant involvement of the S2'S2 loop (M526-H536) and the S2 flap (F311-G314). This results in partial closure of the active site with widespread interactions in both the prime (S') and the non-prime (S) sites of PMX. The catalytic aspartate residues D266 and D467 directly interact with the HEA pharmacophore. Docking of a 7k derivative, compound 7a, highlights a region in the S3 pocket near the S3 flexible loop (H242-F248) that may be key for ligand stabilisation. The dynamic nature of PMX and its propensity to undergo distinct types of induced fit upon inhibitor binding enables generation of potent inhibitors that target this essential malarial aspartic protease.

We previously reported that the Plasmodium falciparum putative serine/threonine protein phosphatase 7 (PP7) is a high-confidence substrate of the cAMP-dependent protein kinase (PKA). Here we explore the function of PP7 in asexual P. falciparum blood stage parasites. We show that conditional disruption of PP7 leads to a severe growth arrest. We show that PP7 is a calcium-dependent phosphatase that interacts with calmodulin and calcium-dependent protein kinase 1 (CDPK1), consistent with a role in calcium signaling. Notably, PP7 was found to be dispensable for erythrocyte invasion, but was crucial for ring-stage development, with PP7-null parasites arresting shortly following invasion and showing no transition to ameboid forms. Phosphoproteomic analysis revealed that PP7 may regulate certain PKAc substrates. Its interaction with calmodulin and CDPK1 further emphasizes a role in calcium signaling, while its impact on early ring development and PKAc substrate phosphorylation underscores its importance in parasite development. IMPORTANCE:Plasmodium falciparum causes malaria and is responsible for more than 600,000 deaths each year. Although effective drugs are available to treat disease, the spread of drug-resistant parasites endangers their future efficacy. It is hoped that a better understanding of the biology of malaria parasites will help us to discover new drugs to tackle the resistance problem. Our work is focused on the cell signaling mechanisms that control the development of the parasite throughout its complex life cycle. All signal transduction pathways are ultimately regulated by reversible protein phosphorylation by protein kinase and protein phosphatase enzymes. In this study, we investigate the function of calcium-dependent protein phosphatase PP7 and show that it is essential for the development of ring-stage parasites following the invasion of human erythrocytes. Our results contribute to the understanding of the erythrocytic stages of the parasite life cycle that cause malaria pathology.

Plasmodium falciparum subtilisin-like serine protease 1 (PfSUB1) is essential for egress of invasive merozoite forms of the parasite, rendering PfSUB1 an attractive antimalarial target. Here, we report studies aimed to improve drug-like properties of peptidic boronic acid PfSUB1 inhibitors including increased lipophilicity and selectivity over human proteasome (H20S). Structure-activity relationship investigations revealed that lipophilic P-3 amino acid side chains as well as N-capping groups were well tolerated in retaining PfSUB1 inhibitory potency. At the P-1 position, replacing the methyl group with a carboxyethyl substituent led to boralactone PfSUB1 inhibitors with remarkably improved selectivity over H20S. Combining lipophilic end-capping groups with the boralactone reduced the selectivity over H20S. However, compound 4c still showed >60-fold selectivity versus H20S and low nanomolar PfSUB1 inhibitory potency. Importantly, this compound inhibited the growth of a genetically modified P. falciparum line expressing reduced levels of PfSUB1 13-fold more efficiently compared to a wild-type parasite line.

BACKGROUND:The malaria parasite Plasmodium falciparum replicates within red blood cells, then ruptures the cell in a process called egress in order to continue its life cycle. Egress is regulated by a proteolytic cascade involving an essential parasite subtilisin-like serine protease called SUB1. Maturation of SUB1 initiates in the parasite endoplasmic reticulum with autocatalytic cleavage of an N-terminal prodomain (p31), which initially remains non-covalently bound to the catalytic domain, p54. Further trafficking of the p31-p54 complex results in formation of a terminal p47 form of the SUB1 catalytic domain. Recent work has implicated a parasite aspartic protease, plasmepsin X (PMX), in maturation of the SUB1 p31-p54 complex through controlled cleavage of the prodomain p31. METHODS:Here we use biochemical and enzymatic analysis to examine the activation of SUB1 by PMX. RESULTS:We show that both p31 and p31-p54 are largely dimeric under the relatively acidic conditions to which they are likely exposed to PMX in the parasite. We confirm the sites within p31 that are cleaved by PMX and determine the order of cleavage. We find that cleavage by PMX results in rapid loss of the capacity of p31 to act as an inhibitor of SUB1 catalytic activity and we directly demonstrate that exposure to PMX of recombinant p31-p54 complex activates SUB1 activity. CONCLUSIONS:Our results confirm that precise, PMX-mediated cleavage of the SUB1 prodomain activates SUB1 enzyme activity. GENERAL SIGNIFICANCE:Our findings elucidate the role of PMX in activation of SUB1, a key effector of malaria parasite egress.

The malaria parasite needs nearly half of its genes to propagate normally within red blood cells. Inducible ways to interfere with gene expression like the DiCre-lox system are necessary to study the function of these essential genes. However, existing DiCre-lox strategies are not well-suited to be deployed at scale to study several genes simultaneously. To overcome this, we have developed SHIFTiKO (frameshift-based trackable inducible knockout), a novel scaleable strategy that uses short, easy-to-construct, barcoded repair templates to insert loxP sites around short regions in target genes. Induced DiCre-mediated excision of the flanked region causes a frameshift mutation resulting in genetic ablation of gene function. Dual DNA barcodes inserted into each mutant enables verification of successful modification and induced excision at each locus and collective phenotyping of the mutants, not only across multiple replication cycles to assess growth fitness but also within a single cycle to identify specific phenotypic impairments. As a proof of concept, we have applied SHIFTiKO to screen the functions of malarial rhomboid proteases, successfully identifying their blood stage-specific essentiality. SHIFTiKO thus offers a powerful platform to conduct inducible phenotypic screens to study essential gene function at scale in the malaria parasite.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. One major barrier for developing antimalarial drugs is the ability of the parasite to rapidly generate resistance. We previously demonstrated that salinipostin A (SalA), a natural product, potently kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism that results in a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a small library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent antiparasitic potencies that enabled the identification of therapeutically relevant targets. The active compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor orlistat and shows synergistic killing with orlistat. Our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are promising, synthetically tractable antimalarials.

The cGMP-dependent protein kinase (PKG) is the sole cGMP sensor in malaria parasites, acting as an essential signalling hub to govern key developmental processes throughout the parasite life cycle. Despite the importance of PKG in the clinically relevant asexual blood stages, many aspects of malarial PKG regulation, including the importance of phosphorylation, remain poorly understood. Here we use genetic and biochemical approaches to show that reduced cGMP binding to cyclic nucleotide binding domain B does not affect in vitro kinase activity but prevents parasite egress. Similarly, we show that phosphorylation of a key threonine residue (T695) in the activation loop is dispensable for kinase activity in vitro but is essential for in vivo PKG function, with loss of T695 phosphorylation leading to aberrant phosphorylation events across the parasite proteome and changes to the substrate specificity of PKG. Our findings indicate that Plasmodium PKG is uniquely regulated to transduce signals crucial for malaria parasite development.

For its replication within red blood cells, the malaria parasite depends on a highly active and regulated lipid metabolism. Enzymes involved in lipid metabolic processes such as phospholipases are, therefore, potential drug targets. Here, using reverse genetics approaches, we show that only 1 out of the 19 putative phospholipases expressed in asexual blood stages of Plasmodium falciparum is essential for proliferation in vitro, pointing toward a high level of redundancy among members of this enzyme family. Using conditional mislocalization and gene disruption techniques, we show that this essential phosphoinositide-specific phospholipase C (PI-PLC, PF3D7_1013500) has a previously unrecognized essential role during intracellular parasite maturation, long before its previously perceived role in parasite egress and invasion. Subsequent lipidomic analysis suggests that PI-PLC mediates cleavage of phosphatidylinositol bisphosphate (PIP2) in schizont-stage parasites, underlining its critical role in regulating phosphoinositide levels in the parasite. IMPORTANCE The clinical symptoms of malaria arise due to repeated rounds of replication of Plasmodium parasites within red blood cells (RBCs). Central to this is an intense period of membrane biogenesis. Generation of membranes not only requires de novo synthesis and acquisition but also the degradation of phospholipids, a function that is performed by phospholipases. In this study, we investigate the essentiality of the 19 putative phospholipase enzymes that the human malaria parasite Plasmodium falciparum expresses during its replication within RBCs. We not only show that a high level of functional redundancy exists among these enzymes but, at the same time, also identify an essential role for the phosphoinositide-specific phospholipase C in parasite development and cleavage of the phospholipid phosphatidylinositol bisphosphate.

The Plasmodium falciparum aspartic protease plasmepsin X (PMX) is essential for the egress of invasive merozoite forms of the parasite. PMX has therefore emerged as a new potential antimalarial target. Building on peptidic amino alcohols originating from a phenotypic screening hit, we have here developed a series of macrocyclic analogues as PMX inhibitors. Incorporation of an extended linker between the S1 phenyl group and S3 amide led to a lead compound that displayed a 10-fold improved PMX inhibitory potency and a 3-fold improved half-life in microsomal stability assays compared to the acyclic analogue. The lead compound was also the most potent of the new macrocyclic compounds in in vitro parasite growth inhibition. Inhibitor 7k cleared blood-stage P. falciparum in a dose-dependent manner when administered orally to infected humanized mice. Consequently, lead compound 7k represents a promising orally bioavailable molecule for further development as a PMX-targeting antimalarial drug.

The lack of a long-term in vitro culture method has severely restricted the study of Plasmodium vivax, in part because it limits genetic manipulation and reverse genetics. We used the recently optimized Plasmodium cynomolgi Berok in vitro culture model to investigate the putative P. vivax drug resistance marker MDR1 Y976F. Introduction of this mutation using clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) increased sensitivity to mefloquine, but had no significant effect on sensitivity to chloroquine, amodiaquine, piperaquine, and artesunate. To our knowledge, this is the first reported use of CRISPR-Cas9 in P. cynomolgi, and the first reported integrative genetic manipulation of this species.

To ensure their survival in the human bloodstream, malaria parasites degrade up to 80% of the host erythrocyte hemoglobin in an acidified digestive vacuole. Here, we combine conditional reverse genetics and quantitative imaging approaches to demonstrate that the human malaria pathogen Plasmodium falciparum employs a heteromultimeric V-ATPase complex to acidify the digestive vacuole matrix, which is essential for intravacuolar hemoglobin release, heme detoxification, and parasite survival. We reveal an additional function of the membrane-embedded V-ATPase subunits in regulating morphogenesis of the digestive vacuole independent of proton translocation. We further show that intravacuolar accumulation of antimalarial chemotherapeutics is surprisingly resilient to severe deacidification of the vacuole and that modulation of V-ATPase activity does not affect parasite sensitivity toward these drugs.

Malaria parasite release (egress) from host red blood cells involves parasite-mediated membrane poration and rupture, thought to involve membrane-lytic effector molecules such as perforin-like proteins and/or phospholipases. With the aim of identifying these effectors, we disrupted the expression of two Plasmodium falciparum perforin-like proteins simultaneously and showed that they have no essential roles during blood stage egress. Proteomic profiling of parasite proteins discharged into the parasitophorous vacuole (PV) just prior to egress detected the presence in the PV of a lecithin:cholesterol acyltransferase (LCAT; PF3D7_0629300). Conditional ablation of LCAT resulted in abnormal egress and a reduced replication rate. Lipidomic profiles of LCAT-null parasites showed drastic changes in several phosphatidylserine and acylphosphatidylglycerol species during egress. We thus show that, in addition to its previously demonstrated role in liver stage merozoite egress, LCAT is required to facilitate efficient egress in asexual blood stage malaria parasites.

ABSTRACT Plasmodium parasites rely on a functional electron transport chain (ETC) within their mitochondrion for proliferation, and compounds targeting mitochondrial functions are validated antimalarials. Here, we localize Plasmodium falciparum patatin-like phospholipase 2 ( Pf PNPLA2, PF3D7_1358000) to the mitochondrion and reveal that disruption of the Pf PNPLA2 gene impairs asexual replication. Pf PNPLA2-null parasites are hypersensitive to proguanil and inhibitors of the mitochondrial ETC, including atovaquone. In addition, Pf PNPLA2-deficient parasites show reduced mitochondrial respiration and reduced mitochondrial membrane potential, indicating that disruption of Pf PNPLA2 leads to a defect in the parasite ETC. Lipidomic analysis of the mitochondrial phospholipid cardiolipin (CL) reveals that loss of Pf PNPLA2 is associated with a moderate shift toward shorter-chained and more saturated CL species, implying a contribution of Pf PNPLA2 to CL remodeling. Pf PNPLA2-deficient parasites display profound defects in gametocytogenesis, underlining the importance of a functional mitochondrial ETC during both the asexual and sexual development of the parasite. IMPORTANCE For their proliferation within red blood cells, malaria parasites depend on a functional electron transport chain (ETC) within their mitochondrion, which is the target of several antimalarial drugs. Here, we have used gene disruption to identify a patatin-like phospholipase, Pf PNPLA2, as important for parasite replication and mitochondrial function in Plasmodium falciparum . Parasites lacking Pf PNPLA2 show defects in their ETC and become hypersensitive to mitochondrion-targeting drugs. Furthermore, Pf PNPLA2-deficient parasites show differences in the composition of their cardiolipins, a unique class of phospholipids with key roles in mitochondrial functions. Finally, we demonstrate that parasites devoid of Pf PNPLA2 have a defect in gametocyte maturation, underlining the importance of a functional ETC for parasite transmission to the mosquito vector.

In model organisms P4-ATPases require cell division control protein 50 (CDC50) chaperones for their phospholipid flipping activity. In the malaria parasite, P. falciparum, guanylyl cyclase alpha (GCα) is an integral membrane protein that is essential for release (egress) of merozoites from their host erythrocytes. GCα is unusual in that it contains both a C-terminal cyclase domain and an N-terminal P4-ATPase domain of unknown function. We sought to investigate whether any of the three CDC50 orthologues (denoted A, B and C) encoded by P. falciparum are required for GCα function. Using gene tagging and conditional gene disruption, we demonstrate that both CDC50B and CDC50C are expressed in the clinically important asexual blood stages and that CDC50B is a binding partner of GCα whereas CDC50C is the binding partner of another putative P4-ATPase, ATP2. Our findings indicate that CDC50B has no essential role for intraerythrocytic parasite maturation but modulates the rate of parasite egress by interacting with GCα for optimal cGMP synthesis. In contrast, CDC50C is essential for blood stage trophozoite maturation. Additionally, we find that the CDC50C-ATP2 complex may influence parasite endocytosis of host cell haemoglobin and consequently hemozoin formation.

Widespread resistance to many antimalarial therapies currently in use stresses the need for the discovery of new classes of drugs with new modes of action. The subtilisin-like serine protease SUB1 controls egress of malaria parasites (merozoites) from the parasite-infected red blood cell. As such, SUB1 is considered a prospective target for drugs designed to interrupt the asexual blood stage life cycle of the malaria parasite. Inhibitors of SUB1 have potential as wide-spectrum antimalarial drugs, as a single orthologue of SUB1 is found in the genomes of all known Plasmodium species. This mini-perspective provides a short overview of the function and structure of SUB1 and summarizes all of the published SUB1 inhibitors. The inhibitors are classified by the methods of their discovery, including both rational design and screening.

The malaria parasite Plasmodium falciparum synthesizes significant amounts of phospholipids to meet the demands of replication within red blood cells. De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway is essential, requiring choline that is primarily sourced from host serum lysophosphatidylcholine (lysoPC). LysoPC also acts as an environmental sensor to regulate parasite sexual differentiation. Despite these critical roles for host lysoPC, the enzyme(s) involved in its breakdown to free choline for PC synthesis are unknown. Here, we show that a parasite glycerophosphodiesterase (PfGDPD) is indispensable for blood stage parasite proliferation. Exogenous choline rescues growth of PfGDPD-null parasites, directly linking PfGDPD function to choline incorporation. Genetic ablation of PfGDPD reduces choline uptake from lysoPC, resulting in depletion of several PC species in the parasite, whilst purified PfGDPD releases choline from glycerophosphocholine in vitro. Our results identify PfGDPD as a choline-releasing glycerophosphodiesterase that mediates a critical step in PC biosynthesis and parasite survival.

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Basigin is an essential host receptor for invasion of Plasmodium falciparum into human erythrocytes, interacting with parasite surface protein PfRH5. PfRH5 is a leading blood-stage malaria vaccine candidate and a target of growth-inhibitory antibodies. Here, we show that erythrocyte basigin is exclusively found in one of two macromolecular complexes, bound either to plasma membrane Ca2+-ATPase 1/4 (PMCA1/4) or to monocarboxylate transporter 1 (MCT1). PfRH5 binds to each of these complexes with a higher affinity than to isolated basigin ectodomain, making it likely that these are the physiological targets of PfRH5. PMCA-mediated Ca2+ export is not affected by PfRH5, making it unlikely that this is the mechanism underlying changes in calcium flux at the interface between an erythrocyte and the invading parasite. However, our studies rationalise the function of the most effective growth-inhibitory antibodies targeting PfRH5. While these antibodies do not reduce the binding of PfRH5 to monomeric basigin, they do reduce its binding to basigin-PMCA and basigin-MCT complexes. This indicates that the most effective PfRH5-targeting antibodies inhibit growth by sterically blocking the essential interaction of PfRH5 with basigin in its physiological context. Editor's evaluation This elegantly performed and rigorous study generates new and conceptually important insights into the interaction between an essential malaria parasite invasion ligand (and vaccine candidate) called PfRH5, and its erythrocyte surface integral membrane receptor basigin. The authors provide compelling evidence based on rigorous biochemical assays that erythrocyte basigin is predominantly expressed in a complex with one of two distinct erythrocyte membrane proteins called PMCA and MCT1 and that PfRH5 binds to these complexes better than to isolated basigin. Certain invasion-inhibitory antibodies, that do not prevent binding of PfRH5 to isolated basigin, do in contrast prevent binding to the basigin complexes, explaining the mode of action of these previously enigmatic antibodies and providing valuable data towards the improved design of vaccines based on PfRH5. https://doi.org/10.7554/eLife.83681.sa0 Decision letter Reviews on Sciety eLife's review process Introduction Malaria is still one of the most deadly parasitic diseases to affect humans, with Plasmodium falciparum as the causative agent of the most severe cases (Sato, 2021). The clinical symptoms of malaria occur as the merozoite developmental form of the parasite invades and replicates within human red blood cells (RBCs) (Venugopal et al., 2020). Vaccines or therapeutics which block erythrocyte invasion therefore have the potential to contribute to reduction and elimination of malaria. Erythrocyte invasion is driven by a series of molecular interactions between merozoite ligands and their receptors on erythrocyte surfaces (Cowman et al., 2017). While many of these interactions are redundant, the binding of merozoite P. falciparum reticulocyte homologue 5 (PfRH5) to erythrocyte basigin is essential for invasion by all tested P. falciparum strains (Crosnier et al., 2011), making PfRH5 one of the most promising blood-stage malaria vaccine candidates. PfRH5 is part of the five-component PfPCRCR complex, containing PfRH5, PfCyRPA, PfRIPR, PfPTRAMP, and PfCSS, and each component is essential for invasion and is a target of growth-inhibitory antibodies or nanobodies (Farrell et al., 2023; Reddy et al., 2015; Scally et al., 2022; Wong et al., 2019). Vaccination of human volunteers with PfRH5 elicits strain-transcending anti-malarial antibodies (Payne et al., 2017) and, in a human challenge model, reduces the rate of parasite growth (Minassian et al., 2021). Structural studies of PfRH5 have revealed that basigin binds towards one of the tips of the ‘kite-like’ structure of PfRH5 (Wright et al., 2014). Structures of PfRH5 in complex with Fab fragments of monoclonal antibodies have identified a number of important epitopes across PfRH5, including those for human and mouse growth-inhibitory antibodies (R5.004, R5.016, 9AD4, and QA1) (Alanine et al., 2019; Wright et al., 2014), as well as the epitope for an antibody which potentiates the effects of growth-inhibitory antibodies (R5.011) (Alanine et al., 2019). This insight has guided development of improved PfRH5-containing vaccines (Campeotto et al., 2017). However, mysteries remain, with the most effective growth-inhibitory antibodies, 9AD4 and R5.016 not able to block basigin binding. How do they prevent invasion? Basigin is a type I transmembrane protein of the immunoglobulin (Ig) superfamily, with the most common isoform presenting an ectodomain consisting of two highly glycosylated extracellular Ig domains (Muramatsu, 2016), both of which contact PfRH5 (Wright et al., 2014). More recently, the transmembrane segment of basigin has been shown to mediate formation of heteromeric complexes which contain basigin and either monocarboxylate transporters (MCTs) or plasma membrane calcium ATPases (PMCAs) (Muramatsu, 2016; Supper et al., 2016). MCTs are involved in proton-coupled exchange of lactate or pyruvate across plasma membranes (Felmlee et al., 2020). Both MCT1 and MCT4 interact with basigin in multiple eukaryotic cell lines (Kirk et al., 2000), affecting their function and stability (Kirk et al., 2000) and MCT1 is the primary subtype in human erythrocytes (Juel et al., 2003). PMCAs also play a role in membrane transport, with four subtypes of PMCA removing Ca2+ from the cell cytosol to maintain intracellular Ca2+ homeostasis and modulate cell signalling (Stafford et al., 2017). Either basigin, or its close paralog neuroplastin, are essential subunits of PMCA, influencing expression and function of PMCAs in rodent neurons, with human erythrocyte PMCAs complexed with basigin (Schmidt et al., 2017). Both basigin-MCT1 and neuroplastin-PMCA1 complexes have been structurally characterised, revealing interactions mediated by transmembrane helices (Gong et al., 2018; Wang et al., 2021), with the PfRH5 binding site on the basigin ectodomain remaining accessible. Proteomics studies have confirmed that PMCA1, PMCA4, and MCT1 are all expressed on human RBC plasma membrane (Ravenhill et al., 2019) and, intriguingly, the actions of both MCTs and PMCAs have been linked to severe malaria (Bedu-Addo et al., 2013; Mariga et al., 2014; Timmann et al., 2012). In the case of MCT1, cerebral malaria is associated with increased lactate concentrations, which may damage the blood-brain barrier. However, it is hard to envisage a mechanism by which modulation of MCT1 function by PfRH5 during the rapid process of erythrocyte invasion could contribute to the symptoms of malaria. In contrast, PMCA polymorphisms have been linked to the development of severe malaria in a genome-wide association study, suggesting that calcium homeostasis across the erythrocyte membrane may affect the outcomes of malaria. Spikes of increased intra-erythrocytic calcium have been observed during the parasite invasion process and these are dependent on the interaction between PfRH5 and basigin (Volz et al., 2016; Weiss et al., 2015), raising the question of whether localised blockage of PMCA-mediated calcium efflux may cause these calcium spikes. In this study, we therefore asked whether PfRH5 can interact with basigin which is complexed with MCT1 and PMCAs, whether this modulates PMCA function and whether this explains the function of growth-inhibitory antibodies. Results Basigin in human erythrocyte is primarily found in complex with PMCAs and MCTs We first aimed to determine whether basigin is mostly free when extracted from human erythrocyte membranes or is predominantly in complex with PMCAs or MCTs. We solubilised human erythrocyte ghosts using a mild detergent mixture of n-dodecyl-β-D-maltose and cholesterol hemisuccinate (DDM:CHS), fractionated this extract by size exclusion chromatography and used western blotting to assess where basigin, PMCAs, and MCT1 elute (Figure 1a). Probing individual fractions with a basigin-binding antibody revealed a broad band indicating the presence of heterogeneously glycosylated basigin. Fractions rich in basigin also showed strong signals for both PMCAs and MCT1, indicating that the majority of basigin co-migrates with PMCAs and MCT1 (Figure 1a). The three components predominantly eluted in a region of the migration profile between standards of mass 157 kDa and 200 kDa, compatible with the mass of detergent-solubilised basigin-MCT1 and basigin-PMCA complexes (Figure 1a). No basigin was found in later fractions in the region of the profile which would correspond to the mass of a single basigin in a detergent micelle. Figure 1 with 2 supplements see all Download asset Open asset Basigin from human erythrocytes is found in complex with plasma membrane calcium ATPase (PMCA) or monocarboxylate transporter 1 (MCT1). (a) The upper panel shows the trace from size exclusion chromatography of n-dodecyl-β-D-maltose and cholesterol hemisuccinate (DDM/CHS)-solubilised ghost membrane proteins fractionated on a Superdex 200 increase 10/300 column (blue) and for full-length basigin purified from insect cells in CHAPS and exchanged into DDM:CHS (green). The elution volumes of molecular weight standards are indicated above the trace. Fractions collected are demarcated by vertical red dotted lines. The lower panel shows western blots of fractions 2–14. Representative blots for basigin (40–65 kDa, upper panel), PMCA (~138 kDa, upper middle panel), and MCT1 (~45 kDa, lower middle panel) show that all three proteins co-elute predominantly in fractions 7–9. The lower panel shows the equivalent blot for full-length basigin (FL-BSG), related to the green trace. Data shown are representative from n=2 biological replicates. (b) Western blot analysis of 2D blue native PAGE/SDS-PAGE separations of human erythrocyte membrane solubilisates before (upper panel) and after pre-incubation with anti-basigin antibody (lower panel). Blot membranes were stained with antibodies specific for PMCA1/4, basigin and MCT1. Markers of apparent complex size indicate the positions of known mitochondrial respiratory chain (super)complexes (Schägger and Pfeiffer, 2000) run in a separate gel lane. Binding of the antibody led to a full size-shift of both PMCA1/4-basigin and MCT1-basigin complexes, whereas no signal of free basigin could be observed in the low molecular weight range, even after overexposure of the blot. Data shown are representative from n=2 biological replicates. (c) The left-hand panel shows representative western blot images depicting sequential depletion of PMCA (upper panel) and MCT1 (upper-middle panel). Depletion of both transporters leads to reduced basigin levels, while levels of glycophorin C are unaffected in each fraction, again confirming that basigin is in complex with PMCA or MCT1. The remaining panels show densitometry plots obtained from inverted images of the western blots. Mean integrated band densities are shown with error bars as the standard error of the mean (n=3) and represent technical replicates. (d) Bar diagram depicting molecular abundances (abundancenormspec values) of the indicated proteins in depleting affinity purifications with anti-basigin and anti-neuroplastin (NPTN) antibodies from mildly solubilised human erythrocyte membranes as determined by mass spectrometry. The abundance for all proteins was 0 after affinity purification with an IgG control. Data shown are representative from n=8 biological replicates for basigin and n=5 biological replicates for neuroplastin. Figure 1—source data 1 Data associated with Figure 1. https://cdn.elifesciences.org/articles/83681/elife-83681-fig1-data1-v2.xlsx Download elife-83681-fig1-data1-v2.xlsx Figure 1—source data 2 Gels and blots associated with Figure 1. https://cdn.elifesciences.org/articles/83681/elife-83681-fig1-data2-v2.zip Download elife-83681-fig1-data2-v2.zip To calibrate this experiment, we expressed full-length basigin in baculovirus-infected Sf9 cells. This was extracted and purified using either CHAPS or DDM:CHS. The DDM:CHS-extracted protein purified with other protein components, migrated as a broad peak on a size exclusion column and showed complex binding kinetics for PfRH5 (Figure 1—figure supplement 1a, b). In contrast, protein extracted with CHAPS appeared pure on a gel, showed an elution profile indicating it to be monodispersed on a size exclusion column and bound to PfRH5, as determined by surface plasmon resonance, with binding kinetics which matched those of basigin ectodomain (Figure 1—figure supplement 1a, b). We therefore concluded that insect cell expressed full-length basigin also associates with binding partners in Sf9 cells which co-purify in DDM:CHS but are removed using the harsher detergent CHAPS. To generate a sample suitable to calibrate our size exclusion experiment, we therefore purified full-length basigin after solubilisation with CHAPS and then detergent exchanged this sample into DDM:CHS, producing monomeric full-length basigin in a detergent micelle which matched that of our basigin-containing complexes purified from erythrocytes (Figure 1—figure supplement 1a, b). This full-length basigin eluted at ~11.2 ml from a size exclusion column, later than the basigin-containing complexes extracted from erythrocytes (Figure 1a). These data suggest that erythrocyte basigin is predominantly found in complexes which co-elute with MCT1 and PMCA. To more closely investigate the association of basigin with PMCAs and MCT1, we performed antibody shift assays in two different settings. First, we used size exclusion chromatography to assess whether antibodies alter the mobility of basigin-MCT1 complexes (Figure 1—figure supplement 2). A basigin-rich solubilised ghost extract was applied to a size exclusion column either in the presence or absence of an MCT1-targeting monoclonal antibody and the resultant fractions were probed on western blots for MCT1, basigin, or a glycophorin C control. While there was no shift of glycophorin C, the addition of the MCT1-specific antibody caused a shift in elution profile of the majority of the MCT1 and ~53% of basigin, suggesting them to be in complex (Figure 1—figure supplement 2). However, ~47% of the basigin did not shift. This identifies pools of basigin which are found in complex with and without MCT1. Next, we performed antibody shift experiments on detergent-solubilised human erythrocyte membrane extracts resolved by 2D blue native PAGE (Figure 1b, Figure 1—figure supplement 1c). Western blotting these 2D gels and staining with basigin, PMCA, and MCT1 antibodies indicated two separate populations of basigin-containing complexes, one of which co-migrated with PMCA and one of which co-migrated with MCT1. Inclusion of an antibody which quantitatively binds to basigin shifted both populations to higher molecular weight (Figure 1b). Signals at the appropriate molecular weight for free basigin were not observed in either the presence or absence of the basigin-binding antibody (Figure 1b, Figure 1—figure supplement 1c), even upon prolonged exposure of the blots. In parallel, we conducted a depletion experiment. PMCAs and MCT1 were sequentially depleted from solubilised ghosts. PMCAs were first depleted by affinity capture on a calmodulin (CaM) resin, which binds to a regulatory site on the cytoplasmic tails of PMCAs (Stafford et al., 2017), while a monoclonal antibody was used to subsequently immunodeplete MCT1. Western blotting was used to assess the fraction of PMCA, MCT1, and basigin depleted at each stage and these were quantified by densitometry (Figure 1c, Figure 1—figure supplement 1d). Sequential depletions of both PMCA and MCT1 resulted in almost complete elimination of basigin from the solubilised ghost membrane proteome, confirming that nearly all basigin is complexed with one of these transporters. Densitometric measurements also showed greater reduction in basigin levels upon MCT1 depletion, with ~72% of basigin complexed with MCT1 and ~28% of basigin complexed with PMCA. No basigin was depleted using beads alone (Figure 1—figure supplement 1e). As there are multiple PMCA subtypes in human cells, we next used mass spectrometry analysis to identify which subtypes co-purify with basigin from human erythrocytes. This revealed PMCA4 to be the major partner of basigin in human erythrocytes, while also identifying PMCA1 (Figure 1d). Also identified were MCTs, with MCT1 as the primary subtype and MCT2 and -4 also detected (Figure 1d). Collectively, these data show that native basigin in human erythrocytes is found only in heteromeric complexes, either with PMCA (predominantly PMCA4) or MCT1. As the interaction between PfRH5 and basigin has been shown to be essential for erythrocyte invasion by P. falciparum, this raises the question of whether PfRH5 can bind to either the basigin-PMCA and/or basigin-MCT1 complexes. PfRH5 binds to the basigin-PMCA complex with greater affinity than to monomeric basigin We next investigated the binding of PfRH5 to basigin-PMCA complexes. The structure of the neuroplastin-PMCA1 complex shows the neuroplastin ectodomain to be presented above the extracellular loops of PMCA, with the surface of neuroplastin equivalent to that used by basigin to bind to PfRH5 being exposed, making it likely that PfRH5 can bind to PMCA-bound basigin (Figure 2a; Gong et al., 2018). To test this, we purified basigin-PMCA complexes from detergent solubilised human erythrocyte ghost cells using CaM affinity chromatography. Elution fractions showed a prominent band at ~138 kDa on a Coomassie-stained SDS-PAGE gel, corresponding to the mass of PMCA (Figure 2—figure supplement 1a). Western blotting confirmed the co-purification of basigin with PMCA (Figure 2—figure supplement 1a). Basigin and PMCA eluted as a single peak on a size exclusion column, with an elution profile matching a 200 kDa standard, suggesting complex formation (Figure 2b). The purified complex showed Ca2+-dependent ATP hydrolysis activity, confirming that active basigin-PMCA complexes had been isolated (Figure 2—figure supplement 1b). Figure 2 with 2 supplements see all Download asset Open asset P. falciparum reticulocyte homologue 5 (PfRH5) binds to basigin-plasma membrane calcium ATPase (PMCA) without affecting calcium pumping activity. (a) A structural model showing a complex of basigin (blue) and PMCA (pink) (based on PDB: 6A69, Gong et al., 2018) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, Wright et al., 2014) has been docked. (b) A size exclusion profile obtained for calmodulin affinity chromatography-purified PMCA from human erythrocyte ghosts separated on a Superdex 200 increase 10/300 column. The inset shows a western blot probed with an anti-basigin monoclonal antibody indicating co-migration of basigin with PMCA. Data shown are representative from n=3 biological replicates. (c) A surface plasmon resonance (SPR) sensogram showing the binding of a concentration series of full-length basigin (twofold dilutions from 4 μM) to immobilised PfRH5. Black lines show data and dashed red lines show fitting to a 1:1 binding model, with a dissociation constant of 0.56 μM. Data shown are representative from n=3 biological replicates. (d) An SPR sensogram showing the binding of a concentration series of basigin-PMCA (twofold dilutions from 400 nM) to immobilised PfRH5. Black lines show data and dashed red lines show fitting to a two-state binding model. The derived rate and affinity constants are presented in Supplementary files 1 and 2. Data shown are representative from n=2 biological replicates. (e) Activation curves of BKCa channels recorded in Chinese hamster ovary (CHO) cells expressing BKCa channels alone (grey), or cells also transfected with human PMCA4b and basigin with (red) and without (black) addition of PfRH5 at 2 μM concentration. Data shown are from n=3 biological replicates and error bars show standard error of mean. (f) Concentration-response curves for the inhibition of Ca2+-ATPase activity, as determined by measuring inorganic phosphate production, of purified basigin-PMCA complex by known inhibitor caloxin 1c2 (red) and PfRH5 (blue). Each data point represents the mean and error bars represent the standard error of the mean of technical replicates (n=3, technical replicates). Figure 2—source data 1 Data associated with Figure 2. https://cdn.elifesciences.org/articles/83681/elife-83681-fig2-data1-v2.xlsx Download elife-83681-fig2-data1-v2.xlsx Figure 2—source data 2 Gels and blots associated with Figure 2. https://cdn.elifesciences.org/articles/83681/elife-83681-fig2-data2-v2.zip Download elife-83681-fig2-data2-v2.zip Binding of purified basigin-PMCA complexes to PfRH5 was measured by surface plasmon resonance (SPR) analysis. PfRH5 was chemically biotinylated on lysine residues and was immobilised on a streptavidin-coated chip and either 0.4 μM basigin-PMCA or 4 μM of basigin ectodomain were injected. Basigin ectodomain bound with similar kinetics to those previously observed (Figure 2—figure supplement 1c; Crosnier et al., 2011; Wright et al., 2014). Basigin-PMCA complexes bound PfRH5 with a sensogram profile indicating slower on- and off-rates (Figure 2—figure supplement 1d). To confirm that these responses are specific, we pre-incubated surface-immobilised PfRH5 with monoclonal antibody R5.004, which occludes the basigin binding site (Alanine et al., 2019). This blocked the binding of both basigin ectodomain and basigin-PMCA to PfRH5, as expected (Figure 2—figure supplement 1c and d). To measure binding kinetics, we produced PfRH5 carrying an N-terminal biotin acceptor peptide (BAP) tag. This was captured at low density on a streptavidin-coated SPR chip and binding was studied by injecting twofold dilution series of either basigin ectodomain (Figure 2—figure supplement 1e), full-length basigin (purified in CHAPS and exchanged into DDM:CHS) (Figure 2c), or basigin-PMCA (Figure 2d). Kinetic binding data for full-length basigin could be fitted to a 1:1 binding model with an affinity of 0.56 µM (χ2=2.44), in good agreement with the affinity of ~1 µM measured for basigin ectodomain in previous studies (Figure 2—figure supplement 1e; Crosnier et al., 2011; Wright et al., 2014). In contrast, the kinetic data for the basigin-PMCA complexes did not fit well to a 1:1 binding model (χ2=7.71) (Figure 2—figure supplement 1f), but the fit improved significantly (χ2=1.85) when using a two-state binding model (Figure 2—figure supplement 1f, Supplementary files 1 and 2). This model is consistent with an interaction which involves two separate, successive events, such as an initial lower affinity capture event, followed by a second binding event which increases the overall affinity, or a conformational change which follows an initial binding event. PfRH5 therefore bound to basigin-PMCA ~9-fold more tightly than to full-length basigin, primarily due to a lower dissociation rate constant (Supplementary files 1 and 2). PfRH5 binding does not modulate calcium transport mediated by basigin-PMCA complexes We next investigated whether PfRH5 binding can modulate the activity of PMCA, perhaps accounting for changes in calcium flux at the junction between the erythrocyte and invading merozoite. To test this, we used an established system in which current recordings from BKCa-type Ca2+-activated K+ channels were used as a readout of apparent intracellular Ca2+ concentration (Schmidt et al., 2017). The expression of PMCA4b and basigin, together with BKCa, in this system caused reduced intracellular Ca2+ concentrations, as demonstrated by a right shift in the BKCa activation curve. We confirmed that the PMCA4-basigin complex formed in these cells using 2D blue native PAGE western blot analysis of solubilised membranes (Figure 2—figure supplement 2a). We next added PfRH5 at a concentration of 2 μM which caused no further change in the BKCa activation curve, indicating that PfRH5 binding did not modulate PMCA-basigin function and did not change the intracellular Ca2+ concentration (Figure 2e). As PfRH5 is normally part of the three-component PfRCR complex, we also added PfRCR at a concentration of 1 μM to the same system. Here too, we observed no effect on intracellular Ca2+ concentration (Figure 2—figure supplement 2b and c). Finally, we studied the effect of PfRH5 on Ca2+-dependent ATPase activity of purified basigin-PMCA by following inorganic phosphate release through a colorimetric assay. While the extracellular peptide inhibitor caloxin 1c2 inhibited calcium-dependent ATP hydrolysis at high micromolar concentrations, the addition of PfRH5 did not alter the ATPase activity of PMCA (Figure 2f). We therefore see no evidence that PfRH5 binding affects the activity of the basigin-PMCA calcium pump. PfRH5 binds to the basigin-MCT1 complex with greater affinity than for monomeric basigin As a large fraction of erythrocyte basigin is associated with MCT1, we also tested whether the basigin-MCT1 complex can bind to PfRH5, as the PfRH5-binding site of basigin is exposed in the structure of the basigin-MCT1 complex (Wang et al., 2021; Figure 3a). We first studied the binding of PfRH5 to solubilised ghosts and their depleted forms using SPR. Flowing solubilised ghost membrane extract over a chip surface coated with PfRH5 produced a binding response (Figure 3b). This reduced ~22% after depletion of PMCA-containing complexes, and the binding response was nearly absent for samples depleted for both PMCAs and MCT1 (Figure 3b). This suggests that basigin in complex with either MCT1 or PMCAs can bind to PfRH5. Figure 3 with 2 supplements see all Download asset Open asset P. falciparum reticulocyte homologue 5 (PfRH5) binds to basigin-monocarboxylate transporter 1 (MCT1). (a) A structural model showing a complex of basigin (blue) and MCT1 (cyan) (based on PDB: 6LYY, Wang et al., 2021) onto which the complex of PfRH5 (yellow) and basigin (blue) (PDB:4U0Q, Wright et al., 2014) has been docked. (b) Surface plasmon resonance (SPR) traces after flowing detergent solubilised erythrocyte membrane basigin-rich fractions (red) and membrane fractions depleted for plasma membrane calcium ATPase (PMCA) (pink) and both PMCA and MCT1 (orange) over a PfRH5-coated surface. (c) An SPR sensogram showing the binding of a concentration series of basigin-MCT1 (twofold dilutions from 1600 nM) to immobilised PfRH5. Black lines show data and dotted red lines show fitting to a two-state binding model. Data shown are representative from n=3 technical replicates. (d) Purified PfRH5 and basigin-MCT1 were assayed for complex formation through size exclusion chromatography using a Superdex 200 increase 10/300 column. The elution profile of basigin-MCT1 alone (blue) and in the presence of PfRH5 (red) are shown. The inset SDS-PAGE gel shows the protein species present in the fractions indicated by stars in the elution trace. (e) Growth inhibition assays assessed the effect of antibodies targeting basigin (at 1 μg/ml) and MCT-1 and PMCAs (both at 75 μg/ml) on P. falciparum growth in human blood culture. Five mM EDTA was used as a positive control (100% growth inhibition) while complete media was used as a negative control (0% growth inhibition). Data are the mean and standard deviation of three technical replicates, with a single example from three biological repeats shown. Figure 3—source data 1 Data associated with Figure 3. https://cdn.elifesciences.org/articles/83681/elife-83681-fig3-data1-v2.xlsx Download elife-83681-fig3-data1-v2.xlsx Figure 3—source data 2 Gels and blots associated with Figure 3. https://cdn.elifesciences.org/articles/83681/elife-83681-fig3-data2-v2.zip Download elife-83681-fig3-data2-v2.zip We next expressed the basigin-MCT1 complex in Sf9 cells and tested its binding to PfRH5. To measure the affinity of the interaction, monobiotinylated PfRH5 was coupled to an SPR chip before injection of a twofold dilution series of purified basigin-MCT1 complex. Again, the kinetic data could be best described by a two-state binding model (χ2=3.09) with a KD value of 93 nM (Figure 3c, Figure 3—figure supplement 1a, Supplementary files 1 and 2). One of the predictions of a two-state binding model is that longer periods of incubation of PfRH5 with the basigin-MCT1 complex allow a larger fraction of interacting species to form stable complexes due to completion of the second binding event, and lead to slower off-rates. When we injected basigin-MCT1 over a chip decorated with biotinylated PfRH5, we indeed observed slower off-rates to be associated with longer incubation times, confirming a two-state interaction (Figure 3—figure supplement 1b). We also mixed basigin-MCT1 with PfRH5 for analysis by size exclusion chromatography, and again observed formation of a stable complex (Figure 3d). Therefore, PfRH5 binds to both basigin-PMCA and basigin-MCT1 with similar affinities, both more tightly than the interaction with the isolated basigin ectodomain. Two antibodies that bind to PMCAs and MCT1 do not inhibit parasite growth We next assessed whether polyclonal antibodies that bind to PMCAs and MCT1 can prevent the growth of P. falciparum in human blood culture. We obtained the only two commercially available antibodies which bind to the extracellular regions of either PMCAs or MCT1, together with the growth-inhibitory basigin-binding monoclonal antibody, TRA-1–85. We first assesse

All intracellular pathogens must escape (egress) from the confines of their host cell to disseminate and proliferate. The malaria parasite only replicates in an intracellular vacuole or in a cyst, and must undergo egress at four distinct phases during its complex life cycle, each time disrupting, in a highly regulated manner, the membranes or cyst wall that entrap the parasites. This Cell Science at a Glance article and accompanying poster summarises our current knowledge of the morphological features of egress across the Plasmodium life cycle, the molecular mechanisms that govern the process, and how researchers are working to exploit this knowledge to develop much-needed new approaches to malaria control.